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WO2024094216A1 - 一种促进泛素蛋白酶体系统和自噬溶酶体系统对病理性蛋白清除的方法和药物 - Google Patents

一种促进泛素蛋白酶体系统和自噬溶酶体系统对病理性蛋白清除的方法和药物 Download PDF

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Publication number
WO2024094216A1
WO2024094216A1 PCT/CN2023/130018 CN2023130018W WO2024094216A1 WO 2024094216 A1 WO2024094216 A1 WO 2024094216A1 CN 2023130018 W CN2023130018 W CN 2023130018W WO 2024094216 A1 WO2024094216 A1 WO 2024094216A1
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plasminogen
protein
plasmin
mice
kringle
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PCT/CN2023/130018
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English (en)
French (fr)
Inventor
李季男
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泰伦基国际有限公司
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Publication of WO2024094216A1 publication Critical patent/WO2024094216A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present application relates to the use of plasminogen activation pathway-related compounds such as plasminogen or plasmin for promoting the clearance of pathological proteins by the ubiquitin proteasome system and the autophagy lysosome system.
  • Chronic neurodegenerative diseases are a disease state of neuronal loss in the brain and spinal cord, including Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, etc. Studies have found that it is due to lesions caused by ubiquitin-dependent processes, including ubiquitination degradation by the 26S proteasome and abnormal autophagy through the lysosomal pathway, which have serious effects on neural development, homeostasis and the occurrence of diseases. Therefore, ubiquitination degradation and autophagy are essential for neural activity and are involved in synapse formation and cell-cell interactions.
  • the ubiquitin proteasome system (UPS) and the autophagy-lysosome system are protein degradation systems in cells, and their interaction can promote the degradation of pathological proteins. People hope to find substances that can promote the interaction between the ubiquitin proteasome system (UPS) and the autophagy-lysosome system, thereby promoting the clearance of pathological proteins that cause disease in cells.
  • plasminogen can promote the interaction between the ubiquitin proteasome system (UPS) and the autophagy lysosome system to some extent, and promote the clearance of pathological proteins inside and outside the cell.
  • UPS ubiquitin proteasome system
  • the present application relates to the following items:
  • a method for promoting the clearance of pathological proteins by the ubiquitin proteasome system and the autophagy lysosome system comprises administering to a subject an effective amount of one or more compounds selected from the following: 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.
  • the components of the plasminogen activation pathway are selected from plasminogen, recombinant human plasmin, 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 activator, tPA and uPA.
  • any one of items 1-3 wherein the compound has one or more of the following activities: directly clearing different types of pathological proteins outside the cell and/or directly entering the cell and/or the cell nucleus, promoting the clearance of pathological proteins by the ubiquitin proteasome system, promoting the clearance of pathological proteins by the autophagy-lysosome system, regulating to optimize the expression and/or activity of members of the ubiquitin system, regulating to optimize the expression and/or activity of LC3, regulating to optimize the expression and/or activity of members of the autophagy-lysosome system, and regulating to optimize (especially promote) the expression of LAMP2.
  • plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2 and has lysine binding activity and/or proteolytic activity of plasminogen.
  • plasminogen comprises one or more selected from the following:
  • Kringle domain selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4 and Kringle 5;
  • a Kringle domain that is at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identical to one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5 and retains lysine binding activity.
  • plasminogen is selected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogen or variants thereof that retain the proteolytic activity of plasminogen.
  • plasminogen comprises the amino acid sequence shown in sequence 2, 6, 8, 10, or 12, or comprises a conservatively substituted variant of the amino acid sequence shown in sequence 2, 6, 8, 10, or 12.
  • plasminogen is administered by nasal inhalation, nebulized inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery) or intramuscular administration.
  • the present application also relates to:
  • 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 the following: 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.
  • the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasmin, Lys-plasminogen, Glu-plasminogen, plasmin, one or more plasminogen and plasmin plasminogen and plasmin variants and analogs having a kringle domain and a protease domain, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activator, tPA and uPA.
  • plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2 and has lysine binding activity and/or proteolytic activity of plasminogen.
  • plasminogen comprises one or more selected from the following:
  • Kringle domain selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4 and Kringle 5;
  • a Kringle domain that is at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identical to one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5 and retains lysine binding activity.
  • plasminogen is selected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogen or variants thereof that retain the proteolytic activity of plasminogen.
  • plasminogen comprises the amino acid sequence shown in sequence 2, 6, 8, 10, or 12, or comprises a conservatively substituted variant of the amino acid sequence shown in sequence 2, 6, 8, 10, or 12.
  • any one of items 1-12 wherein the plasminogen is administered by nasal inhalation, nebulized inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery) or intramuscular administration.
  • the method of item 12. 11, wherein the other treatment methods include cell therapy (including stem cell therapy), gene therapy, supportive therapy and physical therapy.
  • plasminogen is administered by nasal inhalation, nebulized inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery) or intramuscular administration.
  • the above-mentioned diseases caused by pathological protein aggregation include: Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion-like disease (Creutzfeldt-Jakob disease), Gerstmann syndrome, diffuse or familial fatal insomnia and kuru, Huntington's disease, spinocerebellar ataxia type 3, dentate-rubral-pallidular Lewy body atrophy, diabetes, spongiform encephalopathy, macular degeneration, atherosclerosis, familial British dementia, familial Danish dementia, Down syndrome, hemodialysis-related amyloid deposition disease, amyloid cardiomyopathy, systemic amyloidosis, corneal dystrophy, glomerulonephritis, and amyloid bodies.
  • Alzheimer's disease Parkinson's disease, amyotrophic lateral sclerosis, prion-like disease (Creutzfeldt-Jakob disease), Gerstmann syndrome, diffuse or familial fatal insomnia and kuru
  • the 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 disease, Crohn's disease, Alzheimer's disease, breast cancer and ovarian cancer, muscular amyloliquefaciens disease, amylopectinosis, amyotrophic lateral sclerosis, spinal muscular atrophy, systemic lupus erythematosus, childhood ataxia, X-linked parkinsonism with spasticity, multisystem disorder, diabetes mellitus, multiple sclerosis, cystinosis, Vici syndrome, Gaucher's disease, frontotemporal dementia (heterozygous) or neuronal ceroid lipofuscinosis (homozygous), Danon
  • the plasminogen pathway activator is administered in combination with one or more other drugs and/or treatments, preferably, the treatments include cell therapy (e.g., stem cell therapy) and gene therapy, such as antisense RNA, small molecule splicing modifiers.
  • cell therapy e.g., stem cell therapy
  • gene therapy such as antisense RNA, small molecule splicing modifiers.
  • the plasminogen pathway activator is a component of the plasminogen activation pathway, such as plasminogen.
  • the plasminogen comprises or has an amino acid sequence 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 has plasminogen activity and/or lysine binding activity.
  • the plasminogen is a protein having plasminogen activity and/or lysine binding activity, with addition, deletion and/or substitution of 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids on the basis of sequence 2, 6, 8, 10 or 12.
  • the plasminogen activity The activity is the proteolytic activity of plasminogen.
  • the plasminogen is a protein comprising a plasminogen active fragment and having plasminogen activity and/or lysine binding activity.
  • the plasminogen activity is the proteolytic activity of plasminogen.
  • the plasminogen active fragment comprises or has a plasminogen serine protease domain or a plasminogen protease domain.
  • the amino acid sequence of the plasminogen active fragment is as shown in sequence 14.
  • the plasminogen is selected from Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (human full-length plasminogen after cleavage between amino acids 76-77), mini-plasminogen (comprising Kringle 5 (K5) and serine protease domain), micro-plasminogen (comprising serine protease domain), delta-plasminogen (comprising Kringle 1 and serine protease domain) or variants thereof retaining plasminogen activity.
  • the plasminogen is a human full-length plasminogen, or a variant or fragment thereof that still retains plasminogen activity and/or lysine binding activity.
