Methods and agents for promoting the removal of pathological proteins by the ubiquitin-proteasome system and the autophagy-lysosome system

Abstract

A method for regulating and optimizing the ubiquitin proteasome system and / or the autophagy lysosome system to promote the degradation of pathological proteins inside and outside the cells, as well as a drug that promotes the degradation of pathological proteins inside and outside the cells and its use.

Description

[Technical Field]

[0001] The present application relates to the use of plasminogen activation pathway-related compounds, such as plasminogen or plasmin, to promote the removal of pathological proteins by the ubiquitin-proteasome and autophagy-lysosomal systems. [Background technology]

[0002] Chronic neurodegenerative diseases are pathologies characterized by neuronal loss in the brain and spinal cord, including Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy. Research has shown that these pathologies are caused by ubiquitin-dependent processes, such as ubiquitin degradation by the 26S proteasome and aberrant autophagy via the lysosomal pathway, which have significant effects on neuronal development, homeostasis, and disease pathogenesis. Therefore, ubiquitin degradation and autophagy are essential for neural activity and are involved in synapse formation and cell-cell interactions. In the adult central and peripheral nervous systems, protein ubiquitination and deubiquitination are crucial for neuronal survival in non-dividing cells. Most age-related chronic neurodegenerative diseases result from the accumulation of total proteins, often present as nuclear contents.

[0003] The ubiquitin proteasome system (UPS) and the autophagy-lysosome system are intracellular protein degradation systems, and their interaction can promote the degradation of pathological proteins. It is hoped that substances that can promote the interaction between the ubiquitin proteasome system (UPS) and the autophagy-lysosome system, thereby facilitating the removal of pathogenic proteins from cells, will be discovered. Summary of the Invention

[0004] The present application has discovered through research that plasminogen can promote the interaction between the ubiquitin proteasome system (UPS) and the autophagy-lysosome system to a certain extent, thereby promoting the removal of pathological proteins inside and outside the cells. Specifically, the present application relates to the following provisions:

[0005] 1. A method for promoting the removal of pathological proteins by the ubiquitin proteasome system and the autophagy lysosome system, comprising administering to a subject an effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

[0006] 2. The method of claim 1, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA, and uPA.

[0007] 3. The method according to item 1, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, such as an antibody.

[0008] 4. The method according to any one of items 1 to 3, wherein the compound has one or more of the following activities: direct removal of different types of pathological proteins from outside the cell and / or directly from the cytoplasm and / or nucleus; promotion of removal of pathological proteins by the ubiquitin-proteasome system; promotion of removal of pathological proteins by the autophagy-lysosomal system; modulation for optimizing the expression and / or activity of members of the ubiquitin system; modulation for optimizing the expression and / or activity of LC3; modulation for optimizing the expression and / or activity of members of the autophagy-lysosomal system; and modulation for optimizing (particularly promoting) the expression of LAMP2.

[0009] 5. The method according to any one of items 1 to 4, wherein the compound is plasminogen or plasmin.

[0010] 6. The method according to any one of items 1 to 5, wherein the plasminogen is Glu-plasminogen, Lys-plasminogen, or a conservatively substituted variant thereof.

[0011] 7. The method according to any one of items 1 to 6, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2 and has lysine-binding activity and / or proteolytic activity of plasminogen.

[0012] 8. The method according to any one of items 1 to 7, wherein the plasminogen comprises one or more selected from the group consisting of: 1) the serine protease domain set forth in SEQ ID NO: 14; 2) a serine protease domain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 and retaining proteolytic activity; 3) one or more Kringle domains selected from the group consisting of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5; and 4) A Kringle domain that has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to one or more selected from the group consisting of Kringle 1, Kringle 2, Kringle 3, Kringle 4 and Kringle 5 and retains lysine-binding activity.

[0013] 9. The method according to any one of items 1 to 8, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or a mutant thereof that retains the proteolytic activity of plasminogen.

[0014] 10. The method according to any one of items 1 to 9, wherein the plasminogen comprises the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12, or comprises a conservatively substituted variant of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12.

[0015] 11. The method according to any one of items 1 to 10, wherein the plasminogen is used in combination with one or more other therapeutic methods or drugs.

[0016] 12. The method according to item 11, wherein the other treatment methods include cell therapy (including stem cell therapy), gene therapy, supportive therapy, and physical therapy.

[0017] 13. The method according to any one of items 1 to 12, wherein the plasminogen is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), or intramuscular administration.

[0018] In another aspect, the present application also relates to the following provisions:

[0019] 1. A method for preventing or treating a disease caused by pathological protein aggregation or ubiquitin / lysosomal dysfunction, comprising administering to a subject an effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

[0020] 2. The method of claim 1, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA, and uPA.

[0021] 3. The method according to item 1, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, such as an antibody.

[0022] 4. The method according to any one of items 1 to 3, wherein the compound has one or more of the following activities: direct removal of different types of pathological proteins from outside the cell and / or directly from the cytoplasm and / or nucleus; promotion of removal of pathological proteins by the ubiquitin-proteasome system; promotion of removal of pathological proteins by the autophagy-lysosomal system; modulation for optimizing the expression and / or activity of members of the ubiquitin system; modulation for optimizing the expression and / or activity of LC3; modulation for optimizing the expression and / or activity of members of the autophagy-lysosomal system; and modulation for optimizing (particularly promoting) the expression of LAMP2.

[0023] 5. The method according to any one of items 1 to 4, wherein the compound is plasminogen or plasmin.

[0024] 6. The method according to any one of items 1 to 5, wherein the plasminogen is Glu-plasminogen, Lys-plasminogen, or a conservatively substituted variant thereof.

[0025] 7. The method according to any one of items 1 to 6, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2 and has lysine-binding activity and / or proteolytic activity of plasminogen.

[0026] 8. The method according to any one of items 1 to 7, wherein the plasminogen comprises one or more selected from the group consisting of: 1) containing the serine protease domain set forth in SEQ ID NO: 14; 2) a serine protease domain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 and retaining proteolytic activity; 3) one or more Kringle domains selected from the group consisting of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5; and 4) A Kringle domain that has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to one or more selected from the group consisting of Kringle 1, Kringle 2, Kringle 3, Kringle 4 and Kringle 5 and retains lysine-binding activity.

[0027] 9. The method according to any one of items 1 to 8, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or a mutant thereof that retains the proteolytic activity of plasminogen.

[0028] 10. The method according to any one of items 1 to 9, wherein the plasminogen comprises the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12, or comprises a conservatively substituted variant of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12.

[0029] 11. The method according to any one of items 1 to 10, wherein the plasminogen is used in combination with one or more other therapeutic methods or drugs.

[0030] 12. The method according to item 11, wherein the other treatment methods include cell therapy (including stem cell therapy), gene therapy, supportive therapy, and physical therapy.

[0031] 13. The method of any one of items 1 to 12, wherein the plasminogen is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), or intramuscular administration. In some embodiments, the method of item 12, wherein the other treatment method comprises cell therapy (including stem cell therapy), gene therapy, supportive care, and physical therapy.

[0032] 14. The method of any one of items 1 to 12, wherein the plasminogen is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), or intramuscular administration.

[0033] In some embodiments, the diseases caused by pathological protein aggregation include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion-like diseases (Creutzfeldt-Jakob disease), Gerstmann's syndrome, diffuse or familial fatal insomnia and kuru, Huntington's disease, spinocerebellar ataxia type 3, dentatorubral-pallidal atrophy with Lewy body, diabetes, spongiform encephalopathy, macular degeneration, atherosclerosis, familial British dementia, familial Danish dementia, Down's syndrome, hemodialysis-associated amyloidosis, amyloid cardiomyopathy, systemic amyloidosis, corneal dystrophies, glomerulonephritis, and amyloid bodies.

[0034] In some embodiments, diseases caused by ubiquitin / lysosomal dysfunction include Fanconi anemia, Xeroderma pigmentosum, Cockayne syndrome, cancer, Cowden syndrome, Parkinson's disease, genomic instability, metabolic syndrome, muscular dystrophy, Von Hippel-Lindau syndrome, multiple myeloma, RIDDLE syndrome, Huntington's disease, X-linked lymphoproliferative disorders, Crohn's disease, Alzheimer's disease, breast and ovarian cancer, muscular amylopectinosis, amyotrophic lateral sclerosis, spinal muscular atrophy, systemic lupus erythematosus, and the like. erythematosus, infantile ataxia, X-linked parkinsonism with spasticity, multisystem disorders, diabetes, multiple sclerosis, cystinosis, Vici syndrome, Gaucher's disease, frontotemporal dementia (heterozygous) or neuronal ceroid lipofuscinosis (homozygous), Danon cardiomyopathy, cortical atrophy, epilepsy, autosomal recessive spinocerebellar ataxia, hereditary spastic paraplegia, beta-propeller protein-associated neurodegeneration (BPAN), autism spectrum disorder, frontotemporal dementia (FTD), inflammatory bowel disease, nonalcoholic fatty liver disease, tuberculosis, Rett syndrome, osteopetrosis, Charcot-Marie-Tooth type 2B disease disease, juvenile arthritis, Snyder-Robinson syndrome (SRS), Wiskott-Aldrich syndromesyndrome, primary microcephaly, hereditary sensory and autonomic neuropathy type II, primary open-angle glaucoma (POAG), Paget's disease of the bone (PGD), colorectal cancer, lung cancer, brain tumors, autosomal recessive and sporadic early-onset Parkinson's disease, Zellweger syndrome spectrum disorders, distal myopathy, ulcerative colitis, familial Mediterranean fever, spastic ataxia, Fabry disease, Gaucher disease, lysosomal acid lipase deficiency, mucopolysaccharidoses, and Angelman syndrome.

[0035] In certain embodiments, the plasminogen pathway activator is administered in combination with one or more other agents and / or therapeutic methods, preferably including cell therapy (e.g., stem cell therapy) and gene therapy (e.g., antisense RNA, small molecule splicing modifiers).

[0036] In certain embodiments, the plasminogen pathway activator is a component of the plasminogen activation pathway, such as plasminogen, which comprises or has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12, and has plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen is a protein having 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid added, deleted, and / or substituted based on SEQ ID NO: 2, 6, 8, 10, or 12, and having plasminogen activity and / or lysine-binding activity. In some specific embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In some specific embodiments, the plasminogen is a protein comprising a plasminogen activity fragment and having plasminogen activity and / or lysine-binding activity. In some specific embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In certain embodiments, the plasminogen active fragment comprises or has a plasminogen serine protease domain or a plasminogen protease domain. In certain embodiments, the amino acid sequence of the plasminogen active fragment is set forth in SEQ ID NO: 14. In certain embodiments, the plasminogen is Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (human full-length plasminogen after cleavage between amino acids 76 and 77), miniplasminogen (comprising Kringle 5 (K5) and a serine protease domain), microplasminogen (comprising a serine protease domain), delta-plasminogen (comprising Kringle 1 and a serine protease domain), or a variant thereof that retains plasminogen activity.In certain embodiments, the plasminogen is human full-length plasminogen, or a variant or fragment that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen is an ortholog of human plasminogen derived from a primate or rodent, or a variant or fragment that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen comprises the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human native plasminogen.

[0037] In some specific embodiments, the plasminogen pathway activator is administered systemically or locally, for example, intravenously, intramuscularly, as a nasal inhalation spray, or in the form of nasal drops. In some embodiments, the subject is human. In some embodiments, the subject has a plasminogen deficiency or deficiency. In some embodiments, the deficiency or deficiency is congenital, inherited, and / or localized. In some embodiments, the plasminogen is administered at a daily dose of 0.0001-2000 mg / kg, 0.001-800 mg / kg, 0.01-600 mg / kg, 0.1-400 mg / kg, 1-200 mg / kg, 1-100 mg / kg, 10-100 mg / kg (calculated per kilogram of body weight), or 0.0001-2000 mg / cm. 2 , 0.001~800mg / cm 2 , 0.01 to 600 mg / cm 2 , 0.1 to 400 mg / cm 2 , 1-200mg / cm 2 , 1-100mg / cm 2 , 10-100mg / cm 2 (calculated per square centimeter of body surface area) administered daily, every two days, or every three days continuously.

[0038] In one aspect, the present application also relates to pharmaceutical compositions, medicaments, formulations, kits, and articles of manufacture for use in the above methods, comprising the above plasminogen pathway activators, e.g., the above plasminogens.

[0039] In some embodiments, the pharmaceutical compositions, medicaments, and formulations comprise a pharmaceutically acceptable carrier and a component of the plasminogen activation pathway, e.g., a plasminogen pathway activator such as plasminogen. In some embodiments, the kits and articles of manufacture comprise one or more containers containing the pharmaceutical composition, medicament, or formulation. In some embodiments, the kit or article of manufacture also further comprises a label or protocol instructing use of the plasminogen pathway activator, e.g., a component of the plasminogen activation pathway, such as plasminogen, in the above-described methods. In some embodiments, the kit or article of manufacture also further comprises one or more additional containers containing one or more other agents.

[0040] In one aspect, the present application also relates to a plasminogen pathway activator, such as the plasminogen described above, for the uses described above.

[0041] In one aspect, the present application also relates to the use of a therapeutically effective amount of the above-described plasminogen pathway activator in the manufacture of a pharmaceutical composition, medicament, formulation, kit, or article of manufacture for use in the above-described method.

[0042] In some embodiments, the plasminogen pathway activator is one or more selected from a component of the plasminogen activation pathway, a compound that can activate plasminogen directly or indirectly by activating an upstream component of the plasminogen activation pathway, a compound that mimics the activity of plasminogen or plasmin, a compound that can upregulate the expression of plasminogen or plasminogen activators, a plasminogen analog, a plasmin analog, a tPA or uPA analog, and an antagonist of a fibrinolysis inhibitor.

[0043] In certain embodiments, the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasmin, plasminogen activators, tPA, and uPA. In certain embodiments, the antagonist of the fibrinolysis inhibitor is an antagonist of PAI-1, complement C1 inhibitor, α2-antiplasmin, or α2-macroglobulin, e.g., an antibody against PAI-1, complement C1 inhibitor, α2-antiplasmin, or α2-macroglobulin.

[0044] In certain embodiments, the plasminogen pathway activator is administered in combination with one or more other agents and / or therapeutic methods, preferably including cell therapy (e.g., stem cell therapy) and gene therapy (e.g., antisense RNA, small molecule splicing modifiers).

[0045] In certain embodiments, the plasminogen pathway activator is a component of the plasminogen activation pathway, such as plasminogen, which comprises or has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12, and has plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen is a protein having 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid added, deleted, and / or substituted based on SEQ ID NO: 2, 6, 8, 10, or 12, and having plasminogen activity and / or lysine-binding activity. In some specific embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In some specific embodiments, the plasminogen is a protein comprising a plasminogen activity fragment and having plasminogen activity and / or lysine-binding activity. In some specific embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In certain embodiments, the plasminogen active fragment comprises or has a plasminogen serine protease domain or a plasminogen protease domain. In certain embodiments, the amino acid sequence of the plasminogen active fragment is set forth in SEQ ID NO: 14. In certain embodiments, the plasminogen is Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (human full-length plasminogen after cleavage between amino acids 76 and 77), miniplasminogen (comprising Kringle 5 (K5) and a serine protease domain), microplasminogen (comprising a serine protease domain), delta-plasminogen (comprising Kringle 1 and a serine protease domain), or a variant thereof that retains plasminogen activity.In certain embodiments, the plasminogen is human full-length plasminogen, or a variant or fragment that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen is an ortholog of human plasminogen derived from a primate or rodent, or a variant or fragment that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen comprises the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human native plasminogen.

[0046] In some specific embodiments, the plasminogen pathway activator, e.g., a component of the plasminogen activation pathway, e.g., plasminogen, is administered in combination with one or more other drugs and / or therapeutic methods. In some embodiments, the plasminogen pathway activator, e.g., a component of the plasminogen activation pathway, e.g., plasminogen, is administered intravenously, intramuscularly, intrathecally, intranasally, aerosolized, as nose drops, or as eye drops.

[0047] In some embodiments, the pharmaceutical compositions, medicaments, and formulations comprise a pharmaceutically acceptable carrier and a component of the plasminogen activation pathway, e.g., a plasminogen pathway activator such as plasminogen. In some embodiments, the kits and articles of manufacture comprise one or more containers containing the pharmaceutical composition, medicament, or formulation. In some embodiments, the kit or article of manufacture also further comprises a label or protocol instructing use of the plasminogen pathway activator, e.g., a component of the plasminogen activation pathway, such as plasminogen, in the above-described methods.

[0048] In some embodiments, the kit or article of manufacture also further comprises one or more separate containers containing one or more other agents.

[0049] The present invention explicitly covers all combinations of the technical features belonging to the embodiments of the present invention, and the technical configurations after these combinations are expressly disclosed in this application as if the above technical configurations were expressly disclosed separately. Furthermore, the present invention also explicitly covers the combinations between each embodiment and their elements, and the technical configurations after the combinations are expressly disclosed in this specification. [Brief explanation of the drawings]

[0050] [Figure 1] Schematic diagram of how plasminogen interacts with the ubiquitin-proteasome and autophagy-lysosomal systems to promote the degradation of pathological proteins in the central nervous system. Blood-brain barrier, basement membrane, endothelial cell, plasminogen (Plg), plasminogen receptor (PLgR), tissue-type plasminogen activator (tPA), conformationally abnormal proteins (CAP), plasmin (Plm), plasmin-generated protein fragments (PGPF), plasmin degradation products (PDP), lysosome, ubiquitin (UBI), ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin ligase (E3), proteasome, nucleus. Previous studies have shown that plasminogen can enter cells and interact with the intracellular protein degradation systems, the ubiquitin proteasome system (UPS) and the autophagy-lysosome system. Therefore, plasminogen can promote the degradation of pathological proteins abnormally deposited in the central nervous system and improve degenerative diseases of the central nervous system. [Figure 2]This figure shows the localization of plasminogen and the colocalization of plasminogen and ubiquitin in the spinal cord tissue of amyotrophic lateral sclerosis (ALS) model mice. The results showed that the positive staining of plasminogen (green fluorescence) in the spinal cord tissue of the drug-treated group was significantly higher than that of the vehicle group, indicating that the administered plasminogen can enter and concentrate in the spinal cord tissue. Plasminogen is also present in the cytoplasm and nucleus. Plasminogen and ubiquitin (red fluorescence) colocalized in the cytoplasm (as indicated by triangles) (Figure 2). The ubiquitin expression level in the spinal cord tissue of the drug-treated group appeared to be lower than that of the vehicle group. This indicates that in ALS model mice, plasminogen can enter the spinal cord tissue, enter cells, colocalize with ubiquitin, and reduce ubiquitin expression. This suggests that plasminogen may interact with ubiquitin. [Figure 3] This figure shows the localization of plasminogen and the colocalization of plasminogen and LC3B in the spinal cord tissue of amyotrophic lateral sclerosis (ALS) model mice. The results showed that the positive staining of plasminogen (green fluorescence) in the spinal cord tissue of the drug-treated group was significantly higher than that of the vehicle group, indicating that the administered plasminogen was able to enter and concentrate in the spinal cord tissue. Plasminogen was also present in the cytoplasm and nucleus. Plasminogen and LC3B (red fluorescence) were colocalized in the cytoplasm (as indicated by triangles), and the LC3B expression level in the spinal cord tissue of the drug-treated group appeared to be lower than that of the vehicle group. This indicates that in ALS model mice, plasminogen enters the spinal cord tissue, enters cells, and colocalizes with LC3B. This suggests that plasminogen may interact with LC3B. [Figure 4]This figure shows the localization of plasminogen and the colocalization of plasminogen and LAMP2 in the spinal cord tissue of an amyotrophic lateral sclerosis model mouse. The results showed that the positive staining of plasminogen (green fluorescence) in the spinal cord tissue of the drug-treated group was significantly higher than that of the vehicle group, indicating that the administered plasminogen was able to enter and concentrate in the spinal cord tissue. Plasminogen was also present in the cytoplasm and nucleus. Plasminogen and LAMP2 (red fluorescence) were colocalized in the cytoplasm (as indicated by triangles). LAMP2 expression levels in the spinal cord tissue of the drug-treated group appeared to be higher than in the vehicle group, and they entered the cells and colocalized with LAMP2, promoting LAMP2 expression and lysosomal function. [Figure 5] This figure shows that administration of plasminogen promotes LC3 expression in the spinal cord tissue of amyotrophic lateral sclerosis model mice. As a result, LC3 expression in the drug-treated group was significantly higher than that in the vehicle group, and the difference was statistically significant (* indicates P<0.05). These results indicate that plasminogen promotes LC3 gene transcription in amyotrophic sclerosis model mice, thereby contributing to the regulation of the autophagy process. [Figure 6] This figure shows the localization of plasminogen and the colocalization of plasminogen and ubiquitin in the substantia nigra and striatum of Parkinson's disease model mice. The results showed significantly more positive staining for plasminogen (green fluorescence) in the substantia nigra and striatum of the drug-treated group than in the vehicle group, indicating that the administered plasminogen can enter and concentrate in the substantia nigra and striatum. Plasminogen is also present in the cytoplasm and nucleus. Plasminogen colocalizes with ubiquitin (red fluorescence) in the cytoplasm (as indicated by triangles). This indicates that in Parkinson's disease model mice, plasminogen can enter the substantia nigra and striatum, enter cells, and colocalize with ubiquitin. This suggests that plasminogen may interact with ubiquitin. [Figure 7]This figure shows the localization of plasminogen and the colocalization of plasminogen and LC3B in the substantia nigra and striatum of Parkinson's disease model mice. The results showed significantly more positive staining for plasminogen (green fluorescence) in the substantia nigra and striatum of the drug-treated group than in the vehicle group, indicating that the administered plasminogen can enter and concentrate in the substantia nigra and striatum. Plasminogen is also present in the cytoplasm and nucleus. Plasminogen colocalizes with LC3B (red fluorescence) in the cytoplasm (as indicated by triangles). This indicates that in Parkinson's disease model mice, plasminogen can enter the substantia nigra and striatum, enter cells, and colocalize with LC3B. This suggests that plasminogen may interact with LC3B. [Figure 8] This figure shows the localization of plasminogen and the colocalization of plasminogen and LAMP2 in the substantia nigra and striatum of Parkinson's disease model mice. The results showed significantly more positive staining for plasminogen (green fluorescence) in the substantia nigra and striatum of the drug-treated group than in the vehicle group, indicating that the administered plasminogen can enter and concentrate in the substantia nigra and striatum. Plasminogen is also present in the cytoplasm and nucleus. Plasminogen colocalizes with LAMP2 (red fluorescence) in the cytoplasm (as indicated by triangles). This indicates that in Parkinson's disease model mice, plasminogen can enter the substantia nigra and striatum, enter cells, and colocalize with LAMP2. This suggests that plasminogen may interact with LAMP2. [Figure 9]This figure shows the localization of plasminogen and the colocalization of plasminogen and ubiquitin in the hippocampal tissue of Alzheimer's disease model mice. The results show that the positive staining of plasminogen (green fluorescence) in the hippocampal tissue of the drug-treated group was significantly higher than that of the vehicle group, indicating that the administered plasminogen can enter and concentrate in the hippocampal tissue. Plasminogen is also present in the cytoplasm and nucleus. Plasminogen colocalizes with ubiquitin (red fluorescence) in the cytoplasm (as indicated by triangles). This indicates that plasminogen can enter the hippocampal tissue and cells in Alzheimer's disease model mice. This suggests that plasminogen may interact with ubiquitin. [Figure 10] This figure shows the localization of plasminogen and the colocalization of plasminogen and LC3B in the hippocampal tissue of Alzheimer's disease model mice. The results show that the positive staining of plasminogen (green fluorescence) in the hippocampal tissue of the drug-treated group was significantly higher than that of the vehicle group, indicating that the administered plasminogen can enter and concentrate in the hippocampal tissue. Plasminogen is also present in the cytoplasm and nucleus. Plasminogen colocalizes with LC3B (red fluorescence) in the cytoplasm (as indicated by triangles). This indicates that plasminogen can enter the hippocampal tissue and cells in Alzheimer's disease model mice. This suggests that plasminogen may interact with LC3B. [Figure 11]This figure shows the localization of plasminogen and the colocalization of plasminogen and LAMP2 in the hippocampal tissue of Alzheimer's disease model mice. The results show that the positive staining of plasminogen (green fluorescence) in the hippocampal tissue of the drug-treated group was significantly higher than that of the vehicle group, indicating that the administered plasminogen can enter and concentrate in the hippocampal tissue. Plasminogen is also present in the cytoplasm and nucleus. Plasminogen colocalizes with LAMP2 (red fluorescence) in the cytoplasm (as indicated by triangles). This indicates that plasminogen can enter the hippocampal tissue and cells in Alzheimer's disease model mice. This suggests that plasminogen may interact with LAMP2. [Figure 12] Figures A-B show the results of Western blot quantitative analysis of huntingtin protein in cardiac tissue from Huntington's disease model mice 28 days after plasminogen administration. A is a Western blot image, and B is the result of quantitative analysis of the optical density of the huntingtin protein band. The results show that cardiac HTT levels in the vehicle group mice were significantly higher than in the normal control group mice, and cardiac HTT levels in the drug-treated group mice were significantly lower than in the vehicle group mice, with the difference being statistically significant (* indicates P<0.05). This suggests that plasminogen can promote the degradation of huntingtin protein in cardiac tissue from Huntington's disease model mice. [Figure 13]Figure 1 shows the results of WB detection of TDP-43 levels in the cytoplasm (A-B) and nucleus (C-D) of okadaic acid-treated NSC34 cells after plasminogen administration. A and C are Western blot images, and B and D are quantitative analysis results of the TDP-43 band. The results show that TDP-43 levels in the cytoplasm and nucleus of the drug-treated group were significantly lower than those in the nucleus of the vehicle group, indicating that the addition of EACA completely inhibited the effect of plasminogen on TDP-43. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. These results suggest that plasminogen can promote the degradation of TDP-43 in the cytoplasm and nucleus, and that this effect of plasminogen is closely related to the lysine-binding site in its structure. [Figure 14] Figure 1 shows the effects of plasminogen administration on phosphorylated tau protein (A-B), total tau protein, and the ratio of phosphorylated tau protein to total tau protein in okadaic acid-treated NSC34 cells. A and C are Western blot images, while B, D, and E are the results for phosphorylated tau protein, total tau protein, and the ratio of phosphorylated tau protein to total tau protein, respectively. The results show that the phosphorylated tau protein levels in the drug-treated group were significantly lower than those in the vehicle group, indicating that the addition of EACA completely inhibited the effect of plasminogen on phosphorylated tau protein. There was no significant difference in total tau protein levels between the drug-treated and vehicle groups, and the ratio of phosphorylated tau protein to total tau protein in the drug-treated group was significantly lower than that in the vehicle group, indicating that the addition of EACA completely inhibited this effect of plasminogen. * indicates P<0.05, *** indicates P<0.001. These results suggest that plasminogen can promote the degradation of phosphorylated tau protein in cells, and that this effect of plasminogen is closely related to the lysine-binding site in its structure. [Figure 15]Figures A-D show the results of measuring plasminogen and plasmin activity levels in the cytoplasm and nucleus of okadaic acid-treated NSC34 cells after plasminogen administration. Figure A shows the ELISA results for cytoplasmic plasminogen levels, Figure B shows the ELISA results for nuclear plasminogen levels, Figure C shows the enzyme substrate kinetics of cytoplasmic plasmin activity levels, and Figure D shows the enzyme substrate kinetics of nuclear plasmin activity levels. The results show that the levels of human plasminogen and plasmin activity in the cytoplasm and nucleus of the drug-treated group were significantly higher than those of the vehicle group, with a statistically significant difference. The addition of EACA completely inhibited these effects of plasminogen (** indicates P<0.01, *** indicates P<0.001). This suggests that plasminogen can enter cells and nuclei to enhance plasmin activity, and that plasmin entry into cells and nuclei is closely related to its lysine-binding activity. [Figure 16] Figures 16A-B show the results of Western blot quantitative analysis of huntingtin protein in kidney tissue from Huntington's disease model mice 28 days after plasminogen administration. A is a Western blot image, and B is the result of quantitative analysis of the optical density of the huntingtin protein band. The results showed that renal HTT levels in the vehicle-treated mice were significantly higher than those in the normal control group, and that renal HTT levels in the drug-treated mice were significantly lower than those in the vehicle group (Figures 16A-B). The differences were statistically significant (* indicates P<0.05, *** indicates P<0.001). This suggests that plasminogen can promote the degradation of huntingtin protein in kidney tissue from Huntington's disease model mice. [Figure 17]Figures A and B show the results of Western blot quantitative analysis of huntingtin protein in brain tissue from Huntington's disease model mice cultured in vitro two days after plasminogen administration. A is a Western blot image, and B is the result of quantitative analysis of the optical density of the huntingtin protein band. The results show that brain tissue HTT levels in the drug-treated mice were significantly lower than those in the vehicle-treated mice, and the difference was statistically significant (* indicates P<0.05). This suggests that plasminogen can promote the degradation of huntingtin protein in brain tissue from Huntington's disease model mice. DETAILED DESCRIPTION OF THE INVENTION