  • the plasminogen is a human plasminogen straight homologue from a primate or rodent, or a variant or fragment thereof that still retains plasminogen activity and/or lysine binding activity.
  • the plasminogen comprises an amino acid sequence as shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is human natural plasminogen.
  • the plasminogen pathway activator is administered systemically or locally, for example, intravenously, intramuscularly, by nasal inhalation, by nebulization inhalation, or by nasal drops.
  • the subject is a human.
  • the subject lacks or is deficient in plasminogen.
  • the deficiency or deficiency is congenital, secondary, and/or local.
  • the plasminogen is administered at a 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/ cm2 , 0.001-800 mg/ cm2 , 0.01-600 mg/ cm2 , 0.1-400 mg/ cm2 , 1-200 mg/ cm2 , 1-100 mg/ cm2 , 10-100 mg/ cm2 (calculated per square centimeter of body surface area) per day, every other day, or every three days continuously.
  • the present application also relates to a pharmaceutical composition, a drug, a preparation, a kit, and a product for use in the above method, comprising the above-mentioned plasminogen pathway activator, such as the above-mentioned plasminogen.
  • the pharmaceutical composition, medicament, or formulation comprises a pharmaceutically acceptable carrier and a plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as a fibrinolytic
  • the kits and articles of manufacture comprise one or more containers containing the pharmaceutical composition, drug or formulation.
  • the kits or articles of manufacture further comprise a label or instructions for use, which label or instructions for use indicate the use of a plasminogen pathway activator, such as a component of a plasminogen activation pathway, such as plasminogen for the above methods.
  • the kits or articles of manufacture further comprise one or more additional containers containing one or more other drugs.
  • the present application also relates to a plasminogen pathway activator for the above-mentioned use, such as the plasminogen described above.
  • the present application also relates to the use of a therapeutically effective amount of the above-mentioned plasminogen pathway activator in the preparation of a pharmaceutical composition, a drug, a preparation, a kit, or a product for the above-mentioned method.
  • the plasminogen pathway activator is selected from one or more of the following: 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.
  • the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasmin, 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 activator, tPA and uPA.
  • the antagonist of the fibrinolytic inhibitor is an antagonist of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin, such as antibodies to PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin.
  • the plasminogen pathway activator is administered in combination with one or more other drugs and/or treatments, preferably, the treatments include cell therapy (e.g., stem cell therapy) and gene therapy, such as antisense RNA, small molecule splicing modifiers.
  • cell therapy e.g., stem cell therapy
  • gene therapy such as antisense RNA, small molecule splicing modifiers.
  • the plasminogen pathway activator is a component of the plasminogen activation pathway, such as plasminogen.
  • the plasminogen comprises or has at least 75%, 10%, or 20% of the amino acid sequence of SEQ ID NO: 2, 6, 8, 10, or 12. 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequence, and has plasminogen activity and/or lysine binding activity.
  • the plasminogen is based on sequence 2, 6, 8, 10 or 12, adding, deleting and/or replacing 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids, and has plasminogen activity and/or lysine binding activity of the protein.
  • the plasminogen activity is the proteolytic activity of plasminogen.
  • the plasminogen is a protein comprising a plasminogen active fragment and having plasminogen activity and/or lysine binding activity.
  • the plasminogen activity is the proteolytic activity of plasminogen.
  • the plasminogen active fragment comprises or has a plasminogen serine protease domain or a plasminogen protease domain.
  • the amino acid sequence of the plasminogen active fragment is as shown in sequence 14.
  • the plasminogen is selected from Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (human full-length plasminogen after cleavage between amino acids 76-77), mini-plasminogen (comprising Kringle 5 (K5) and serine protease domain), micro-plasminogen (comprising serine protease domain), delta-plasminogen (comprising Kringle 1 and serine protease domain) or variants thereof that retain plasminogen activity.
  • the plasminogen is human full-length plasminogen, or a variant or fragment thereof that still retains plasminogen activity and/or lysine binding activity.
  • the plasminogen is a human plasminogen ortholog from a primate or rodent or a variant or fragment thereof that still retains plasminogen activity and/or lysine binding activity.
  • the plasminogen comprises an amino acid sequence as shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is human native plasminogen.
  • the plasminogen pathway activator e.g., a component of the plasminogen activation pathway, e.g., plasminogen
  • the plasminogen pathway activator e.g., a component of the plasminogen activation pathway, e.g., plasminogen
  • the pharmaceutical composition, drug, or formulation comprises a pharmaceutically acceptable carrier and a plasminogen pathway activator, such as a component of a plasminogen activation pathway, such as plasminogen.
  • the kits and articles of manufacture comprise one or more containers containing the pharmaceutical composition, drug, or formulation.
  • the reagent The kit or article of manufacture further comprises a label or instructions for use directing the use of the plasminogen pathway activator, eg, a component of the plasminogen activation pathway, eg, plasminogen, for the above uses.
  • the kit or article of manufacture further comprises one or more additional containers containing one or more other drugs.
  • the present invention explicitly covers all combinations of technical features belonging to the embodiments of the present invention, and the technical solutions after such combinations have been explicitly disclosed in this application, just as the above technical solutions have been individually and explicitly disclosed.
  • the present invention also explicitly covers the combination between the various embodiments and their elements, and the technical solutions after such combinations are explicitly disclosed in this article.
  • FIG 1 Schematic diagram of the interaction between plasminogen and the ubiquitin proteasome system and the autophagy-lysosome system to promote the degradation of pathological proteins in the central nervous system in cells.
  • Blood-brain barrier Basement membrane, Endothelial cells, Plasminogen (Plg), Plasminogen receptor (PlgR), Tissue-type plasminogen activator (tPA), Conformationally abnormal proteins (CAP), Plasmin (Plm), Plasmin generated protein fragments (PGPFs), Plasmin degradation products (PDP), Lysosome, Ubiquitin (UBI), Ubiquitin activating enzyme (E1), Ubiquitin conjugating enzyme (E2), Ubiquitin ligase (E3), Proteasome, Nucleus.
  • Plg Plasminogen receptor
  • tPA Tissue-type plasminogen activator
  • CAP Conformationally abnormal proteins
  • Plm Plasmin
  • PGPFs Plasmin generated protein fragments
  • Plasminogen can enter cells and interact with the intracellular protein degradation system - the ubiquitin proteasome system (UPS) and the autophagy lysosome system. Plasminogen can thus promote the degradation of abnormally deposited central nervous system pathological proteins in cells and improve central nervous system degenerative diseases.
  • UPS ubiquitin proteasome system
  • FIG. 2 Localization of plasminogen in the spinal cord tissue of mice with amyotrophic lateral sclerosis model and co-localization of plasminogen and ubiquitin.
  • 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 the spinal cord tissue and be enriched in the spinal cord tissue.
  • plasminogen exists in the cytoplasm and nucleus.
  • Plasminogen and ubiquitin co-localize in the cytoplasm (as shown in ⁇ ) ( Figure 2), and the ubiquitin expression level in the spinal cord tissue of the drug-treated group seems to be lower than that of the vehicle group. This indicates that in the amyotrophic lateral sclerosis model mice In mice, plasminogen can enter the spinal cord tissue, enter the cells, co-localize with ubiquitin, and reduce the expression of ubiquitin, suggesting that plasminogen may interact with ubiquitin.
  • Figure 3 shows the localization of plasminogen in the spinal cord tissue of mice with amyotrophic lateral sclerosis model and the co-localization of plasminogen and LC3B.
  • the results showed that the positive staining of plasminogen (green fluorescence) in the spinal cord tissue of the drug-treated group was significantly more than that of the vehicle group, indicating that the drug-treated plasminogen can enter the spinal cord tissue and be enriched in the spinal cord tissue.
  • plasminogen is present in the cytoplasm and nucleus.