[0051] [Detailed Description of the Invention] The fibrinolytic system, also known as the fibrinolytic system, is a system of chemicals involved in the process of fibrinolysis (fibrinolysis). These chemicals include plasminogen (also called PLG), plasmin, plasminogen activators, and fibrinolysis inhibitors. Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA is a serine protease synthesized by vascular endothelial cells. t-PA activates plasminogen, a process that primarily occurs with fibrin. Urokinase-type plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells and can directly activate plasminogen without requiring fibrin as a cofactor. Plasminogen (PLG) is synthesized in the liver. During blood clot formation, PLG is absorbed in large quantities into the fibrin network and activated to plasmin by t-PA or u-PA, promoting fibrinolysis. Plasminase (PL) is a serine protease that degrades fibrin and fibrinogen, hydrolyzes various coagulation factors (e.g., V, VIII, X, VII, XI, and II), converts plasminogen to plasmin, and hydrolyzes complement. Fibrinolysis inhibitors include plasminogen activator inhibitor (PAI) and α2-antiplasmin (α2-AP). PAIs exist in two forms, PAI-1 and PAI-2, which specifically bind to t-PA in a 1:1 ratio, thereby inactivating it and activating PLG. α2-AP is synthesized in the liver and binds to PL in a 1:1 ratio to form a complex, thereby inhibiting PL activity. FXIII covalently binds α2-AP to fibrin, thereby reducing its susceptibility to PL. Substances that inhibit the activity of the fibrinolytic system in vivo include PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin.

[0052] The term "plasminogen pathway activator" or "fibrin plasminogen" of the present invention covers components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolysis inhibitors.

[0053] As used herein, the term "component of the fibrin plasminogen activation pathway" or "component of the plasminogen activation pathway" refers to a 1. Plasminogen, Lys-plasminogen, Glu-plasminogen, microplasminogen, delta-plasminogen, their variants or analogs; 2. Plasmin and its variants or analogs; and 3. Plasminogen activators, such as tPA and uPA, as well as tPA or uPA variants and analogs that contain one or more domains of tPA or uPA, such as one or more kringle domains and proteolytic domains.

[0054] The term "antagonist of a fibrinolysis inhibitor" covers antagonists of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, for example antibodies of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0055] The above-mentioned "variants" of plasminogen, plasmin, tPA and uPA include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as proteins having, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid added, deleted and / or substituted and still having plasminogen activity, plasmin activity, tPA or uPA activity. For example, a "variant" of plasminogen activity, plasmin activity, tPA, or uPA includes, for example, mutants of these proteins resulting from substitution of 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 conservative amino acid.

[0056] The "plasminogen variant" of the present invention includes or covers proteins having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, or 12, and having plasminogen activity and / or lysine-binding activity. For example, the "plasminogen variant" of the present invention may be a protein in which 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is added, deleted, and / or substituted based on SEQ ID NO: 2, 6, 8, 10, or 12, and still has plasminogen activity and / or lysine-binding activity. In particular, the plasminogen variants of the present invention include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as mutants of these proteins obtained by conservative substitution of, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acids.

[0057] The plasminogen of the present invention may be an orthologue of human plasminogen derived from a primate or rodent, or a variant that still retains plasminogen activity and / or lysine-binding activity, such as the plasminogen shown in SEQ ID NO: 2, 6, 8, 10 or 12, e.g., the human native plasminogen shown in SEQ ID NO: 2.

[0058] The above "analogs" of plasminogen, plasmin, tPA and uPA include compounds that provide substantially the same effects as plasminogen, plasmin, tPA or uPA, respectively.

[0059] The above-mentioned "variants" and "analogs" of plasminogen, plasmin, tPA, and uPA include "variants" and "analogs" of plasminogen, plasmin, tPA, and uPA that contain one or more domains (e.g., one or more kringle domains and proteolytic domains). For example, "variants" and "analogs" of plasminogen include plasminogen variants and analogs, such as mini-plasminogen, that contain one or more plasmin domains (e.g., one or more kringle (k) domains and proteolytic domains (also called serine protease domains or plasminogen protease domains)). "Variants" and "analogs" of plasmin include plasmin "variants" and "analogs," such as mini-plasmin and delta-plasmin, that contain one or more plasmin domains (e.g., one or more kringle domains and proteolytic domains).

[0060] Whether the above-mentioned "variants" or "analogs" of plasminogen, plasmin, tPA, or uPA have the activity of plasminogen, plasmin, tPA, or uPA, respectively, or whether they provide substantially the same effect as plasminogen, plasmin, tPA, or uPA, respectively, can be measured by measuring the level of activated plasmin activity using methods known in the art, such as enzymography, ELISA (enzyme-linked immunosorbent assay), and FACS (fluorescence-activated cell sorting). For example, this can be measured by referring to the methods described in the following documents: Ny, A., Leonardsson, G., Hagglund, AC, Hagglof, P., Ploplis, VA, Carmeliet, P. and Ny, T. (1999). 1984). “Complex formation of platelet thrombospondin with plasminogen.Modulation of activation by tissue activator”.J.Clin.Invest.74(5):1625-33;Gravanis I,Tsirka SE (February 2008).“Tissue-type plasminogen activator as a therapeutic target in stroke”.Expert Opinion on Therapeutic Targets.12(2):159-70;Geiger M,Huber K, Wojta J, Stingl L, Espana F, Griffin JH, Binder BR (Aug 1989). “Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo”. Blood. 74(2):722-8.

[0061] In some embodiments of the present invention, the "component of the plasminogen activation pathway" of the present invention is plasminogen, and is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or a variant thereof that retains plasminogen activity. In some embodiments, the plasminogen is natural or synthetic human plasminogen, or a conservative mutant or fragment thereof that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen is an ortholog of human plasminogen derived from a primate or rodent, or a conservative mutant or fragment thereof that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the amino acid sequence of the plasminogen comprises or has the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human full-length plasminogen. In some embodiments, the plasminogen is human full-length plasminogen as set forth in SEQ ID NO:2.

[0062] "A compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway" refers to any compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway, including, for example, tPA, uPA, streptokinase, saruplase, alteplase, reteplase, tenecteplase, anistreplase, monteplase, lanoteplase, pamiteplase, and staphylokinase.

[0063] The "antagonist of fibrinolysis inhibitors" of the present invention is a compound that antagonizes, weakens, blocks, or prevents the action of fibrinolysis inhibitors, such as PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin. The antagonists are antibodies to PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or antisense RNA or mini-RNA that, for example, blocks or downregulates the expression of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that occupy the binding site of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin but do not have the function of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that block the binding and / or activation domains of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0064] Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease capable of hydrolyzing several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans. Plasmin can also activate some pro-matrix metalloproteinases (pro-MMPs) to form active matrix metalloproteinases (MMPs). Thus, plasmin is an important upstream regulator of extracellular proteolysis. Plasmin is formed by the proteolysis of plasminogen from two physiological PAs: tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). Because plasminogen is present at relatively high levels in plasma and other body fluids, it has traditionally been thought that regulation of the PA system is primarily achieved by PA synthesis and activity levels. The synthesis of PA system components is tightly regulated by various factors, such as hormones, growth factors, and cytokines. In addition, there are specific physiological inhibitors of plasmin and PAs. The main inhibitor of plasmin is α2-antiplasmin. The activity of PAs is simultaneously inhibited by plasminogen activator inhibitor-1 (PAI-1), which inhibits uPA and tPA, and is regulated by plasminogen activator inhibitor-2 (PAI-2), which primarily inhibits uPA. Some cells have a uPA-specific cell surface receptor (uPAR) that is active in direct hydrolysis.

[0065] Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. It is synthesized primarily in the liver and present in large amounts in extracellular fluids. Plasminogen contains approximately 2 μM of plasminogen in plasma. Therefore, plasminogen is a significant potential source of proteolytic activity in tissues and body fluids. Two molecular forms of plasminogen exist: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). The naturally secreted and undegraded form of plasminogen contains a single amino-terminal (N-terminal) glutamic acid and is therefore called glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys77. Compared with glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PA at a higher rate. The Arg560-Val561 peptide bond in these two forms of plasminogen is cleaved by uPA or tPA, resulting in the formation of the disulfide-linked double-chain protease plasmin. The amino-terminal portion of plasminogen contains five homologous tricyclic rings, or kringles, and the carboxyl-terminal portion contains the protease domain. Some kringles contain lysine-binding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor, α2-AP. The most recently discovered 38-kDa fibrin-plasminogen fragment, kringlel-4, is a potent inhibitor of angiogenesis. This fragment, named angiostatin, is generated by hydrolysis of plasminogen by several proteases.

[0066] The main substrate of plasmin is fibrin, and fibrin lysis is key to preventing the formation of pathological thrombi. Plasmin also has substrate specificity for several components of the extracellular matrix (ECM), including laminin, fibronectin, proteoglycans, and gelatin, indicating that plasmin plays an important role in ECM remodeling. Indirectly, plasmin also degrades other components of the ECM by converting several protease precursors, including MMP-1, MMP-2, MMP-3, and MMP-9, into active proteases. Therefore, plasmin has been proposed to be an important upstream regulator of extracellular protein hydrolysis. Plasmin also has the ability to activate several latent forms of growth factors. In vitro, plasmin can also hydrolyze components of the complement system to release chemotactic complement fragments.

[0067] "Plasmin" is a very important enzyme present in blood, which hydrolyzes fibrin clots into fibrin degradation products and D-dimers.

[0068] "Plasminogen" is the proenzyme form of plasmin, and based on the sequence in Swiss prot, the amino acid sequence of natural human plasminogen (Sequence 4), including the signal peptide, is calculated to consist of 810 amino acids, has a molecular weight of approximately 90 kD, and is a glycoprotein that is synthesized mainly in the liver and can circulate in the blood; the cDNA sequence encoding this amino acid sequence is shown in Sequence 3. Full-length plasminogen contains seven domains: a serine protease domain located at the C-terminus, a Pan Apple (PAp) domain located at the N-terminus, and five Kringle domains (Kringles 1-5). According to the sequence in swissprot, the signal peptide contains residues Met1-Gly19, PAp contains residues Glu20-Val98, Kringle1 contains residues Cys103-Cys181, Kringle2 contains residues Glu184-Cys262, Kringle3 contains residues Cys275-Cys352, Kringle4 contains residues Cys377-Cys454, and Kringle5 contains residues Cys481-Cys560. According to NCBI data, the serine protease domain contains residues Val581-Arg804.

[0069] Glu-plasminogen is natural, full-length plasminogen, consisting of 791 amino acids (excluding the 19-amino acid signal peptide). The cDNA sequence encoding this sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2. Lys-plasminogen, formed in vivo by further hydrolysis of Glu-plasminogen at the 76th and 77th amino acids, exists, and is shown, for example, in SEQ ID NO: 6, and the cDNA sequence encoding this amino acid sequence is shown in SEQ ID NO: 5. Delta-plasminogen (δ-plasminogen) is a fragment of full-length plasminogen lacking the Kringle 2 to Kringle 5 structures, and contains only Kringle 1 and a serine protease domain (also called the proteolytic domain or plasminogen protease domain). The amino acid sequence of δ-plasminogen (SEQ ID NO: 8) has been reported in a literature, and a cDNA sequence encoding this amino acid sequence is shown, for example, in SEQ ID NO: 7. Mini-plasminogen consists of Kringle 5 and serine protease domains, and the literature reports residues Val443-Asn791 (the starting amino acid is the Glu residue of the Glu-plasminogen sequence without the signal peptide). The amino acid sequence is shown in Sequence 10, and the cDNA sequence encoding this amino acid sequence is shown in Sequence 9. However, literature reports that microplasminogen contains only a serine protease domain, and its amino acid sequence is Ala543-Asn791 (the Glu residue in the Glu-plasminogen sequence without the signal peptide is the starting amino acid), and patent document CN102154253A discloses that it contains Lys531-Asn791 (the Glu residue in the Glu-plasminogen sequence without the signal peptide is the starting amino acid). For the sequence of this patent, please refer to patent document CN102154253A, whose amino acid sequence is shown in Sequence 12, and the cDNA sequence encoding this amino acid sequence is shown in Sequence 11.

[0070] In the present invention, "plasmin", "fibrinplasmin", and "fibrous protein plasmin" can be used interchangeably and have the same meaning. "Plasminogen", "fibrinplasmin", and "fibrinplasminogen" can be used interchangeably and have the same meaning.

[0071] In this application, the term "insufficiency" of plasminogen refers to the content or activity of plasminogen in a subject's body being lower than that of a normal person and being low enough to affect the subject's normal physiological functions. The term "deficiency" of plasminogen refers to the content or activity of plasminogen in a subject's body being significantly lower than that of a normal person, with only trace activity or expression, and normal physiological functions being maintained only by external supply.

[0072] Those skilled in the art will understand as follows: All technical features of plasminogen in the present invention can be applied to plasmin, and therefore, the technical features described in the present invention cover both plasminogen and plasmin. During the circulation process, plasminogen is in a closed, inactive conformation. Upon binding to a thrombus or cell surface, it becomes active plasmin with an open conformation under the intervention of a plasminogen activator (PA). Active plasmin further hydrolyzes fibrin clots into fibrin degradation products and D-dimers, thereby dissolving the thrombus. The PAp domain of plasminogen is the key epitope that keeps plasminogen in the closed, inactive conformation, while the KR domain can bind to lysine residues on receptors and substrates. Several enzymes are known as plasminogen activators, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and clotting factor XII (Hagemann factor).

[0073] The term "active plasminogen fragment" in the present application refers to: 1) an active fragment (also referred to as a lysine-binding fragment) capable of binding to a target sequence of a substrate in a plasminogen protein, for example, a fragment containing Kringle 1, Kringle 2, Kringle 3, Kringle 4, and / or Kringle 5 (for the structure of plasminogen, see the description in Aisina RB, Mukhametova L I. Structure and function of plasminogen / plasmin system [J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6):590-605); 2) an active fragment that exerts a proteolytic function in a plasminogen protein, for example, a fragment having the plasminogen activity (proteolytic function) shown in SEQ ID NO: 14; and 3) a fragment having both the activity of binding to a target sequence of a substrate (lysine-binding activity) and the plasminogen activity (proteolytic function) in a plasminogen protein. In some embodiments of the present application, the plasminogen is a protein comprising a plasminogen active fragment set forth in SEQ ID NO: 14. In some embodiments of the present application, the plasminogen is a protein comprising a lysine-binding fragment of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and / or Kringle 5. In some embodiments, the plasminogen active fragment of the present application includes a protein comprising SEQ ID NO: 14 or comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% homology to SEQ ID NO: 14. Thus, plasminogen according to the present invention includes proteins comprising a plasminogen active fragment and still retaining plasminogen activity.In some embodiments, the plasminogen of the present application comprises Kringle 1, Kringle 2, Kringle 3, Kringle 4, and / or Kringle 5, or a protein having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% homology to Kringle 1, Kringle 2, Kringle 3, Kringle 4, or Kringle 5 and still having lysine-binding activity.

[0074] Currently, methods for measuring plasminogen and its activity in blood include tissue fibrin plasminogen activator activity (t-PAA), plasma tissue plasminogen activator antigen (t-PAAg), plasma tissue plasminogen activity (plgA), plasma tissue plasminogen antigen (plgAg), plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator inhibitor antigen, and plasma plasmin-antiplasmin complex (PAP). The most common method is the chromogenic substrate method: streptokinase (SK) and a luminescent substrate are added to the subject's plasma. PLG in the subject's plasma is converted into PLM under the action of SK, which then reacts with the luminescent substrate. The increase in absorbance is then measured using a spectrophotometer; the increase in absorbance is directly proportional to the activity of plasminogen. In addition, fibrin plasminogen activity in blood can be measured using immunochemical methods, gel electrophoresis, immunoturbidimetry, radial immunodiffusion, and the like.

[0075] "Ortholog" refers to a homologue between different species, including both protein and DNA homologues, also known as a direct gene. It specifically refers to a protein or gene evolved from the same ancestral gene between different species. The plasminogen of the present invention includes human native plasminogen, and further includes orthologs or orthologs of plasminogens with plasminogen activity derived from different species.

[0076] "Conservative substitution variants" refer to polypeptides or enzymes in which a single, specific amino acid residue has been altered without altering the overall conformation and function of the protein or enzyme. This includes, but is not limited to, substitutions of amino acids with similar properties (e.g., acidic, alkaline, hydrophobic, etc.) for amino acids in the amino acid sequence of the parent protein. Amino acids with similar properties are known. For example, arginine, histidine, and lysine are hydrophilic, alkaline amino acids and can be substituted for each other. Similarly, isoleucine is a hydrophobic amino acid and can be substituted by leucine, methionine, or valine. Therefore, the similarity between two functionally similar proteins or amino acid sequences may vary. For example, they may have 70% to 99% similarity (identity) based on the MEGALIGN algorithm. "Conservative substitution variants" also include polypeptides or enzymes with 60% or more amino acid identity based on the BLAST or FASTA algorithm, preferably 75% or more, most preferably 85% or more, and even more preferably 90% or more, and still have the same or essentially similar properties or functions as the native or parent protein or enzyme.

[0077] "Isolated" plasminogen refers to plasminogen protein that has been separated and / or recovered from its natural environment. In some embodiments, the plasminogen is (1) purified to greater than 90%, greater than 95%, or greater than 98% purity (by weight), e.g., as determined by the Lowry method, e.g., greater than 99% purity (by weight); (2) purified to the extent that at least 15 residues of N-terminal or internal amino acid sequence are obtained using a spinning cup sequencer; or (3) purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions with Coomassie brilliant blue or silver staining. Isolated plasminogen includes plasminogen produced from recombinant cells by bioengineering techniques and separated by at least one further purification step.

[0078] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term also encompasses fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions containing leader sequences of heterologous and homologous origin (with or without an N-terminal methionine residue); and the like.

[0079] The "percentage (%) amino acid sequence identity" of a reference peptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and excluding any conservative substitutions that are not considered part of the sequence identity. Alignment for purposes of determining percentage amino acid sequence identity can be accomplished by several means within the skill of the art, including publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm that achieves the maximum comparison required for the full length of the sequences being compared. However, for purposes of the present invention, the percentage amino acid sequence identity is determined by the sequence comparison computer program ALIGN-2.

[0080] When comparing amino acid sequences by using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (also referred to as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or for a given amino acid sequence B) is calculated as follows: Fraction X / Y×100

[0081] where X is the number of amino acid residues that the sequence alignment program ALIGN-2 evaluates as identical and matching in its alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood as follows: if the lengths of amino acid sequence A and amino acid sequence B are not equal, the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are as described in the preceding paragraph and are generated by the ALIGN-2 computer program.

[0082] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0083] A "therapeutically effective amount" or "effective amount" is an amount of plasminogen that, when administered to a mammal or other subject to treat a disease, can prevent and / or treat the disease. The "therapeutically effective amount" varies depending on the plasminogen used, the severity of the disease and / or symptoms, and the age, weight, etc., of the subject to be treated.

[0084] The term "treatment" of a disease state includes inhibiting or arresting the progression of said disease state or its clinical symptoms, or alleviating said disease state or symptoms such that said disease state or its clinical symptoms are diminished, either temporarily or permanently.

[0085] Preparation of the Plasminogen of the Present Invention Plasminogen may be isolated and purified from nature for further therapeutic use, or it may be synthesized by standard chemical peptide synthesis techniques. When polypeptides are synthesized by chemical methods, synthesis can be performed in liquid phase or solid phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support and the remaining amino acids in the sequence are sequentially coupled, is suitable for chemically synthesizing plasminogen. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen. The techniques used in solid-phase synthesis are described in Barany and Solid-Phase Peptide Synthesis; p. 3-284; The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al., J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6: 3-10 and Camarero JA et al., 2005 Protein Pept Lett. 12: 723-8. Briefly, small insoluble porous beads are prepared with functional units onto which peptide chains are constructed. After repeated coupling / deprotection cycles, a single N-protected amino acid unit is coupled to the free N-terminal amine of the attached solid phase. The unit is then deprotected, revealing a new N-terminal amine for linking with another amino acid. The peptide remains immobilized on the solid phase, and is then excised.

[0086] The plasminogen of the present invention is produced by standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector and operably linked to control sequences in the expression vector. Expression control sequences include, but are not limited to, a promoter (e.g., a naturally associated promoter or a heterologous promoter), a signal sequence, an enhancer element, and a transcription termination sequence. Expression can be controlled by a eukaryotic promoter system in a vector that is transformed or transfected into eukaryotic host cells (e.g., COS or CHO cells). Once the vector is introduced into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for the collection and purification of the plasminogen.

[0087] Suitable expression vectors typically replicate in the host either episomes or as an integral part of the host chromosomal DNA. Expression vectors usually contain selectable markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline-resistance, kanamycin-resistance, or neomycin-resistance) that are useful for detecting in vitro those cells transformed with the desired DNA sequences.

[0088] Escherichia coli is an example of a prokaryotic host cell that can be used to clone a polynucleotide encoding plasminogen. Other suitable microbial hosts include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors can be generated in these prokaryotic hosts and typically contain expression control sequences (e.g., an origin of replication) compatible with the host cell. Many known promoters are available, including the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, and the phage lambda-derived promoter system. Promoters generally control expression and, if necessary, may contain ribosome binding sequences to initiate transcription and translation of the controlled gene sequence.

[0089] Other microorganisms, such as yeast, can also be used for expression. Yeast (e.g., Saccharomyces (S. cerevisiae) and Pichia are examples of suitable yeast host cells, in which suitable carriers include expression control sequences (e.g., promoters), origins of replication, termination sequences, etc., as needed. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters for alcohol dehydrogenase, isocytochrome C, and enzymes for maltose and galactose utilization.

[0090] In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in in vitro cell culture) can also be used to express and produce the plasminogen of the present invention (e.g., a polynucleotide encoding plasminogen). See, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for use in these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from rabbit immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0091] Once synthesized (chemically or recombinantly), the plasminogen described in the present invention can be purified by standard procedures in the art, such as ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, etc. The plasminogen can be essentially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure or even purer, e.g., free from contaminants, such as cellular debris, large molecules other than plasminogen, etc.

[0092] Drug combinations Plasminogen of the desired purity is mixed with pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)) as needed to form a lyophilized preparation or aqueous solution to obtain a therapeutic formulation. Acceptable carriers, excipients, and stabilizers are non-toxic to subjects at the required doses and concentrations, and further include buffers such as phosphates, citrates, and other organic acids. Antioxidants include ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethylenediamine chloride; benzalkonium chloride). chloride), benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parahydroxybenzoate esters, such as methyl or propyl parahydroxybenzoate ester; pyrocatechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptides (having at least 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, fucose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants, such as TWEENT™, PLURONICS™, or polyethylene glycol (PEG).

[0093] The combination preparation of the present invention may contain one or more active compounds required for the specific symptoms requiring treatment, preferably compounds with complementary activities and no adverse effects, such as antihypertensives, antiarrhythmics, and antidiabetic drugs.

[0094] The plasminogen of the present invention can be encapsulated in microcapsules made, for example, by aggregation techniques or interfacial polymerization, and can be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into hydroxymethylcellulose or gel microcapsules and poly(methyl methacrylate) microcapsules in coarse emulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0095] The plasminogen of the present invention to be administered internally must be sterile, which is readily accomplished by filtration through sterile filtration membranes, prior to or after lyophilization and recombination.

[0096] The plasminogen of the present invention can be prepared in a buffered formulation. Suitable examples of buffered formulations include semipermeable matrices of solid hydrophobic polymers having a defined shape and containing the glycoprotein, such as membranes or microcapsules. Examples of buffer matrices include polyesters, aqueous gels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981); Langer, Chem. Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S. Patent 3,773,919, EP 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer et al., supra), or degradable lactic acid-hydroxyacetic acid copolymers, e.g., Lupron. These include Depot™ (injectable microspheres composed of lactic acid-hydroxyacetic acid copolymer and leuprolide acetate), and poly(D-(-)-3-hydroxybutyrate). Polymers such as ethylene-ethyl acetate and lactic acid-hydroxyacetic acid can release molecules sustainedly for over 100 days, whereas some aqueous gels release proteins for a relatively short period of time. Rational strategies for protein stabilization can be designed depending on the mechanism involved. For example, if the aggregation mechanism involves the exchange of sulfur disulfide bonds to form intermolecular disulfide bonds, stabilization can be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions.

[0097] Dosage and dosage The administration of the pharmaceutical compositions of the present invention can be achieved by different ways, such as intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (eg, via the carotid artery), intramuscular administration.

[0098] Preparations used for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte supplements, and the like. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, inert gases, and the like, may also be present.

[0099] A medical professional can determine dosage recommendations based on various clinical factors. For example, as is well known in the medical arts, the dosage for any given patient depends on several factors, including the patient's body size, body surface area, age, the specific compound being administered, sex, frequency and route of administration, overall health, and other concurrently administered medications. The dosage range for the plasminogen-containing pharmaceutical compositions of the present invention can be about 0.0001 to 2000 mg / kg of subject body weight daily, or about 0.001 to 500 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 10 mg / kg, 50 mg / kg, etc.) of subject body weight. For example, dosages can be 1 mg / kg body weight or 50 mg / kg body weight, or in the range of 1-50 mg / kg, or at least 1 mg / kg. Doses higher or lower than this exemplary range are also encompassed, particularly when the aforementioned factors are taken into consideration. Intermediate doses within the above ranges are also within the scope of the present invention. Subjects can receive such doses daily, every other day, weekly, or according to any schedule determined by empirical analysis. An exemplary dosing schedule includes administration of 1-10 mg / kg for several consecutive days. During the administration of the agents of the present invention, real-time evaluation of therapeutic efficacy and safety is required.