  • Plasminogen and LC3B are co-localized in the cytoplasm (as shown in ⁇ ), and the expression level of LC3B in the spinal cord tissue of the drug-treated group seems to be less than that of the vehicle group. This shows that in mice with amyotrophic lateral sclerosis model, plasminogen can enter the spinal cord tissue, enter the cell, and co-localize with LC3B, suggesting that there is an interaction between plasminogen and LC3B.
  • Figure 4 shows the localization of plasminogen in the spinal cord tissue of mice with amyotrophic lateral sclerosis model and the co-localization of plasminogen and LAMP2.
  • the results showed that the positive staining of plasminogen (green fluorescence) in the spinal cord tissue of the drug-treated group was significantly more than that of the vehicle group, indicating that the drug-treated plasminogen can enter the spinal cord tissue and accumulate in the spinal cord tissue.
  • plasminogen is present in the cytoplasm and nucleus.
  • Plasminogen and LAMP2 are co-localized in the cytoplasm (as shown in ⁇ ), and the expression level of LAMP2 in the spinal cord tissue of the drug-treated group seems to be more than that of the vehicle group, entering the cell, co-localizing with LAMP2, and promoting the expression of LAMP2, promoting lysosomal action.
  • FIG. 5 Administration of plasminogen promotes the expression of LC3 in the spinal cord tissue of mice with amyotrophic lateral sclerosis model.
  • the results show that the expression of LC3 in the drug administration group is significantly higher than that in the vehicle group, and the statistical difference is significant (* indicates P ⁇ 0.05). This indicates that plasminogen can promote the transcription of the LC3 gene in mice with amyotrophic lateral sclerosis model, thereby participating in the regulation of the autophagy process.
  • Figure 6 shows the localization of plasminogen in the substantia nigra and striatum tissues of Parkinson's model mice and the co-localization of plasminogen and ubiquitin.
  • the results showed that the positive staining of plasminogen (green fluorescence) in the substantia nigra and striatum tissues in the drug-treated group was significantly more than that in the vehicle group, indicating that the drug-treated plasminogen can enter the substantia nigra and striatum tissues and is enriched in the substantia nigra and striatum tissues.
  • plasminogen is present in the cytoplasm and nucleus.
  • Plasminogen and ubiquitin are co-localized in the cytoplasm (as shown in ⁇ ). This shows that in Parkinson's model mice, plasminogen can enter the substantia nigra and striatum tissues, enter the cells, and co-localize with ubiquitin. This suggests that plasminogen may interact with ubiquitin.
  • FIG. 7 Localization of plasminogen in the substantia nigra and striatum of Parkinson's model mice and co-localization of plasminogen and LC3B.
  • the results showed that the positive staining of plasminogen (green fluorescence) in the substantia nigra and striatum of the drug-treated group was significantly higher than that in the vehicle group, indicating that the administration of plasminogen can promote It enters the substantia nigra and striatum tissues and is enriched in the substantia nigra and striatum tissues.
  • plasminogen is present in the cytoplasm and nucleus.
  • Plasminogen and LC3B co-localize in the cytoplasm (as shown in ⁇ ). This indicates that plasminogen can enter the substantia nigra and striatum tissues, enter the cells, and co-localize with LC3B in Parkinson's model mice. This suggests that plasminogen may interact with LC3.
  • Figure 8 shows the localization of plasminogen in the substantia nigra and striatum tissues of Parkinson's model mice and the co-localization of plasminogen and LAMP2.
  • the results showed that the positive staining of plasminogen (green fluorescence) in the substantia nigra and striatum tissues in the drug-treated group was significantly more than that in the vehicle group, indicating that the drug-treated plasminogen can enter the substantia nigra and striatum tissues and is enriched in the substantia nigra and striatum tissues.
  • plasminogen is present in the cytoplasm and nucleus.
  • Plasminogen and LAMP2 are co-localized in the cytoplasm (as shown). It shows that in Parkinson's model mice, plasminogen can enter the substantia nigra and striatum tissues, enter the cells, and co-localize with LAMP2. It suggests that plasminogen may interact with LAMP2.
  • Figure 9 shows the localization of plasminogen in the hippocampus of Alzheimer's model mice and the co-localization of plasminogen and ubiquitin.
  • the results showed that the positive staining of plasminogen (green fluorescence) in the hippocampus of the drug-treated group was significantly more than that in the vehicle group, indicating that the drug-treated plasminogen was able to enter the hippocampus and be enriched in the hippocampus.
  • plasminogen was present in the cytoplasm and nucleus. Plasminogen and ubiquitin (red fluorescence) were co-localized in the cytoplasm (as shown in ⁇ ). This indicates that plasminogen can enter the hippocampus and enter the cells in Alzheimer's model mice. This suggests that plasminogen may interact with ubiquitin.
  • Figure 10 shows the localization of plasminogen in the hippocampus of Alzheimer's model mice and the co-localization of plasminogen and LC3B.
  • the results showed that the positive staining of plasminogen (green fluorescence) in the hippocampus of the drug-treated group was significantly more than that in the vehicle group, indicating that the drug-treated plasminogen can enter the hippocampus and accumulate in the hippocampus.
  • plasminogen is present in the cytoplasm and nucleus. Plasminogen and LC3B (red fluorescence) are co-localized in the cytoplasm (as shown in ⁇ ). This indicates that plasminogen can enter the hippocampus and enter the cells in Alzheimer's model mice. This suggests that plasminogen may interact with LC3B.
  • FIG 11 Localization of plasminogen in the hippocampus of Alzheimer's model mice and co-localization of plasminogen and LAMP2.
  • the results showed that the positive staining of plasminogen (green fluorescence) in the hippocampus of the drug-treated group was significantly higher than that in the vehicle group, indicating that the drug-treated plasminogen can enter the hippocampus and accumulate in the hippocampus.
  • plasminogen exists in the cytoplasm and nucleus. Plasminogen and LAMP2 (red fluorescence) co-localize in the cytoplasm (as shown in ⁇ ). This indicates that plasminogen can enter the hippocampal tissue and enter the cells in Alzheimer's model mice, suggesting that plasminogen may interact with LAMP2.
  • Figure 12A-B Results of western blot quantitative analysis of Huntington protein in heart tissue of Huntington model mice 28 days after plasminogen administration.
  • A is a Western blot image
  • B is the result of quantitative analysis of optical density of Huntington protein bands.
  • the results showed that the heart HTT level of mice in the vehicle group was significantly higher than that of mice in the normal control group, and the heart HTT level of mice in the drug administration group was significantly lower than that of mice in the vehicle group, and the statistical difference was significant (* represents P ⁇ 0.05). This suggests that plasminogen can promote the degradation of Huntington protein in the heart tissue of Huntington model mice.
  • FIG. 13 WB test results of TDP-43 levels in the cytoplasm (A-B) and nucleus (C-D) of NSC34 cells treated with okadaic acid and given plasminogen.
  • a and C are Western blot images, and C and D are the results of quantitative analysis of TDP-43 bands.
  • the results showed that the levels of TDP-43 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 could completely inhibit the effect of plasminogen on TDP-43.
  • * represents P ⁇ 0.05
  • ** represents P ⁇ 0.01
  • *** represents P ⁇ 0.001. This suggests that plasminogen can promote the degradation of TDP-43 in the cytoplasm and nucleus, and this effect of plasminogen is closely related to the lysine binding site in its structure.
  • FIG. 14 Phosphorylated Tau protein (A-B), total Tau protein and the ratio of phosphorylated Tau protein to total Tau protein in NSC34 cells treated with okadaic acid after administration of plasminogen.
  • a and C are Western blot images
  • B, D and E are the results of phosphorylated Tau protein, total Tau protein and the ratio of phosphorylated Tau protein to total Tau protein, respectively.
  • Figure 15A-D Results of the detection of plasminogen and plasmin activity levels in the cytoplasm and nucleus of NSC34 cells treated with plasminogen okadaic acid.