[0100] Product or Kit One embodiment of the present invention relates to a product or kit containing the plasminogen or plasmin of the present invention, which can be used to treat cardiovascular disease and related diseases caused by diabetes. The product preferably includes a container, label, or protocol. Suitable containers include bottles, vials, syringes, etc. The container can be made of various materials, such as glass or plastic. The container contains a composition that effectively treats the disease or condition of the present invention and has a sterile access point (e.g., the container is an intravenous infusion pack or vial and includes a stopper that can be pierced by a hypodermic needle). At least one activator in the composition is plasminogen or plasmin. A label on or attached to the container explains that the composition is used to treat cardiovascular disease and related diseases caused by diabetes as described in the present invention. The product may further include a second container containing a pharmaceutical buffer, such as phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials required from a commercial and user perspective, such as other buffers, diluents, filters, needles, and syringes. The product also includes a protocol with instructions for use, for example, instructing a user of the composition to administer the plasminogen composition and other agents to a patient for the treatment of the associated disorder.

[0101] In some embodiments, the diseases caused by pathological protein aggregation include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion-like diseases (Creutzfeldt-Jakob disease), Gerstmann's syndrome, diffuse or familial fatal insomnia and kuru, Huntington's disease, spinocerebellar ataxia type 3, and dentatorubral-pallidal atrophy with Lewy body disease.

[0102] In some embodiments, diseases caused by ubiquitin / lysosomal dysfunction include Fanconi anemia, Xeroderma pigmentosum, Cockayne syndrome, cancer, Cowden syndrome, Parkinson's disease, genomic instability, metabolic syndrome, muscular dystrophy, Von Hippel-Lindau syndrome, multiple myeloma, RIDDLE syndrome, Huntington's disease, X-linked lymphoproliferative disorders, Crohn's disease, Alzheimer's disease, breast and ovarian cancer, muscular amylopectinosis, amyotrophic lateral sclerosis, spinal muscular atrophy, systemic lupus erythematosus, and the like. erythematosus, lupus erythematosus, infantile ataxia, X-linked parkinsonism with spasticity, multisystem disorders, diabetes mellitus, multiple sclerosis, cystinosis, Vici syndrome, Gaucher's disease, frontotemporal dementia (heterozygous) or neuronal ceroid lipofuscinosis (homozygous), Danon cardiomyopathy, cortical atrophy, epilepsy, autosomal recessive spinocerebellar ataxia, hereditary spastic paraplegia, beta-propeller protein-associated neurodegeneration (BPAN), autism spectrum disorder, frontotemporal dementia (FTD), inflammatory bowel disease, nonalcoholic fatty liver disease, tuberculosis, Rett syndrome, osteopetrosis, Charcot-Marie-Tooth type 2B disease disease, juvenile arthritis, Snyder-Robinson syndrome (SRS), Wiskott-Aldrich syndromesyndrome, primary microcephaly, hereditary sensory and autonomic neuropathy type II, primary open-angle glaucoma (POAG), Paget's disease of the bone (PGD), colorectal cancer, lung cancer, brain tumors, autosomal recessive and sporadic early-onset Parkinson's disease, Zellweger syndrome spectrum disorders, distal myopathy, ulcerative colitis, familial Mediterranean fever, spastic ataxia, Fabry disease, Gaucher disease, lysosomal acid lipase deficiency, mucopolysaccharidoses, and Angelman syndrome. [Example]

[0103] The human plasminogen used in the following examples was derived from donor plasma and has been described in the following literature: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry, 1965, 240(1):541-550; Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J Biol Chem. 1976 Jun 25; 251(12):3693-9; Hagan JJ, Ablondi F, De Renzo EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 Based on the method described in [1-3], the process was optimized and purified from human donor plasma, resulting in over 98% human Lys-plasminogen (Lys-plasminogen) and Glu-plasminogen (Glu-plasminogen).

[0104] Example Example 1 Plasminogen is concentrated in the spinal cord tissue of ALS model mice and co-localizes with ubiquitin Transgenic mutant SOD1 exhibits histopathological features observed in the clinical setting of sporadic and familial amyotrophic lateral sclerosis (ALS). The ALS model mice used in this study were B6.Cg-Tg(SOD1-G93A)1Gur / J transgenic mice (abbreviated as SOD1-G93A) (pedigree number: 004435) purchased from the Jackson Laboratory. Animal experiments were performed under an SPF environment. SOD1-G93A model mice are widely used in research into ALS mechanisms and preclinical trials for new drug development.

[0105] Five wild-type male mice and nine SOD1-G93A male mice of similar age were used. Wild-type mice served as a blank control group, while SOD1-G93A mice were observed and recorded from the time of hindlimb tremors at 14 weeks of onset. The time of onset for each mouse was recorded, and drug administration began 14 days after onset. All mice were randomly assigned to a vehicle or drug-treatment group based on the onset of symptoms. Five mice in the vehicle group received daily injections of 0.1 ml of vehicle (sodium citrate buffer) via the tail vein, while four mice in the drug-treatment group received daily injections of 1 mg of plasminogen per 0.1 ml via the tail vein. These mice were continuously administered in an SPF environment, and samples were collected near the end of life. The maximum treatment period was 61 days. Spinal cord tissue was fixed in formalin. Fixed tissue was dehydrated through an alcohol gradient, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, rehydrated, and then washed once with water. The sections were immersed in an antigen retrieval solution (0.01 M sodium citrate buffer) and microwaved for 5 minutes, followed by 2 minutes on high heat and 15 minutes on low heat. The tissue was circled with a PAP marker and incubated with 3% hydrogen peroxide for 15 minutes, followed by two 5-minute washes with 0.01 M PBS. The sections were then blocked with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 minutes. The serum was then discarded, and autologous anti-plasminogen antibody (autologous) was added and incubated overnight at 4°C. The sections were then incubated with a goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two 5-minute washes with 0.01 M PBS. Green fluorescent staining with the corresponding anti-plasminogen secondary antibody was performed according to the protocol of the XTSA520 IHC kit (Alpha X Biotech, AXT6202500). After washing three times with PBS, each time for 5 minutes, the above antigen retrieval and blocking procedures were repeated, followed by staining with anti-ubiquitin antibody (Abcam, ab7780) and incubation at 37°C for 1 hour. After washing three times with PBS, each time for 5 minutes, the cells were stained with anti-ubiquitin antibody (Abcam, ab7780).The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. Red fluorescent staining with the corresponding anti-ubiquitin secondary antibody was performed according to the protocol of the XTSA520 IHC kit (Alpha X Biotech, AXT6202500). The sections were washed three times with PBS for 5 minutes each time. Nuclear staining with DAPI (BOSTER, 11K16B77) was performed. The sections were dehydrated through an alcohol gradient, cleared with xylene, and mounted in neutral rubber. Sections were observed and photographed under a light microscope at 400x magnification.

[0106] Ubiquitin (Ub) is a highly conserved, low-molecular-weight protein with a molecular weight of approximately 8500. It consists of 76 amino acids and is widely distributed in eukaryotic cells. The main function of ubiquitin is to participate in the degradation of most proteins in eukaryotic cells, and the proteasome is the site of ubiquitin-mediated protein degradation. The ubiquitin-proteasome pathway is one of the important pathways for "digesting" intracellular proteins.

[0107] The results showed that positive staining for plasminogen (green fluorescence) in the spinal cord tissue of the drug-treated group was significantly higher than that of the vehicle group, indicating that the administered plasminogen can enter and concentrate in spinal cord tissue. Plasminogen also exists in the cytoplasm and nucleus. Plasminogen and ubiquitin (red fluorescence) colocalized in the cytoplasm (as indicated by triangles) (Figure 2). The ubiquitin expression level in the spinal cord tissue of the drug-treated group appeared to be lower than that of the vehicle group. This indicates that in the amyotrophic lateral sclerosis model mice, plasminogen can enter spinal cord tissue, enter cells, colocalize with ubiquitin, and reduce ubiquitin expression. This suggests that plasminogen may interact with ubiquitin.

[0108] Example 2 Plasminogen is concentrated in the spinal cord tissue of ALS model mice and co-localizes with LC3B Five wild-type male mice and nine SOD1-G93A male mice of similar age were used. Wild-type mice served as a blank control group, while SOD1-G93A mice were observed and recorded from the time of hindlimb tremors at 14 weeks of onset. The time of onset for each mouse was recorded, and drug administration began 14 days after onset. All mice were randomly assigned to a vehicle or drug-treatment group based on the onset of symptoms. Five mice in the vehicle group received daily injections of 0.1 ml of vehicle (sodium citrate buffer) via the tail vein, while four mice in the drug-treatment group received daily injections of 1 mg of plasminogen per 0.1 ml via the tail vein. These mice were continuously administered in an SPF environment, and samples were collected near the end of life. The maximum treatment period was 61 days. Spinal cord tissue was fixed in formalin. Fixed tissue was dehydrated through an alcohol gradient, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, rehydrated, and then washed once with water. The sections were immersed in an antigen retrieval solution (0.01 M sodium citrate buffer) and microwaved for 5 minutes, followed by 2 minutes on high heat and 15 minutes on low heat. The tissue was circled with a PAP marker and incubated with 3% hydrogen peroxide for 15 minutes, followed by two 5-minute washes with 0.01 M PBS. The sections were then blocked with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 minutes. The serum was then discarded, and autologous anti-plasminogen antibody (autologous) was added and incubated overnight at 4°C. The sections were then incubated with a goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two 5-minute washes with 0.01 M PBS. Green fluorescent staining with the corresponding anti-plasminogen secondary antibody was performed according to the protocol of the XTSA520 IHC kit (Alpha X Biotech, AXT6202500). After washing three times with PBS for 5 minutes each time, the above antigen retrieval and blocking procedures were repeated, followed by staining with anti-LC3B antibody (Proteintech, 18725-1-AP) and incubation at 37°C for 1 hour. After washing three times with PBS for 5 minutes each time, the cells were stained with anti-LC3B antibody (Proteintech, 18725-1-AP).The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. Red fluorescent staining with the corresponding anti-LC3B secondary antibody was performed according to the protocol of the XTSA520 IHC kit (Alpha X Biotech, AXT6202500). The sections were washed three times with PBS for 5 minutes each time. Nuclear staining with DAPI (BOSTER, 11K16B77) was performed. The sections were dehydrated through an alcohol gradient, cleared with xylene, and mounted in neutral rubber. Sections were observed and photographed under a light microscope at 400x magnification.

[0109] Autophagy is the process of degrading dysfunctional cellular components within cells via lysosomes. Autophagy can degrade and digest biological macromolecules, such as damaged or denatured organelles, proteins, and nucleic acids. It provides raw materials for cellular regeneration and repair and enables the recycling of intracellular materials. LC3 (protein light chain 3) is a marker of the autophagic process. Its function is primarily involved in the formation of autophagosomes. The LC3 precursor molecule is cleaved to remove a C-terminal 5-peptide, forming the cytosolic form LC3-I. It is then activated by APG7L / ATG7 and transferred to ATG3, where it couples with phosphatidylethanolamine (PE) to form the membrane-bound form LC3-II (LC3B), which can then attach to the autophagosome membrane and become a structural protein of the autophagosome.

[0110] The results showed that positive staining for plasminogen (green fluorescence) in the spinal cord tissue of the drug-treated group was significantly higher than that of the vehicle group, indicating that the administered plasminogen was able to enter and concentrate in spinal cord tissue. Plasminogen was also present in the cytoplasm and nucleus. Plasminogen and LC3B (red fluorescence) colocalized in the cytoplasm (as indicated by triangles), and the LC3B expression level in the spinal cord tissue of the drug-treated group appeared to be lower than that of the vehicle group. This indicates that in the amyotrophic lateral sclerosis model mice, plasminogen enters spinal cord tissue, enters cells, and colocalizes with LC3B. This suggests that plasminogen may interact with LC3B.

[0111] Example 3 Plasminogen is concentrated in the spinal cord tissue of ALS model mice and co-localizes with LAMP2 Five wild-type male mice and nine SOD1-G93A male mice of similar age were used. Wild-type mice served as a blank control group, while SOD1-G93A mice were observed and recorded from the time of hindlimb tremors at 14 weeks of onset. The time of onset for each mouse was recorded, and drug administration began 14 days after onset. All mice were randomly assigned to a vehicle or drug-treatment group based on the onset of symptoms. Five mice in the vehicle group received daily injections of 0.1 ml of vehicle (sodium citrate buffer) via the tail vein, while four mice in the drug-treatment group received daily injections of 1 mg of plasminogen per 0.1 ml via the tail vein. These mice were continuously administered in an SPF environment, and samples were collected near the end of life. The maximum treatment period was 61 days. Spinal cord tissue was fixed in formalin. Fixed tissue was dehydrated through an alcohol gradient, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, rehydrated, and then washed once with water. The sections were immersed in an antigen retrieval solution (0.01 M sodium citrate buffer) and microwaved for 5 minutes, followed by 2 minutes on high heat and 15 minutes on low heat. The tissue was circled with a PAP marker and incubated with 3% hydrogen peroxide for 15 minutes, followed by two 5-minute washes with 0.01 M PBS. The sections were then blocked with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 minutes. The serum was then discarded, and autologous anti-plasminogen antibody (autologous) was added and incubated overnight at 4°C. The sections were then incubated with a goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two 5-minute washes with 0.01 M PBS. Green fluorescent staining with the corresponding anti-plasminogen secondary antibody was performed according to the protocol of the XTSA520 IHC kit (Alpha X Biotech, AXT6202500). After washing three times with PBS for 5 minutes each time, the above antigen retrieval and blocking procedures were repeated, followed by staining with anti-LAMP2 antibody (BOSTER, BM4357) and incubation at 37°C for 1 hour. After washing three times with PBS for 5 minutes each time, the cells were stained with anti-LAMP2 antibody (BOSTER, BM4357).The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. Red fluorescence staining with the corresponding anti-LAMP2 secondary antibody was performed according to the protocol of the XTSA520 IHC kit (Alpha X Biotech, AXT6202500). The sections were washed three times with PBS for 5 minutes each time. Nuclear staining was performed with DAPI (BOSTER, 11K16B77). The sections were dehydrated through an alcohol gradient, cleared with xylene, and mounted in neutral rubber. Sections were observed and photographed under a light microscope at 400x magnification.