  • A is the result of ELISA for the level of plasminogen in the cytoplasm
  • B is the result of ELISA for the level of plasminogen in the nucleus
  • C is the result of enzyme substrate kinetics for the level of plasmin activity in the cytoplasm
  • D is the result of enzyme substrate kinetics for the level of plasmin activity in the nucleus.
  • the results showed that the levels of human plasminogen and plasmin in the cytoplasm and nucleus of the drug-treated group were significantly higher than those in the control group.
  • the level of plasmin activity was significantly higher than that of the vehicle group, and the statistical difference was significant; the addition of EACA could completely inhibit the effects of plasminogen (** represents P ⁇ 0.01, *** represents P ⁇ 0.001). This suggests that plasminogen can enter cells and even the nucleus to increase plasmin activity, and the entry of plasminogen into cells and nuclei is closely related to its lysine binding activity.
  • Figure 16A-B shows the results of western blot quantitative analysis of Huntington protein in the kidney tissue of Huntington model mice 28 days after administration of plasminogen.
  • A is a Western blot image
  • B is the result of quantitative analysis of the optical density of the Huntington protein band.
  • the results showed that the HTT level in the kidney of the vehicle group mice was significantly higher than that of the normal control group mice, and the HTT level in the kidney of the drug group mice was significantly lower than that of the vehicle group mice ( Figure 16A-B), and the statistical difference was significant (* represents P ⁇ 0.05, *** represents P ⁇ 0.001). This suggests that plasminogen can promote the degradation of Huntington protein in the kidney tissue of Huntington model mice.
  • Figure 17A-B shows the results of western blot quantitative analysis of Huntington protein in brain tissue of Huntington model mice cultured in vitro 2 days after administration of plasminogen.
  • A is a Western blot image
  • B is the results of quantitative analysis of the optical density of the Huntington protein band.
  • the results showed that the HTT level in the brain tissue of the mice in the drug administration group was significantly lower than that in the vehicle group, and the statistical difference was significant (* represents P ⁇ 0.05). This suggests that plasminogen can promote the degradation of Huntington protein in the brain tissue of Huntington model mice.
  • the fibrinolytic system also known as the fibrinolytic system, is a system composed of a series of chemical substances involved in the fibrinolysis (fibrinolysis) process, mainly including plasminogen (plasminogen), plasmin, plasminogen activators, and fibrinolysis inhibitors.
  • Plasminogen activators include tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA).
  • t-PA tissue plasminogen activator
  • u-PA urokinase plasminogen activator
  • t-PA activates plasminogen, and this process mainly takes place on fibrin;
  • urokinase plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells, and can directly activate plasminogen without the need for fibrin as a cofactor.
  • Plasminogen (PLG) is synthesized by the liver, and when the blood coagulates, PLG A large amount of it is adsorbed on the fibrin network. Under the action of t-PA or u-PA, it is activated into plasmin, which promotes fibrinolysis.
  • Plasmin (PL) is a serine protease that acts as follows: degrades fibrin and fibrinogen; hydrolyzes various coagulation factors V, VIII, X, VII, XI, II, etc.; converts plasminogen into plasmin; hydrolyzes complement, etc.
  • Fibrinolysis inhibitors include plasminogen activator inhibitor (PAI) and ⁇ 2 antiplasmin ( ⁇ 2-AP).
  • PAI mainly has two forms, PAI-1 and PAI-2, which can specifically bind to t-PA in a 1:1 ratio, thereby inactivating it and activating PLG.
  • ⁇ 2-AP is synthesized by the liver and binds to PL in a 1:1 ratio to form a complex, inhibiting PL activity; FXIII allows ⁇ 2-AP to bind to fibrin with a covalent bond, reducing the sensitivity of fibrin to the action of PL.
  • Substances that inhibit the activity of the fibrinolytic system in the body PAI-1, complement C1 inhibitor; ⁇ 2 antiplasmin; ⁇ 2 macroglobulin.
  • plasminogen pathway activator or "plasminogen pathway activator” of the present invention encompass 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.
  • component of the plasminogen activation pathway or “component of the plasminogen activation pathway” according to the present invention encompasses:
  • Plasminogen activators such as tPA and uPA, and tPA or uPA variants and analogs comprising one or more domains of tPA or uPA, such as one or more kringle domains and a proteolytic domain.
  • antagonists of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin such as antibodies to PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin.
  • plasminogen, plasmin, tPA and uPA include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as proteins that have plasminogen, plasmin, tPA or uPA activity by adding, deleting and/or substituting, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids.
  • variants of plasminogen, plasmin, tPA, and uPA include mutational variants of these proteins obtained by, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative amino acid substitutions.
  • the "plasminogen variants" of the present invention encompass proteins that contain or have 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 have plasminogen activity and/or lysine binding activity.
  • the "plasminogen variants" of the present invention can be proteins that add, delete and/or substitute 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids on the basis of SEQ ID NO: 2, 6, 8, 10 or 12, and still have plasminogen activity and/or lysine binding activity.
  • the plasminogen variants of the present invention include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as mutant variants of these proteins obtained by conservative amino acid substitutions, such as 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids.
  • the plasminogen of the present invention can be a human plasminogen ortholog from a primate or rodent or a variant thereof that still retains plasminogen activity and/or lysine binding activity, such as the plasminogen shown in Sequence 2, 6, 8, 10 or 12, such as the human natural plasminogen shown in Sequence 2.
  • analogs of plasminogen, plasmin, tPA and uPA include compounds that provide substantially similar effects as plasminogen, plasmin, tPA or uPA, respectively.
  • variants and analogs of plasminogen, plasmin, tPA and uPA encompass “variants” and “analogs” of plasminogen, plasmin, tPA and uPA comprising one or more domains (e.g., one or more kringle domains and a proteolytic domain).
  • variants and analogs of plasminogen encompass plasminogen variants and analogs comprising one or more plasminogen domains (e.g., one or more kringle (k) domains and a proteolytic domain (or serine protease domain, or plasminogen protease domain), such as mini-plasminogen.
  • “Variants” and “analogs” of plasmin encompass plasmin “variants” and “analogs” comprising one or more plasmin domains (e.g., one or more kringle domains and a proteolytic domain), such as mini-plasmin and delta-plasmin.
  • plasminogen, plasmin, tPA or uPA have the activity of plasminogen, plasmin, tPA or uPA, respectively, or whether they provide substantially similar effects to plasminogen, plasmin, tPA or uPA, respectively, can be detected by methods known in the art, for example, by measuring the level of activated plasmin activity based on enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS (fluorescence-activated cell sorting method), for example, it can be measured by referring to the method described in the following documents: Ny, A., Le Onardsson, G., Hagglund, A.C, Hagglof, P., Ploplis, V.A., Carmeliet, P.and Ny, T.(1999).Ovulation in plasminogen-deficient mice.Endocrinology 140, 5030-5035; Silverstein RL,Le
  • the "component of the plasminogen activation pathway" of the present invention is plasminogen, selected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogen or variants thereof that retain plasminogen activity.
  • the plasminogen is natural or synthetic human plasminogen, or a conservative mutant variant thereof that still retains plasminogen activity and/or lysine binding activity, or a fragment thereof.
  • the plasminogen is a human plasminogen ortholog from a primate or rodent, or a conservative mutant variant thereof that still retains plasminogen activity and/or lysine binding activity, or a fragment thereof.
  • the amino acid sequence of the plasminogen comprises or has an amino acid sequence as shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is a human full-length plasminogen.
  • the plasminogen is a human full-length plasminogen as shown in sequence 2.
  • compound capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway refers to a compound capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway.
  • Any compound of the invention which inhibits the production of an inhibitory agent such as tPA, uPA, streptokinase, saruplase,reteplase, reteplase, tenecteplase, anistreplase, monteplase, lanoteplase, pamiplase, staphylokinase.
  • the "antagonists of fibrinolytic inhibitors" of the present invention are compounds that antagonize, weaken, block, or prevent the effects of fibrinolytic inhibitors.