[0112] Lysosome-associated membrane protein-2 (LAMP2) is a highly abundant lysosomal glycoprotein that functions as a receptor for proteins directly imported into lysosomes and as a mediator of autophagosome / phagosome maturation [4].

[0113] The results showed that the positive staining of plasminogen (green fluorescence) in the spinal cord tissue of the drug-treated group was significantly higher than that of the vehicle group, indicating that the administered plasminogen was able to enter and concentrate in spinal cord tissue. Plasminogen was also present in the cytoplasm and nucleus. Plasminogen and LAMP2 (red fluorescence) colocalized in the cytoplasm (as indicated by triangles), and the LAMP2 expression level in the spinal cord tissue of the drug-treated group appeared to be higher than that of the vehicle group (Figure 4). This indicates that in the amyotrophic lateral sclerosis model mice, plasminogen enters spinal cord tissue, enters cells, colocalizes with LAMP2, promotes LAMP2 expression, and promotes lysosomal function.

[0114] Example 4 Plasminogen promotes LC3 expression in spinal cord tissue of amyotrophic lateral sclerosis model mice Six SOD1-G93A mice aged 10–15 weeks were randomly divided into two groups: three in the vehicle control group and three in the drug-treated group. Mice in the vehicle control group were injected with vehicle at 5 ml / kg via the tail vein, while mice in the drug-treated group were injected with plasminogen (10 mg / ml) at 50 mg / kg body weight via the tail vein. Twenty-four hours after injection, the mice were sacrificed and their spinal cords were collected. After homogenization, RT-PCR detection of LC3 gene transcription was performed, and the C values ​​were recorded. The C values ​​of LC3 gene transcript mRNA in 100 ng of total RNA were calculated.

[0115] As a result, LC3 expression in the drug-treated group was significantly higher than that in the vehicle group, and the difference was statistically significant (* indicates P<0.05) (Figure 5). These results indicate that plasminogen promotes the transcription of the LC3 gene in amyotrophic sclerosis model mice, thereby contributing to the regulation of the autophagy process.

[0116] Example 5 Plasminogen is concentrated in the substantia nigra and striatum of Parkinson's disease model mice and colocalizes with ubiquitin Twelve 9-week-old C57 male mice were weighed one day before modeling and administered 5 mg / ml MPTP solution intraperitoneally at 30 mg / kg body weight for five consecutive days to establish a Parkinson's disease model [5,6]. To prepare the MPTP solution, 10 ml of deionized water was drawn using a syringe and added to 100 mg of MPTP powder (Sigma, M0896) to prepare a 10 mg / ml stock solution. Next, 1 ml of the stock solution was pipetted into an ampoule, and 1 ml of deionized water was added to achieve a final concentration of 5 mg / ml. After modeling, the mice were randomly divided into two groups: a PBS control group and a plasminogen-treated group (six mice each). The administration began on day 1. The plasminogen-treated group received plasminogen solution at 1 mg / 0.1 ml / mouse / day via tail vein injection, while the PBS control group received the same volume of PBS via tail vein injection for 14 consecutive days. On day 15 of administration, mice were sacrificed, and the substantia nigra and striatum were collected and fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissues were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. Co-staining for plasminogen, ubiquitin, and DAPI was performed as described in Example 1. Sections were observed and photographed under a light microscope at 400x magnification.

[0117] The results showed significantly more positive staining for plasminogen (green fluorescence) in the substantia nigra and striatum tissues in the drug-treated group than in the vehicle-treated group, indicating that the administered plasminogen can enter and concentrate in the substantia nigra and striatum tissues. Plasminogen also exists in the cytoplasm and nucleus. Plasminogen colocalizes with ubiquitin (red fluorescence) in the cytoplasm (as indicated by triangles) (Figure 6). This indicates that in Parkinson's disease model mice, plasminogen can enter the substantia nigra and striatum tissues, enter cells, and colocalize with ubiquitin. This suggests that plasminogen may interact with ubiquitin.

[0118] Example 6 Plasminogen is concentrated in the substantia nigra and striatum of Parkinson's disease model mice and co-localizes with LC3B Twelve 9-week-old C57 male mice were weighed one day before modeling and administered 5 mg / ml MPTP solution intraperitoneally at 30 mg / kg body weight for five consecutive days to establish a Parkinson's disease model [5,6]. To prepare the MPTP solution, 10 ml of deionized water was drawn using a syringe and added to 100 mg of MPTP powder (Sigma, M0896) to prepare a 10 mg / ml stock solution. Next, 1 ml of the stock solution was pipetted into an ampoule, and 1 ml of deionized water was added to achieve a final concentration of 5 mg / ml. After modeling, the mice were randomly divided into two groups: a PBS control group and a plasminogen-treated group (six mice each). The administration began on day 1. The plasminogen-treated group received plasminogen solution at 1 mg / 0.1 ml / mouse / day via tail vein injection, while the PBS control group received the same volume of PBS via tail vein injection for 14 consecutive days. On day 15 after administration, the mice were sacrificed, and the substantia nigra and striatum were collected and fixed in 4% paraformaldehyde for 24-48 hours. Co-staining for plasminogen, LC3B, and DAPI was performed as described in Example 2. Sections were observed and photographed under a light microscope at 400x magnification.

[0119] The results showed significantly more positive staining for plasminogen (green fluorescence) in the substantia nigra and striatum tissues of the drug-treated group than in the vehicle-treated group, indicating that the administered plasminogen can enter and concentrate in the substantia nigra and striatum tissues. Plasminogen also exists in the cytoplasm and nucleus. Plasminogen colocalizes with LC3B (red fluorescence) in the cytoplasm (as indicated by triangles) (Figure 7). This indicates that in Parkinson's disease model mice, plasminogen can enter the substantia nigra and striatum tissues, enter cells, and colocalize with LC3B. This suggests that plasminogen may interact with LC3B.

[0120] Example 7 Plasminogen is concentrated in the substantia nigra and striatum of Parkinson's disease model mice and co-localizes with LAMP2 Twelve 9-week-old C57 male mice were weighed one day before modeling and administered 5 mg / ml MPTP solution intraperitoneally at 30 mg / kg body weight for five consecutive days to establish a Parkinson's disease model [5,6]. To prepare the MPTP solution, 10 ml of deionized water was drawn using a syringe and added to 100 mg of MPTP powder (Sigma, M0896) to prepare a 10 mg / ml stock solution. Next, 1 ml of the stock solution was pipetted into an ampoule, and 1 ml of deionized water was added to achieve a final concentration of 5 mg / ml. After modeling, the mice were randomly divided into two groups: a PBS control group and a plasminogen-treated group (six mice each). The administration began on day 1. The plasminogen-treated group received plasminogen solution at 1 mg / 0.1 ml / mouse / day via tail vein injection, while the PBS control group received the same volume of PBS via tail vein injection for 14 consecutive days. On the 15th day after administration, the mice were sacrificed, and the substantia nigra and striatum were collected and fixed in 4% paraformaldehyde for 24-48 hours. Co-staining for plasminogen, LAMP2, and DAPI was performed as described in Example 3. Sections were observed and photographed under a light microscope at 400x magnification.

[0121] The results showed significantly more positive staining for plasminogen (green fluorescence) in the substantia nigra and striatum tissues of the drug-treated group than in the vehicle-treated group, indicating that the administered plasminogen can enter and concentrate in the substantia nigra and striatum tissues. Plasminogen also exists in the cytoplasm and nucleus. Plasminogen colocalized with LAMP2 (red fluorescence) in the cytoplasm (as indicated by triangles) (Figure 8). This indicates that in Parkinson's disease model mice, plasminogen can enter the substantia nigra and striatum tissues, enter cells, and colocalize with LAMP2. This suggests that plasminogen may interact with LAMP2.

[0122] Example 8 Plasminogen is concentrated in the hippocampus of Alzheimer's disease model mice and co-localizes with ubiquitin Twenty 8-week-old male C57 mice were weighed before modeling. Abnormal mice were excluded based on their weight. All mice were randomly divided into two groups: a vehicle group and a drug-treated group (10 mice each). All mice were anesthetized and positioned in the granule cell layer of the hippocampus using a mouse stereotaxic atlas (based on Bregma coordinates: AP -2.0 mm, ML ±1.5 mm, DV 2.0 mm). Each mouse was slowly microinjected bilaterally at a rate of 0.5 μL / min with a volume of 3 μL. Mice in the model group were injected with Aβ 1-42 oligomer solution to establish an Alzheimer's disease model [7], while mice in the model control group were injected with PBS solution. Preparation of Aβ 1-42 oligomer solution (10 μM): β-amyloid (1-42) (ChinaPeptide, 04010011521) was added to cold hexafluoroisopropanol to a concentration of 1 mg / mL. The solution was left at room temperature for 3 days, then dispensed into 45 μL / tube (10 nmol / mL), left overnight in a fume hood, dried in a dry oven at 25°C for 1 hour, and stored at -80°C. For use, each tube was reconstituted with 10 μL of dimethyl sulfoxide solution. For injection, 990 μL of sterile PBS solution was added and the tube was left at 4°C for 24 hours before use. Twenty-one days after the brain-localized injection, treatment with vehicle and drug groups began, marking this as day 1. Mice in the drug-treated group received plasminogen at 1 mg / 0.1 ml / mouse / day via the tail vein, while mice in the vehicle group received vehicle (4% arginine + 2% glycine solution) at 0.1 ml / mouse / day via the tail vein. Treatment continued for 28 days. On day 29, mice were sacrificed, and their brains were collected and fixed in 10% formaldehyde for 24–48 hours. The fixed tissues were dehydrated in an alcohol gradient, permeabilized with xylene, and then embedded in paraffin. Co-staining for plasminogen, ubiquitin, and DAPI was performed as described in Example 1. Sections were observed and photographed under a light microscope at 400x magnification.

[0123] The results showed that positive staining for plasminogen (green fluorescence) in the hippocampal tissue of the drug-treated group was significantly higher than that of the vehicle-treated group, indicating that the administered plasminogen can enter and concentrate in the hippocampal tissue. Plasminogen also exists in the cytoplasm and nucleus. Plasminogen colocalizes with ubiquitin (red fluorescence) in the cytoplasm (as indicated by triangles) (Figure 9). This indicates that plasminogen can enter the hippocampal tissue and cells in Alzheimer's disease model mice. This suggests that plasminogen may interact with ubiquitin.

[0124] Example 9 Plasminogen is concentrated in the hippocampus of Alzheimer's disease model mice and co-localizes with LC3B Twenty 8-week-old male C57 mice were weighed before modeling. Abnormal mice were excluded based on their weight. All mice were randomly divided into two groups: a vehicle group and a drug-treated group (10 mice each). All mice were anesthetized and positioned in the granule cell layer of the hippocampus using a mouse stereotaxic atlas (based on Bregma coordinates: AP -2.0 mm, ML ±1.5 mm, DV 2.0 mm). Each mouse was slowly microinjected bilaterally at a rate of 0.5 μL / min with a volume of 3 μL. Mice in the model group were injected with Aβ 1-42 oligomer solution to establish an Alzheimer's disease model [7], while mice in the model control group were injected with PBS solution. Preparation of Aβ 1-42 oligomer solution (10 μM): β-amyloid (1-42) (ChinaPeptide, 04010011521) was added to cold hexafluoroisopropanol to a concentration of 1 mg / mL. The solution was left at room temperature for 3 days, then dispensed into 45 μL / tube (10 nmol / mL), left overnight in a fume hood, dried in a dry oven at 25°C for 1 hour, and stored at -80°C. For use, each tube was reconstituted with 10 μL of dimethyl sulfoxide solution. For injection, 990 μL of sterile PBS solution was added and the tube was left at 4°C for 24 hours before use. Twenty-one days after the brain-localized injection, treatment with vehicle and drug groups began, marking this as day 1. Mice in the drug-treated group received plasminogen at 1 mg / 0.1 ml / mouse / day via the tail vein, while mice in the vehicle group received vehicle (4% arginine + 2% glycine solution) at 0.1 ml / mouse / day via the tail vein. Treatment continued for 28 days. On day 29, mice were sacrificed, and their brains were collected and fixed in 10% formaldehyde for 24–48 hours. The fixed tissues were dehydrated in an alcohol gradient, permeabilized with xylene, and then embedded in paraffin. Co-staining for plasminogen, LC3B, and DAPI was performed as described in Example 2. Sections were observed and photographed under a light microscope at 400x magnification.

[0125] The results showed that positive staining of plasminogen (green fluorescence) in the hippocampal tissue of the drug-treated group was significantly higher than that of the vehicle group, indicating that the administered plasminogen could enter and concentrate in the hippocampal tissue. Plasminogen was also present in the cytoplasm and nucleus. Plasminogen colocalized with LC3B (red fluorescence) in the cytoplasm (as indicated by triangles) (Figure 10). This indicates that plasminogen can enter the hippocampal tissue and cells in Alzheimer's disease model mice. This suggests that plasminogen may interact with LC3B.