  • the fibrinolytic inhibitors are, for example, PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, and ⁇ 2 macroglobulin.
  • the antagonists are, for example, antibodies to PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, or ⁇ 2 macroglobulin, or antisense RNA or small RNA that blocks or downregulates the expression of, for example, PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, or ⁇ 2 macroglobulin, or compounds that occupy the binding sites of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, or ⁇ 2 macroglobulin but have no function of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, or ⁇ 2 macroglobulin, or compounds that block the binding domains and/or active domains of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, or ⁇ 2 macroglobulin.
  • Plasmin is a key component of the plasminogen activator system (PA system). It is a broad-spectrum protease that can hydrolyze several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans. In addition, plasmin can activate some metalloproteinase precursors (pro-MMPs) to form active metalloproteinases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis. Plasmin is formed by proteolysis of plasminogen by two physiological PAs: tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA).
  • tPA tissue plasminogen activator
  • uPA urokinase plasminogen activator
  • PAI-1 plasminogen activator inhibitor-1
  • PAI-2 plasminogen activator inhibitor-2
  • Plasminogen is a single-chain glycoprotein composed of 791 amino acids with a molecular weight of approximately 92kDa. Plasminogen is mainly synthesized in the liver and exists in large quantities in the extracellular fluid. The content of plasminogen in plasma is about 2 ⁇ M. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids. Plasminogen exists in two molecular forms: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). The naturally secreted and uncleaved form of plasminogen has an amino-terminal (N-terminal) glutamic acid, so it is called glutamate-plasminogen.
  • glutamate-plasminogen is hydrolyzed at Lys76-Lys77 to become lysine-plasminogen.
  • lysine-plasminogen is more sensitive to plasminogen than glutamate-plasminogen.
  • the protein has a higher affinity and can be activated by PAs at a higher rate.
  • the Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide-linked two-chain protease plasmin.
  • the amino-terminal part of plasminogen contains five homologous three rings, the so-called kringles, and the carboxyl-terminal part contains the protease domain. Some kringles contain lysine binding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor ⁇ 2-AP.
  • a newly discovered 38kDa fragment of plasminogen, which includes kringles1-4, is a potent inhibitor of angiogenesis. This fragment, named angiostatin, can be produced by the hydrolysis of plasminogen by several proteases.
  • Plasmin also has substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycans, and gelatin, indicating that plasmin also plays an important role in ECM reconstruction. Indirectly, plasmin can also degrade other components of the ECM, including MMP-1, MMP-2, MMP-3, and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis. In addition, plasmin has the ability to activate certain latent forms of growth factors. In vitro, plasmin can also hydrolyze components of the complement system and release chemotactic complement fragments.
  • Pulmin is a very important enzyme present in the blood, which can hydrolyze fibrin clots into fibrin degradation products and D-dimers.
  • “Plasminogen” is the zymogen form of plasmin. According to the sequence in Swiss prot, the natural human plasminogen amino acid sequence containing a signal peptide (sequence 4) is composed of 810 amino acids, with a molecular weight of about 90kD. It is a glycoprotein mainly synthesized in the liver and can circulate in the blood. The cDNA sequence encoding the amino acid sequence is shown in sequence 3. The full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle1-5).
  • PAp Pan Apple
  • the serine protease domain includes residues Val581-Arg804.
  • Glu-plasminogen is a natural full-length plasminogen, consisting of 791 amino acids (excluding the 19-amino acid signal peptide).
  • the cDNA sequence encoding the sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.
  • Lys-plasminogen is formed by hydrolysis at amino acids, as shown in Sequence 6, and the cDNA sequence encoding the amino acid sequence is shown in Sequence 5.
  • Delta-plasminogen is a fragment of full-length plasminogen lacking the Kringle2-Kringle5 structure, and contains only Kringle1 and a serine protease (structure) domain (also known as a proteolytic domain, or a plasminogen protease domain).
  • structure also known as a proteolytic domain, or a plasminogen protease domain.
  • the amino acid sequence of delta-plasminogen has been reported in the literature (Sequence 8), and the cDNA sequence encoding the amino acid sequence is shown in Sequence 7.
  • Mini-plasminogen consists of Kringle5 and a serine protease domain, and the literature reports that it includes residues Val443-Asn791 (with the Glu residue of the Glu-plasminogen sequence without a signal peptide as the starting amino acid), and its amino acid sequence is shown in Sequence 10, and the cDNA sequence encoding the amino acid sequence is shown in Sequence 9.
  • Micro-plasminogen only contains a serine protease domain.
  • the "plasmin” of the present invention can be used interchangeably with “plasmin” and “fibrinolytic enzyme” and have the same meaning; the "plasminogen” can be used interchangeably with “plasmin” and “fibrinolytic enzyme” and have the same meaning.
  • the meaning or activity of "lack of" plasminogen is that the content of plasminogen in the subject's body is lower than that of a normal person, low enough to affect the normal physiological function of the subject;
  • the meaning or activity of "absence” of plasminogen is that the content of plasminogen in the subject's body is significantly lower than that of a normal person, and even the activity or expression is extremely low, and normal physiological function can only be maintained through exogenous supply.
  • plasminogen adopts a closed inactive conformation, but when bound to a thrombus or cell surface, it is converted into an active plasmin in an open conformation under the mediation of a plasminogen activator (PA).
  • PA plasminogen activator
  • Active plasmin can further hydrolyze fibrin clots into fibrin degradation products and D-dimers, thereby dissolving thrombi.
  • the PAp domain of plasminogen contains an important determinant cluster that maintains plasminogen in an inactive closed conformation, while the KR domain can bind to lysine residues present on receptors and substrates.
  • a variety of enzymes that can act as plasminogen activators are known, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hageman factor), etc.
  • plasminogen active fragment includes 1) an active fragment in the plasminogen protein that is capable of binding to a target sequence in a substrate, also referred to as a lysine binding fragment, such as a fragment comprising Kringle 1, Kringle 2, Kringle 3, Kringle 4 and/or Kringle 5 (the structure of the plasminogen is described in Aisina R B, Mukhametova L I. Structure and function of plasminogen/plasmin system [J].
  • the plasminogen is a protein comprising a plasminogen active fragment shown in sequence 14.
  • the plasminogen is a protein comprising a lysine-binding fragment of Kringle 1, Kringle 2, Kringle 3, Kringle 4 and/or Kringle 5.
  • the plasminogen active fragment of the present application comprises sequence 14, and a protein having an amino acid sequence with at least 80%, 90%, 95%, 96%, 97%, 98%, 99% homology to sequence 14. Therefore, the plasminogen described in the present invention includes a protein containing the plasminogen active fragment and still maintaining the plasminogen activity.
  • the plasminogen of the present application comprises Kringle 1, Kringle 2, Kringle 3, Kringle 4 and/or Kringle 5, or a protein that has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% homology with Kringle 1, Kringle 2, Kringle 3, Kringle 4 or Kringle 5 and still has lysine binding activity.
  • the methods for measuring plasminogen in blood and its activity include: detection of tissue plasminogen activator activity (t-PAA), detection of plasma tissue plasminogen activator antigen (t-PAAg), detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, detection of plasma tissue plasminogen activator inhibitor antigen, and detection of plasma plasmin-antiplasmin complex (PAP).
  • tissue plasminogen activator activity t-PAA
  • t-PAAg tissue plasminogen activator antigen
  • plgA plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma plasmin-antiplasmin complex
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the test plasma, PLG in the test plasma is converted into PLM under the action of SK, and the latter acts on the chromogenic substrate, which is then measured by a spectrophotometer, and the increase in absorbance is proportional to the activity of plasminogen.
  • SK streptokinase
  • immunochemical method, gel electrophoresis, immunoturbidimetry, radial immunodiffusion method, etc. can also be used to measure the activity of plasminogen in blood.
  • orthologs refer to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs and vertical homologs. Specifically refers to proteins or genes evolved from the same ancestral gene in different species.