[0126] Example 10 Plasminogen is concentrated in the hippocampus of Alzheimer's disease model mice and co-localizes with LAMP2 Twenty 8-week-old male C57 mice were weighed before modeling. Abnormal mice were excluded based on their weight. All mice were randomly divided into two groups: a vehicle group and a drug-treated group (10 mice each). All mice were anesthetized and positioned in the granule cell layer of the hippocampus using a mouse stereotaxic atlas (based on Bregma coordinates: AP -2.0 mm, ML ±1.5 mm, DV 2.0 mm). Each mouse was slowly microinjected bilaterally at a rate of 0.5 μL / min with a volume of 3 μL. Mice in the model group were injected with Aβ 1-42 oligomer solution to establish an Alzheimer's disease model [7], while mice in the model control group were injected with PBS solution. Preparation of Aβ 1-42 oligomer solution (10 μM): β-amyloid (1-42) (ChinaPeptide, 04010011521) was added to cold hexafluoroisopropanol to a concentration of 1 mg / mL. The solution was left at room temperature for 3 days, then dispensed into 45 μL / tube (10 nmol / mL), left overnight in a fume hood, dried in a dry oven at 25°C for 1 hour, and stored at -80°C. For use, each tube was reconstituted with 10 μL of dimethyl sulfoxide solution. For injection, 990 μL of sterile PBS solution was added and the tube was left at 4°C for 24 hours before use. Twenty-one days after the brain-localized injection, treatment with vehicle and drug groups began, marking this as day 1. Mice in the drug-treated group received plasminogen at 1 mg / 0.1 ml / mouse / day via the tail vein, while mice in the vehicle group received vehicle (4% arginine + 2% glycine solution) at 0.1 ml / mouse / day via the tail vein. Treatment continued for 28 days. On day 29, mice were sacrificed, and their brains were collected and fixed in 10% formaldehyde for 24–48 hours. The fixed tissues were dehydrated in an alcohol gradient, permeabilized with xylene, and then embedded in paraffin. Co-staining for plasminogen, LAMP2, and DAPI was performed as described in Example 3. Sections were observed and photographed under a light microscope at 400x magnification.

[0127] The results showed that positive staining of plasminogen (green fluorescence) in the hippocampal tissue of the drug-treated group was significantly higher than that of the vehicle-treated group, indicating that the administered plasminogen could enter and concentrate in the hippocampal tissue. Plasminogen was also present in the cytoplasm and nucleus. Plasminogen colocalized with LAMP2 (red fluorescence) in the cytoplasm (as indicated by triangles) (Figure 11). This indicates that plasminogen can enter the hippocampal tissue and cells in Alzheimer's disease model mice. This suggests that plasminogen may interact with LAMP2.

[0128] Example 11 Plasminogen promotes huntingtin protein degradation in the heart of Huntington's disease model mice Ten 14-week-old B6 / JGpt-Tg(hHTT-CAG130)90 / Gpt mice (hereafter referred to as hHTT130 transgenic mice) were selected as the model group, and five C57 mice were selected as the normal control group. The hHTT130 mice were randomly divided into two groups: a vehicle group and a drug-treated group, with five mice in each group. After grouping, all mice were treated. The drug-treated group received 50 mg / kg plasminogen via tail vein injection, while the normal control and vehicle groups received an equal volume of PBS via tail vein injection for 28 consecutive days. After treatment, the mice were sacrificed, and their hearts were harvested. Western blot detection of huntingtin protein (HTT) was performed on cardiac tissue homogenates. 10% SDS-PAGE gels were prepared according to the gel preparation method. Each sample was mixed uniformly with 4x loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated to 100°C for 5 minutes, cooled, centrifuged for 2 minutes, and then 20 μL was loaded. Electrophoresis was performed at 30 V for 45 minutes, followed by electrophoresis at 100 V until the gel bottom was reached. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753). Electrophoresis was performed at 15 V for 2.5 hours. The transferred PVDF membrane was immersed in blocking solution (5% skim milk) and blocked overnight in a refrigerator at 4°C. After washing four times with TBST (0.01M Tris-NaCl, pH 7.6 buffer), rabbit anti-HTT antibody (Abcam, ab109115) and actin antibody were added and incubated at room temperature for 1.5 hours. After washing four times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1 hour. After washing four times with TBST, the PVDF membrane was placed on a clean imaging plate and developed with Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100). Images were captured using a biomolecular imaging system and quantitative analysis was performed using Image J.

[0129] The results showed that cardiac HTT levels in the vehicle-treated mice were significantly higher than in the normal control group, and significantly lower than in the drug-treated mice (Figure 12A-B), with statistically significant differences (* indicates P<0.05). This suggests that plasminogen can promote the degradation of huntingtin protein in the cardiac tissue of Huntington's disease model mice.

[0130] Example 12 Plasminogen promotes degradation of TDP-43 in the cytoplasm and nucleus of okadaic acid-treated NSC34 cells 10 6 NSC34 cells (Otwo Biotech, HTX1846) were cultured in a 9cm 2Cells were seeded onto Petri dishes and cultured in DMEM medium (Gibco, 11965092) containing 10% fetal bovine serum (EVERY GREEN, 11011-8611), then placed in a carbon dioxide incubator at 37.0°C with 5% CO2. After 48 hours of growth and reaching approximately 80%-90% confluence, the medium was replaced and the next experiment was performed. Cells were divided into four groups: a blank control group, a vehicle group, a drug-treated group, and a drug + EACA group. Cells in the blank control group were not treated after the medium change. Cells in the vehicle group, drug-treated group, and drug + EACA group were exposed to 2.5 ng / μL okadaic acid (OA) (Shanghai Yuanye Bio-Technology, S30686-25μg). After 24 hours of stimulation with okadaic acid, the vehicle was added to the cell culture medium of the vehicle group, plasminogen (0.5 mg / mL) was added to the cell culture medium of the drug-treated group, and plasminogen (final concentration 0.5 mg / mL) and aminocaproic acid (EACA) (20 mM) were added to the cell culture medium of the drug + EACA group. After 24 hours of treatment with plasminogen, the cells were harvested. The culture supernatant was aspirated, washed with 1x PBS, and digested with 1 mL of 0.25% pancreatin for 2–3 minutes. When cells were clearly detached, the digestion was stopped with 5–6 mL of DMEM complete medium. The cells were gently pipetted, the suspension was collected in a centrifuge tube, and centrifuged at 1500 rpm for 5 minutes to remove the supernatant. The cells were resuspended in pre-chilled 1x PBS, and then counted. For every 20 μL of cell pellet, 200 μL of plasma protein extraction reagent (2 x 10 6The volume of the cell pellet was approximately 20 μL (or 40 mg) (Solarbio, R0050). The cell pellet was completely dispersed into a single-cell suspension by pipetting or vortexing at high speed for 15 seconds. The mixture was then placed in an ice bath for 10 minutes. The mixture was then vortexed vigorously at maximum speed for 10 seconds and centrifuged at 12,000–16,000 g for 10 minutes at 4°C. The supernatant, which represents the extracted cytoplasmic proteins, was immediately aspirated and transferred to a pre-chilled sample tube for later use. The precipitate, representing the cell nuclei, was then added to the remaining supernatant by aspirating (to avoid contamination with cytoplasmic proteins). The remaining supernatant was then completely aspirated and 50–100 μL of nuclear protein extraction reagent was added. The precipitate was completely dispersed by pipetting or vortexing at high speed for 15 seconds (this can be extended as needed) and then placed in an ice bath for 10 minutes. After vigorously vortexing at maximum speed for 10 seconds, the mixture was centrifuged at 12,000-16,000 g for 10 minutes at 4°C. The supernatant was immediately aspirated and placed in a pre-chilled sample tube. This represents the extracted nuclear proteins. The extracted nuclear proteins were detected by TDP-43 Western blotting.

[0131] Aminocaproic acid (EACA) is a lysine analog that blocks the high-affinity lysine binding site on plasminogen [8].

[0132] The results show that the TDP-43 levels in the cytoplasm and nucleus of the drug-treated group were significantly lower than those in the nucleus of the vehicle group, and the addition of EACA completely inhibited the effect of plasminogen on TDP-43 (Figures S13A-S13D). These results suggest that plasminogen can promote the degradation of TDP-43 in the cytoplasm and nucleus, and that this effect of plasminogen is closely related to the lysine-binding site in its structure.

[0133] Example 13 Plasminogen promotes degradation of TDP-43 in the cytoplasm and nucleus of okadaic acid-treated NSC34 cells 10 6NSC34 cells (Otwo Biotech, HTX1846) were cultured in a 9cm 2 Cells were seeded onto Petri dishes and cultured in DMEM medium (Gibco, 11965092) containing 10% fetal bovine serum (EVERY GREEN, 11011-8611), then placed in a carbon dioxide incubator at 37.0°C with 5% CO2. After 48 hours of growth and reaching approximately 80%-90% confluence, the medium was replaced and the next experiment was performed. Cells were divided into four groups: a blank control group, a vehicle group, a drug-treated group, and a drug + EACA group. Cells in the blank control group were not treated after the medium change. Cells in the vehicle group, drug-treated group, and drug + EACA group were exposed to 2.5 ng / μL okadaic acid (OA) (Shanghai Yuanye Bio-Technology, S30686-25μg). After 24 hours of stimulation with okadaic acid, the vehicle was added to the cell culture medium of the vehicle group, plasminogen (0.5 mg / mL) was added to the cell culture medium of the drug-treated group, and plasminogen (final concentration 0.5 mg / mL) and aminocaproic acid (EACA) (20 mM) were added to the cell culture medium of the drug + EACA group. After 24 hours of treatment with plasminogen, the cells were harvested. The culture supernatant was aspirated, washed with 1x PBS, and digested with 1 mL of 0.25% pancreatin for 2–3 minutes. When cells clearly detached, the digestion was stopped by adding 5–6 mL of DMEM complete medium. The cells were gently pipetted, the suspension was collected in a centrifuge tube, and centrifuged at 1500 rpm for 5 minutes to remove the supernatant. The cells were resuspended in pre-chilled 1x PBS, and then counted. Cell lysis buffer (Solarbio, R0010-100 mL) was added to lyse the cells and extract cellular proteins. Extracted cellular proteins were detected by Western blotting for phosphorylated tau protein (Abcam, ab151559) and total tau protein (Proteintech, 10842-1-AP).

[0134] The results showed that the levels of phosphorylated tau protein in the drug-treated group were significantly lower than in the vehicle group. The addition of EACA completely inhibited the effect of plasminogen on phosphorylated tau protein (Figure 14A-B). There was no significant difference in total tau protein levels between the drug-treated and vehicle groups. The ratio of phosphorylated tau protein to total tau protein in the drug-treated group was significantly lower than in the vehicle group. The addition of EACA completely inhibited this effect of plasminogen (Figure 14C-E). * indicates P<0.05, *** indicates P<0.001. These results suggest that plasminogen can promote the degradation of phosphorylated tau protein within cells, and that this effect of plasminogen is closely related to the lysine-binding site in plasminogen's structure.

[0135] Example 14 Plasminogen promotes increased levels of plasminogen and plasmin activity in the cytoplasm and nucleus of okadaic acid-treated NSC34 cells 10 6 NSC34 cells (Otwo Biotech, HTX1846) were cultured in a 9cm 2Cells were seeded onto Petri dishes and cultured in DMEM medium (Gibco, 11965092) containing 10% fetal bovine serum (EVERY GREEN, 11011-8611), then placed in a carbon dioxide incubator at 37.0°C with 5% CO2. After the cells had grown for 48 hours and reached approximately 80%-90% confluence, the medium was replaced and the next experiment was performed. The cells were divided into three groups: vehicle, drug-treated, and drug + EACA. The vehicle, drug-treated, and drug + EACA groups were exposed to 2.5 ng / μL okadaic acid (OA) (Shanghai Yuanye Bio-Technology, S30686-25μg). After 24 hours of stimulation with okadaic acid, the vehicle was added to the cell culture medium of the vehicle group, plasminogen (0.5 mg / mL) was added to the cell culture medium of the drug-treated group, and plasminogen (final concentration 0.5 mg / mL) and aminocaproic acid (EACA) (20 mM) were added to the cell culture medium of the drug + EACA group. After 24 hours of treatment with plasminogen, the cells were harvested. The culture supernatant was aspirated, washed with 1x PBS, and digested with 1 mL of 0.25% pancreatin for 2–3 minutes. When cells were clearly detached, the digestion was stopped with 5–6 mL of DMEM complete medium. The cells were gently pipetted, the suspension was collected in a centrifuge tube, and centrifuged at 1500 rpm for 5 minutes to remove the supernatant. The cells were resuspended in pre-chilled 1x PBS, and then counted. For every 20 μL of cell pellet, 200 μL of plasma protein extraction reagent (2 x 10 6A volume of approximately 20 μL (40 mg) of nuclear protein extraction reagent (Solarbio, R0050) was added to each cell pellet. The cell pellet was completely dispersed into a single cell suspension by pipetting or vortexing at high speed for 15 seconds. The mixture was then placed in an ice bath for 10 minutes. The mixture was then vortexed vigorously at maximum speed for 10 seconds and centrifuged at 12,000–16,000 g for 10 minutes at 4°C. The supernatant, which represents the extracted cytoplasmic proteins, was immediately aspirated and transferred to a pre-chilled sample tube for later use. The precipitate, representing the cell nuclei, was then added to the remaining supernatant (to avoid contamination with cytoplasmic proteins). 50–100 μL of nuclear protein extraction reagent was added to completely aspirate the remaining supernatant (to avoid contamination with cytoplasmic proteins). The precipitate was completely dispersed by pipetting or vortexing for 15 seconds (this can be extended as needed), and the mixture was then placed in an ice bath for 10 minutes. After vigorously vortexing at maximum speed for 10 seconds, the mixture was centrifuged at 12,000-16,000 g for 10 minutes at 4°C. The supernatant was immediately aspirated and placed in a pre-chilled sample tube. This represents the extracted nuclear proteins.