  • the plasminogen of the present invention includes natural human plasminogen, and also includes orthologs or orthologs of plasminogen derived from different species and having plasminogen activity.
  • Constant substitution variant refers to a variant in which a given amino acid residue is changed but does not change the overall conformation and function of the protein or enzyme, which includes but is not limited to replacing the amino acid in the amino acid sequence of the parent protein with an amino acid of similar properties (such as acidity, basicity, hydrophobicity, etc.).
  • Amino acids with similar properties are well known. For example, arginine, histidine and lysine are hydrophilic basic amino acids and can be interchanged.
  • isoleucine is a hydrophobic amino acid and can be replaced by leucine, methionine or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different.
  • a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm For example, a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm.
  • Constant substitution variant also includes polypeptides or enzymes with more than 60% amino acid identity determined by the BLAST or FASTA algorithm, preferably more than 75%, preferably more than 85%, and even more than 90% is the best, and has the same or substantially similar properties or functions compared to the natural or parent protein or enzyme.
  • Isolated plasminogen refers to plasminogen protein that has been separated and/or recovered from its natural environment.
  • the plasminogen is purified (1) to a purity of greater than 90%, greater than 95%, or greater than 98% (by weight) as determined by the Lowry method, for example, greater than 99% (by weight), (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (3) to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver stain.
  • Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.
  • polypeptide refers to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.
  • the term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without an N-terminal methionine residue); and the like.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as Such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment sequence, including any algorithm required for the maximum comparison of the total length of the compared sequence. However, for purposes of the present invention, the amino acid sequence identity percentage values are generated using the sequence comparison computer program ALIGN-2.
  • the % amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B is calculated as follows:
  • 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.
  • “Therapeutically effective amount” or “effective amount” refers to the amount of plasminogen that is sufficient to achieve the described prevention and/or treatment of the disease when administered to a mammal or other subject for the treatment of a disease.
  • the “therapeutically effective amount” will vary depending on the plasminogen used, the severity of the disease and/or its symptoms of the subject to be treated, and the age, weight, etc.
  • treating includes inhibiting or arresting the development of the disease state or its clinical symptoms, or alleviating the disease state or symptoms such that the disease state or its clinical symptoms regress temporarily or permanently.
  • Plasminogen can be isolated from nature and purified for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is synthesized chemically, the synthesis can be carried out via liquid or solid phase. Solid phase peptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence, is a suitable method for the chemical synthesis of plasminogen. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen.
  • SPPS Solid phase peptide synthesis
  • Fmoc and Boc can be used to synthesize plasminogen.
  • the technique of solid phase synthesis is described in Barany and Solid-Phase Peptide Synthesis; Pages 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol.
  • Standard recombinant methods can be used to produce plasminogen of the present invention.
  • a nucleic acid encoding plasminogen is inserted into an expression vector so that it is operably linked to a regulatory sequence in the expression vector.
  • Expression regulatory sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression regulation can be a eukaryotic promoter system in a vector that is capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of plasminogen.
  • Suitable expression vectors typically replicate in the host organism as an episome or as an integrated part of the host chromosomal DNA.
  • expression vectors typically contain selection markers (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) to facilitate detection of those cells transformed with the desired DNA sequence from an exogenous source.
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone a plasminogen encoding polynucleotide.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • expression vectors can also be generated, which will usually contain expression control sequences (e.g., origins of replication) compatible with the host cell.
  • promoters such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems, or promoter systems from bacteriophage lambda. Promoters will usually control expression, optionally in the case of an operator gene sequence, and have a ribosome binding site sequence, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast e.g., S. cerevisiae
  • Pichia are examples of suitable yeast host cells, with suitable vectors having expression control sequences (e.g., promoters), replication origins, termination sequences, etc. as required.
  • Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization, among others.
  • mammalian cells e.g., mammalian cells cultured in in vitro cell culture
  • the plasminogen of the present invention e.g., a polynucleotide encoding plasminogen.
  • 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 these cells can contain expression control sequences, such as replication origins, promoters and enhancers (Queen et al., Immunol. Rev.
  • RNA splicing sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences.
  • suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).
  • the plasminogen of the present invention can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, etc.
  • the plasminogen is substantially 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 more, e.g., free of contaminants, such as cellular debris, macromolecules other than plasminogen, etc.
  • Therapeutic formulations can be prepared by mixing plasminogen having the desired purity with optional pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)) to form a lyophilized preparation or an aqueous solution.
  • Acceptable carriers, excipients, stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • the formulations of the present invention may also contain more than one active compound required for the specific condition to be treated, preferably those with complementary activities and no side effects between them, for example, antihypertensive drugs, antiarrhythmic drugs, drugs for treating diabetes, etc.
  • the plasminogen of the present invention can be encapsulated in microcapsules prepared by techniques such as coacervation or interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules placed in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • the plasminogen of the present invention for in vivo administration must be sterile. This can be easily achieved by filtering through a sterile filtration membrane before or after lyophilization and reconstitution.
  • the plasminogen of the present invention can be prepared into a sustained-release preparation.
  • sustained-release preparations include solid hydrophobic polymer semipermeable matrices having a certain shape and containing glycoproteins, such as films or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (such as 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 3773919, EP 58,481 copolymers of L-glutamic acid and ⁇ -ethyl-L-glutamic acid (Sidman, et al., Biopolymers 22: 547 (1983)), non-degradable ethylene-vinyl acetate (ethylene-vinyl acetate) (Langer, et al., supra ), or degradable lactic acid-glycolic acid copolymers such as Lupron DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • Lupron DepotTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules continuously for more than 100 days, while some hydrogels release proteins for shorter periods of time.
  • Rational strategies for protein stabilization can be designed based on the relevant mechanisms. For example, if the mechanism of coagulation is found to be the formation of intermolecular S-S bonds through thiodisulfide exchange, stabilization can be achieved by modifying thiol residues, lyophilizing from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions.
  • Administration of the pharmaceutical composition of the invention can be achieved by different ways, for example, intravenously, intraperitoneally, subcutaneously, intracranially, intrathecally, intraarterially (eg via the carotid artery), intramuscularly.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • 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 liquid and nutritional supplements, electrolyte supplements, etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases, etc.
  • the dosage range of the pharmaceutical composition comprising plasminogen of the present invention can be about 0.0001 to 2000 mg/kg per day, 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.) subject body weight.
  • the dosage 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. Dosages higher or lower than this exemplary range are also included, particularly in view of the above-mentioned factors. Intermediate doses in the above range are also included within the scope of the present invention. Subjects can apply such dosages every day, every other day, every week, or according to any other schedule determined by empirical analysis. An exemplary dosage schedule includes 1-10 mg/kg on consecutive days. During the administration of the drug of the present invention, it is necessary to evaluate the therapeutic effect and safety in real time.
  • One embodiment of the present invention relates to a product or kit comprising plasminogen or plasmin of the present invention that can be used to treat cardiovascular disease and related conditions caused by diabetes.
  • the product preferably includes a container, a label or a package insert. 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 can effectively treat the disease or condition of the present invention and has a sterile access port (for example, the container can be an intravenous solution bag or vial containing a stopper that can be penetrated by a hypodermic injection needle). At least one active agent in the composition is plasminogen/plasmin.
  • the label on or attached to the container indicates that the composition is used to treat cardiovascular disease and related conditions caused by diabetes as described in the present invention.
  • the product may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and glucose solution. It may further include what is required from a commercial and user perspective. Other materials required, including other buffers, diluents, filters, needles and syringes.
  • the article of manufacture comprises a package insert with instructions for use, including, for example, instructing the user of the composition to administer the plasminogen composition and other drugs for treating concomitant diseases to the patient.
  • the above-mentioned diseases caused by pathological protein aggregation include: Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion-like disease (CJD), Gerstmann syndrome, diffuse or familial fatal insomnia and kuru, Huntington's disease, spinocerebellar ataxia type 3, and dentatorubral pallidum Lewy body atrophy.
  • the diseases caused by ubiquitin/lysosomal dysfunction include: Fanconi anemia, Xeroderma pigmentosum, Cockayne syndrome, cancer, Cowden syndrome, Parkinson's disease, genomic instability, metabolic syndrome, muscular atrophy, Von Hippel Lindau syndrome, multiple myeloma, RIDDLE syndrome, Huntington's disease, X-linked lymphoproliferative disease, Crohn's disease, Alzheimer's disease, breast cancer and ovarian cancer, muscular amylopectinosis, amyotrophic lateral sclerosis, spinal muscular atrophy, systemic lupus erythematosus, erythematosus), childhood ataxia, X-linked parkinsonism with spasticity, multisystem disorder, diabetes mellitus, multiple sclerosis, cystinosis, Vici syndrome, Gaucher's disease, frontotemporal dementia (heterozygous) or neuronal ceroid lipofuscinosis (homozygous), Danon's cardiomy
  • SRS Wiskott-Aldrich syndrome
  • PAG primary open angle glaucoma
  • PGD Paget's disease of the bone
  • colon cancer lung cancer, brain cancer, autosomal recessive and sporadic early-onset Parkinson's disease, Zellweger syndrome spectrum disorders, distal myopathy, ulcerative colitis, Ulcerative colitis, familial Mediterranean fever, ataxia with spasticity, Fabry disease, Gaucher disease, lysosomal acid lipase deficiency, mucopolysaccharidoses, Angelman syndrome.
  • the human plasminogen used in all the following examples was from donor plasma based on the 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 chromosome
  • the plasma was purified from human donor plasma according to the method described in [1-3] and the process was optimized, in which the content of human Lys-plasminogen (Lys-plasminogen) and Glu-plasminogen (Glu-plasminogen) was >98%.
  • Plasminogen is enriched in the spinal cord tissue of ALS model mice and co-localizes with ubiquitin
  • Transgenic mutant SOD1 has the histopathological characteristics observed in the clinic of sporadic and familial amyotrophic lateral sclerosis (ALS).
  • the ALS model mice in this case are B6.Cg-Tg(SOD1-G93A)1Gur/J transgenic mice (referred to as SOD1-G93A), purchased from Jackson Laboratory, pedigree number: 004435, and animal-related experiments were conducted in an SPF environment.
  • SOD1-G93A model mice have been widely used in the mechanism research of ALS and preclinical experimental research of new drug development.
  • mice Five wild-type male mice and nine male SOD1-G93A mice of similar age were selected.
  • the wild-type mice were used as the blank control group, and the SOD1-G93A mice were observed and recorded from the 14th week when their hind legs trembled.
  • the onset time of each mouse was recorded, and medication was started 14 days after the onset. All mice were randomly divided into a vehicle group and a medication group according to the onset of the disease. There were 5 mice in the vehicle group, and 0.1 ml of vehicle (sodium citrate buffer) was injected into the tail vein every day; there were 4 mice in the medication group, and 1 mg/0.1 ml of plasminogen was injected into the tail vein every day.
  • vehicle sodium citrate buffer
  • the medication was continued under SPF environment, and the samples were collected at the end of life.
  • the longest medication was 61 days.
  • the spinal cord tissue was fixed in formalin fixative.
  • the fixed tissue was dehydrated with alcohol gradient and transparentized with xylene before being embedded in paraffin.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were washed once after dewaxing and rehydration.
  • the sections were immersed in Microwave repair in antigen repair working solution (0.01M sodium citrate buffer), preheat for 5 minutes, high heat for 2 minutes, low heat for 15 minutes. Circle the tissue with a PAP pen, incubate with 3% hydrogen peroxide for 15 minutes, and wash twice with 0.01M PBS for 5 minutes each time.
  • Ubiquitin is a highly conserved small molecular weight protein with a molecular weight of about 8,500. It is composed of 76 amino acids and is widely present 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 ways to "digest" intracellular proteins.
  • Plasminogen is enriched in the spinal cord tissue of ALS model mice and co-localizes with LC3B
  • mice Five wild-type male mice and nine male SOD1-G93A mice of similar age were selected. Wild-type mice were used as the blank control group. SOD1-G93A mice were observed and recorded from the 14th week when their hind legs trembled. The onset time of each mouse was recorded. Drug administration began 14 days after the onset of the disease. All mice were randomly divided into a vehicle group and a drug administration group according to the onset of the disease. Five mice in the vehicle group were injected with 0.1 ml/mouse vehicle (sodium citrate buffer) through the tail vein every day; four mice in the drug administration group were injected with 1 mg/0.1 ml/mouse plasminogen through the tail vein every day.
  • 0.1 ml/mouse vehicle sodium citrate buffer
  • Drug administration was continued under SPF environment, and samples were collected near death. The longest drug administration was 61 days.
  • the spinal cord tissue was fixed in formalin fixative. The fixed tissue was dehydrated by alcohol gradient and transparentized with xylene before paraffin embedding. The thickness of the tissue section was 3 ⁇ m, and the sections were washed once after dewaxing and rehydration. The sections were immersed in the antigen retrieval working solution (0.01M sodium citrate buffer) for microwave repair, preheating for 5 minutes, high heat for 2 minutes, and low heat for 15 minutes. The tissue was circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01M PBS for 5 minutes each time.
  • LC3 refers to protein light chain 3, which is a marker of the autophagic process. Its function is mainly involved in the formation of autophagosomes.
  • the LC3 precursor molecule is cleaved to remove the C-terminal 5-peptide and cleaved to form the cytoplasmic form LC3-I.
  • LC3B membrane-bound form LC3- II (LC3B), which can attach to the membrane of the autophagosome and is a structural protein of the autophagosome.
  • Example 3 Plasminogen is enriched in the spinal cord tissue of ALS model mice and co-localizes with LAMP2
  • mice Five wild-type male mice and nine male SOD1-G93A mice of similar age were selected. Wild-type mice were used as the blank control group. SOD1-G93A mice were observed and recorded from the 14th week when their hind legs trembled. The onset time of each mouse was recorded. Drug administration began 14 days after the onset of the disease. All mice were randomly divided into a vehicle group and a drug administration group according to the onset of the disease. Among them, there were 5 mice in the vehicle group, and 0.1 ml/mouse vehicle (sodium citrate buffer) was injected into the tail vein every day; 4 mice in the drug administration group were injected with 1 mg/0.1 ml/mouse plasminogen into the tail vein every day.
  • 0.1 ml/mouse vehicle sodium citrate buffer
  • the drug administration was continuous under SPF environment, and the samples were collected near death. The longest drug administration was 61 days.
  • the spinal cord tissue was fixed in formalin fixative. The fixed tissue was dehydrated by alcohol gradient and transparentized with xylene before paraffin embedding. The thickness of the tissue section was 3 ⁇ m, and the sections were washed once after dewaxing and rehydration. The sections were immersed in the antigen retrieval working solution (0.01M sodium citrate buffer) for microwave repair, preheating for 5 minutes, high heat for 2 minutes, and low heat for 15 minutes. The tissue was circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01M PBS for 5 minutes each time.
  • Lysosomal associated membrane protein-2 (LAMP2) is a highly abundant lysosomal glycoprotein that serves as a receptor for proteins imported directly into lysosomes and as a mediator of autophagosome/phagosome maturation [4].
  • Example 4 Plasminogen promotes the expression of LC3 in the spinal cord tissue of amyotrophic lateral sclerosis model mice
  • 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 administration group.
  • the vehicle control group mice were injected with the vehicle at 5 ml/kg of tail vein, and the drug administration group mice were injected with plasminogen (10 mg/ml) at 50 mg/kg of body weight of tail vein.
  • the mice were killed 24 hours after administration and the spinal cord was collected.
  • RT-PCR detection of LC3 gene transcription was performed and the CT value was recorded. The CT value of LC3 gene transcription mRNA in 100 ng of total RNA was calculated.