[0136] After cell lysis, detection was performed according to the protocol of the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using the human plasminogen standard included in the kit as an internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per total protein unit for each sample, and statistical analysis was performed.

[0137] Plasmin activity was detected by enzyme substrate kinetics. Seven different concentrations of standard solutions, blanks, and samples were added sequentially to an ELISA plate at 85 μL per well. Then, 15 μL of 20 mM S-2251 solution (Chromogenix, 82033239) was added to each well and incubated at 37°C. Starting at 0 min, the A405 absorbance values ​​were read every 5 min using a multifunction microplate reader until 90 min. All reactions were fitted linearly with time and absorbance values, and the slope of the line was the reaction rate (ΔA405 / min) of the standard / sample. Finally, the titer of the measured sample was calculated using the titer values ​​of the standard and ΔA405 / min as a calibration curve.

[0138] The results showed that the levels of human plasminogen and plasmin activity in the cytoplasm and nucleus of the drug-treated group were significantly higher than those of the vehicle group, with a statistically significant difference. The addition of EACA completely inhibited these effects of plasminogen (Figures 15A-D). This suggests that plasminogen can penetrate into cells and nuclei to enhance plasmin activity, and that the entry of plasminogen into cells and nuclei is closely related to its lysine-binding activity.

[0139] Example 15 Plasminogen promotes huntingtin protein degradation in the kidneys of Huntington's disease model mice Ten 14-week-old B6 / JGpt-Tg(hHTT-CAG130)90 / Gpt mice (hereafter referred to as hHTT130 transgenic mice) were selected as the model group, and five C57 mice were selected as the normal control group. The hHTT130 mice were randomly divided into two groups: a vehicle group and a drug-treated group, with five mice in each group. After grouping, all mice were treated. The drug-treated group received 50 mg / kg plasminogen via tail vein injection, while the normal control and vehicle groups received an equal volume of PBS via tail vein injection for 28 consecutive days. After treatment, the mice were sacrificed, and their kidneys were harvested. Western blot detection of huntingtin protein (HTT) was performed on kidney tissue homogenates. 10% SDS-PAGE gels were prepared according to the gel preparation method. Each sample was mixed uniformly with 4x loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated to 100°C for 5 minutes, cooled, centrifuged for 2 minutes, and then 20 μL was loaded. Electrophoresis was performed at 30 V for 45 minutes, followed by electrophoresis at 100 V until the gel bottom was reached. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753). Electrophoresis was performed at 15 V for 2.5 hours. The transferred PVDF membrane was immersed in blocking solution (5% skim milk) and blocked overnight in a refrigerator at 4°C. After washing four times with TBST (0.01M Tris-NaCl, pH 7.6 buffer), rabbit anti-HTT antibody (Abcam, ab109115) and actin antibody were added and incubated at room temperature for 1.5 hours. After washing four times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1 hour. After washing four times with TBST, the PVDF membrane was placed on a clean imaging plate and developed with Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100). Images were captured using a biomolecular imaging system and quantitative analysis was performed using Image J.

[0140] The results showed that renal HTT levels in the vehicle-treated mice were significantly higher than those in the normal control group, and significantly lower than those in the drug-treated mice (Figures 16A-B), with statistically significant differences (* indicates P<0.05). This suggests that plasminogen can promote the degradation of huntingtin protein in the kidney tissue of Huntington's disease model mice.

[0141] Example 16 Plasminogen promotes huntingtin protein degradation in brain tissue of Huntington's disease model mice Two-week-old B6 / JGpt-Tg(hHTT-CAG130)90 / Gpt mice were sacrificed and brain tissue was collected and placed in pre-chilled 1640 medium (manufacturer: Gibco, catalog number: 31800-105). A 0.8 μm pore size cellulose acetate membrane filter was placed on the bottom of a 6-well plate, and 1 mL of B27 medium (manufacturer: Gibco, catalog number: A3653401) was added to wet the filter. The brain tissue was transferred to a plate containing pre-chilled PBS and rinsed three times with 1x PBS to remove blood from the tissue surface. The brain tissue was then thinly sliced ​​with a blade. The brain slices were divided into six samples on average and spread flat on a membrane filter containing medium, ensuring that the membrane was just submerged in the brain slices. The 6-well plate was placed in a CO2 incubator at 37°C and 5% CO2. The culture medium was replaced the next day, and 0.4 mg / mL plasminogen was added to the drug-treated group (3 samples), while the same volume of saline was added to the vehicle group (3 samples). The culture medium was replaced daily, and the corresponding experimental groups received the same amount of plasminogen or saline as above. After 3 days of culture (2 days for plasminogen), the brain slices and culture medium were aspirated and centrifuged at 1200 RPM for 5 minutes. The supernatant was removed, resuspended in 1x PBS, and centrifuged again to remove the PBS. The brain tissue pellet was saved. Western blot detection of huntingtin protein (HTT) was performed on the homogenate. 10% gels were prepared according to the SDS-PAGE gel preparation method. Each sample was mixed uniformly with 4x loading buffer (TaKaRa, e2139) at a 3:1 volume ratio, heated at 100°C for 5 minutes, cooled, centrifuged for 2 minutes, and 20 μL was then loaded. The electrophoresis conditions were 30 V for 45 minutes, followed by 100 V to the bottom of the gel. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 hours.The transferred PVDF membrane was immersed in blocking solution (5% skim milk) and blocked overnight in a refrigerator at 4°C. After washing four times with TBST (0.01M Tris-NaCl, pH 7.6 buffer), rabbit anti-HTT antibody (Abcam, ab109115) and actin antibody were added and incubated at room temperature for 1.5 hours. After washing four times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1 hour. After washing four times with TBST, the PVDF membrane was placed on a clean imaging plate and developed with Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100). Images were captured using a biomolecular imaging system and quantitative analysis was performed using Image J.

[0142] The results showed that HTT levels in the brain tissue of mice treated with the drug were significantly lower than those in the vehicle group, and the difference was statistically significant (* indicates P<0.05) (Figure 17A-B). This suggests that plasminogen can promote the degradation of huntingtin protein in the brain tissue of Huntington's disease model mice.

[0143] References: [1]KENNETH C.ROBBINS,LOUIS SUMMARIA,DAVID ELWYN et al.Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin.Journal of Biological Chemistry,1965,240(1):541-550. [2] Summaria L, Spitz F, Arzadon L et al.Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins.J Biol Chem.1976 Jun 25;251(12):3693-9. [3]HAGAN JJ,ABLONDI FB,DE RENZO EC.Purification and biochemical properties of human plasminogen.J Biol Chem.1960 Apr;235:1005-10. [4]Eskelinen EL.Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy.Mol Aspects Med.2006 Oct-Dec;27(5-6):495-502 [5]Vernice Jackson-Lewis1 & Serge Przedborski.Protocol for the MPTP mouse model of Parkinson’s Disease [J].Nature protocols VOL.2 NO.1,2007(141). [6]N.A.Tatton and S.J.Kish.In situ detection of apoptotic nuclei in the substantia aigra compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using terminal deoxynucleotidyl transferase labeling and acridine orange staining[J].Neuroscience Vol.77,No.4,pp.1037-1048,1997. [7]Moon M,Choi J G,Kim S Y,et al.Bombycis Excrementum Reduces Amyloid-β Oligomer-Induced Memory Impairments,Neurodegeneration,and Neuroinflammation in Mice[J].Journal of Alzheimer’s disease:JAD, 2014,41(2). [8] Sun Z, Chen YH, Wang P, Zhang J, Gurewich V, Zhang P, Liu JN. The blockage of the high-affinity lysine binding sites of plasminogen by EACA significantly inhibits prourokinase-induced plasminogen activation. Biochim Biophys Acta.2002 Apr 29;1596(2):182-92.

[0144] Sequence Listing SEQ ID NO: 1 (nucleic acid sequence of native plasminogen without signal peptide (Glu-PLG, Glu-plasminogen)): SEQ ID NO: 2 (nucleic acid sequence of native plasminogen without signal peptide (Glu-PLG, Glu-plasminogen)): EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHA HGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKCCQSWSSMTPHRHQKTPE NYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLI SPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN SEQ ID NO: 3 (nucleic acid sequence of native plasminogen (from Swiss Prot) including signal peptide): SEQ ID NO: 4 (amino acid sequence of native plasminogen (from Swiss Prot) including signal peptide): MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMS GLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKCCQSWSSMT PHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFC GGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN SEQ ID NO: 5 (nucleic acid sequence of LYS77-PLG (Lys-plasminogen)): SEQ ID NO: 6 (LYS77-PLG (Lys-plasminogen) amino acid sequence): KVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQ CLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKK CSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPS SYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN SEQ ID NO: 7 (nucleic acid sequence of delta-plg (delta-plasminogen)): SEQ ID NO: 8 (amino acid sequence of delta-plg (delta-plasminogen)): EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGI TCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFG MHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN SEQ ID NO: 9 (nucleic acid sequence of miniplg (miniplasminogen)): SEQ ID NO: 10 (amino acid sequence of Mini-plg (miniplasminogen)): VRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEK SPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN SEQ ID NO: 11 (nucleic acid sequence of Micro-plg (microplasminogen)): gccccttcatttgattgtgggaagcctcaagtggagccgaagaaatgtcctggaagggttgtaggggggtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcacaccaagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagatattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgctgaccggaccgaatgtttcatcactggctggggagaaacccaaggtacttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgagaaataattaa SEQ ID NO: 12 (Amino acid sequence of Micro-plg (microplasminogen)): APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPAC LPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN SEQ ID NO: 13 (nucleic acid sequence of serine protease (structural) domain): gttgtaggggggtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcacacc aagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagatattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgctgaccggaccgaatgtttc360 atcactggctggggagaaacccaaggtacttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgac agttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgaga SEQ ID NO: 14 (amino acid sequence of serine protease (structural) domain): VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVA DRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMR

Claims

1. A method for promoting the removal of pathological proteins by the ubiquitin proteasome system and the autophagy lysosome system, comprising administering to a subject an effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating an upstream component of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

2. A method for removing different types of pathological proteins extracellularly and / or in the cytoplasm and / or nucleus, comprising administering to a subject an effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

3. 3. The method of claim 1, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA, and uPA.

4. 3. The method of claim 1 or 2, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, alpha 2 antiplasmin or alpha 2 macroglobulin, such as an antibody.

5. The method according to any one of claims 1 to 4, wherein the compound has one or more of the following activities: removal of extracellular and / or cytoplasmic and / or intranuclear pathological proteins; promotion of removal of pathological proteins by the ubiquitin proteasome system; promotion of removal of pathological proteins by the autophagy lysosomal system; modulation for optimizing the expression and / or activity of members of the ubiquitin system; modulation for optimizing the expression and / or activity of LC3; modulation for optimizing the expression and / or activity of members of the autophagy lysosomal system; and modulation for optimizing (particularly promoting) the expression of LAMP2.

6. The method of any one of claims 1 to 5, wherein the compound is plasminogen or plasmin.

7. The method according to any one of claims 1 to 6, wherein the plasminogen is Glu-plasminogen, Lys-plasminogen, or a conservatively substituted variant thereof.

8. 8. The method of any one of claims 1 to 7, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2 and has the lysine binding activity and / or proteolytic activity of plasminogen.

9. 9. The method of any one of claims 1 to 8, wherein the plasminogen comprises one or more selected from the group consisting of: 1) a serine protease domain as set forth in SEQ ID NO: 14; 2) a serine protease domain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 and retaining proteolytic activity; 3) one or more Kringle domains selected from the group consisting of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5; and 4) A Kringle domain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with one or more selected from the group consisting of Kringle 1, Kringle 2, Kringle 3, Kringle 4 and Kringle 5, and retaining lysine-binding activity.

10. 10. The method of any one of claims 1 to 9, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or variants thereof that retain the proteolytic activity of plasminogen.

11. The method of any one of claims 1 to 10, wherein the plasminogen comprises the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12, or a conservatively substituted variant of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12.

12. The method of any one of claims 1 to 11, wherein the plasminogen is used in combination with one or more other therapeutic methods or drugs.

13. 13. The method of claim 12, wherein the other treatment methods include cell therapy (including stem cell therapy), supportive therapy, and physical therapy.

14. 14. The method of any one of claims 1 to 13, wherein the plasminogen is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenously, intraperitoneally, subcutaneously, sublingually, intracranially, intrathecally, intraarterially (e.g., via the carotid artery), or intramuscularly.

15. The pathological protein is selected from the group consisting of cystic fibrosis transmembrane conductance regulator (CFTR), α1-antitrypsin, parkin protein, p53 tumor suppressor protein, Wilms' tumor 1 protein (WT1 protein), von Hippel-Lindau (VHL) tumor suppressor protein, merlin protein, Src protein kinase, crystallin, transthyretin, short-chain acyl-CoA dehydrogenase variant (SCAD variant), low-density lipoprotein receptor (LDL) and erythrocyte segregation factor receptor (ECR). receptor), huntingtin, neurofilament protein, peripherin, 1α-internexin, islet amyloid polypeptide, β2-microglobulin, serum amyloid A protein, immunoglobulin light chains, human lysozyme, α-lactalbumin, prothymosin α α), apolipoprotein E, apolipoprotein J, amyloid β-protein (Aβ), tau protein, α-synuclein (α-syn), TAR DNA-binding protein (transactive response DNA-binding protein 43, TDP-43), prion protein, copper-zinc superoxide dismutase (CSP),The method of any one of claims 1 to 14, wherein the protein is selected from one or more of: erythrocyte serine / erythrocyte saccharin (ESS), erythrocyte serine / erythrocyte saccharin (ESC ...

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