  • Plasminogen is enriched in the substantia nigra and striatum of Parkinson's model mice and co-localizes with ubiquitin
  • mice Twelve 9-week-old C57 male mice were weighed one day before modeling. The mice were intraperitoneally injected with 5 mg/ml MPTP solution at 30 mg/kg body weight per day for 5 consecutive days to establish a Parkinson's model [5, 6].
  • Preparation of MPTP solution 10 ml of deionized water was drawn with a syringe and added to 100 mg of MPTP powder (sigma, M0896) to prepare a 10 mg/ml stock solution. Then 1 ml of the stock solution was drawn into an ampoule and 1 ml of deionized water was added to the ampoule to obtain a final concentration of 5 mg/ml.
  • mice were randomly divided into Two groups, 6 mice each for the vehicle PBS control group and the plasminogen group, began to be administered, marked as day 1, the plasminogen group mice were injected with plasminogen solution at 1 mg/0.1 ml/mouse/day through the tail vein, and the vehicle PBS control group was given the same volume of PBS through the tail vein, and the administration continued for 14 days.
  • the mice were killed on the 15th day of administration, and the substantia nigra and striatum of the mice were fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed tissue was dehydrated by alcohol gradient and transparentized with xylene before paraffin embedding. Plasminogen, ubiquitin and DAPI were co-stained as described in Example 1. The sections were observed and photographed under a 400x optical microscope.
  • Example 6 Lysozyme is enriched in the substantia nigra and striatum of Parkinson's model mice and co-localizes with LC3B
  • mice Twelve 9-week-old C57 male mice were weighed 1 day before modeling. The mice were intraperitoneally injected with 5 mg/ml MPTP solution at 30 mg/kg body weight every day for 5 consecutive days to establish a Parkinson's model [5, 6].
  • Preparation of MPTP solution Use a syringe to draw 10 ml of deionized water and add it to 100 mg of MPTP powder (sigma, M0896) to prepare a 10 mg/ml stock solution. Then draw 1 ml of the stock solution into an ampoule and add 1 ml of deionized water to a final concentration of 5 mg/ml.
  • mice were randomly divided into two groups, with 6 mice in each group given a solvent PBS control group and a plasminogen group.
  • Drug administration began, which was recorded as day 1.
  • the plasminogen group mice were injected with plasminogen solution at 1 mg/0.1 ml/mouse/day through the tail vein, and the same volume of PBS was given to the solvent PBS control group through the tail vein.
  • Drug administration continued for 14 days.
  • the mice were killed, and the substantia nigra and striatum of the mice were fixed with 4% paraformaldehyde for 24-48 hours.
  • plasminogen, LC3B and DAPI were co-stained as described in Example 2. The sections were observed and photographed under a 400x optical microscope.
  • Example 7 Plasminogen is enriched in the substantia nigra and striatum tissues of Parkinson's model mice and co-localizes with LAMP2
  • mice Twelve 9-week-old C57 male mice were weighed 1 day before modeling. The mice were intraperitoneally injected with 5 mg/ml MPTP solution at 30 mg/kg body weight every day for 5 consecutive days to establish a Parkinson's model [5, 6].
  • Preparation of MPTP solution Use a syringe to draw 10 ml of deionized water and add it to 100 mg of MPTP powder (sigma, M0896) to prepare a 10 mg/ml stock solution. Then draw 1 ml of the stock solution into an ampoule and add 1 ml of deionized water to a final concentration of 5 mg/ml.
  • mice were randomly divided into two groups, with 6 mice in each group given a solvent PBS control group and a plasminogen group.
  • Drug administration began, which was recorded as day 1.
  • the plasminogen group mice were injected with plasminogen solution at 1 mg/0.1 ml/mouse/day through the tail vein, and the same volume of PBS was given to the solvent PBS control group through the tail vein.
  • Drug administration continued for 14 days.
  • the mice were killed, and the substantia nigra and striatum of the mice were fixed with 4% paraformaldehyde for 24-48 hours.
  • plasminogen, LAMP2 and DAPI co-staining were performed as described in Example 3. The sections were observed and photographed under a 400x optical microscope.
  • Example 8 Plasminogen is enriched in the hippocampal tissue of Alzheimer's model mice and co-localizes with ubiquitin
  • mice Twenty 8-week-old male C57 mice were weighed before modeling. After excluding abnormal mice according to their weight, all mice were randomly divided into two groups, a vehicle group and a drug-treated group, with 10 mice in each group. All mice were anesthetized and positioned in the granule cell layer of the hippocampus according to the mouse stereotaxic atlas (positioned according to the coordinates of the bregma point: AP-2.0mm, ML ⁇ 1.5mm, DV2.0mm). Each mouse was slowly microinjected bilaterally at an injection rate of 0.5 ⁇ L/min and an injection volume of 3 ⁇ L. The model group mice were injected with A ⁇ 1-42 oligomer solution to establish an Alzheimer's model [7], and the model control group mice were injected with PBS solution.
  • mice in the vehicle group and the drug group began to be administered, which was recorded as the first day.
  • the mice in the drug group were injected with plasminogen by tail vein at 1mg/0.1ml/mouse/day, and the mice in the vehicle group were injected with 0.1ml/mouse/day vehicle (4% arginine + 2% glycine solution) by tail vein, and the drug was administered continuously for 28 days.
  • the mice were killed and the brain was fixed in 10% formaldehyde for 24-48 hours.
  • the fixed tissue was dehydrated by alcohol gradient and transparentized by xylene before paraffin embedding. Co-staining of plasminogen, ubiquitin and DAPI was performed as described in Example 1. The sections were observed and photographed under a 400x optical microscope.
  • Example 9 Plasminogen is enriched in the hippocampal tissue of Alzheimer's model mice and co-localizes with LC3B
  • mice Twenty 8-week-old male C57 mice were weighed before modeling. After excluding abnormal mice according to their weight, all mice were randomly divided into two groups, a vehicle group and a drug-treated group, with 10 mice in each group. All mice were anesthetized and located in the granule cell layer of the hippocampus according to the mouse stereotaxic atlas (based on the coordinates of the bregma: AP-2.0mm, ML ⁇ 1.5mm, DV2.0mm). Each mouse was slowly microinjected bilaterally at an injection rate of 0.5 ⁇ L/min and an injection volume of 3 ⁇ L. The model group mice were injected with A ⁇ 1-42 oligomer solution to establish the Alzheimer's model [7], and the model control group mice were injected with PBS solution.
  • a ⁇ 1-42 oligomer solution (10 ⁇ M): Take ⁇ -Amyloid (1-42) (Shanghai Qiangyao Biotechnology Co., Ltd., 04010011521), add cold hexafluoroisopropanol, prepare a concentration of 1mg/ml, place at room temperature for 3 days, divide into 45 ⁇ L/tube, i.e. 10nmol/mL, place in a fume hood overnight, place in a 25°C drying oven to dry for 1 hour, and store at -80°C. When using, add 10 ⁇ l of dimethyl sulfoxide solution to each tube for re-dissolution.
  • mice in the vehicle group and the drug-treated group began to be dosed, recorded as day 1.
  • the mice in the drug-treated group were intravenously injected with 1mg/0.1ml/mouse/day.
  • Plasminogen was injected intravenously, and 0.1 ml/mouse/day of the vehicle (4% arginine + 2% glycine solution) was injected into the tail vein of the mice in the vehicle group for 28 consecutive days.
  • the mice were killed and the brain was fixed in 10% formaldehyde for 24-48 hours.
  • the fixed tissue was dehydrated with alcohol gradient and transparentized with xylene before paraffin embedding. Plasminogen, LC3B and DAPI were co-stained as described in Example 2. The sections were observed and photographed under a 400x optical microscope.
  • Example 10 Plasminogen is enriched in the hippocampal tissue of Alzheimer's model mice and co-localizes with LAMP2
  • mice Twenty 8-week-old male C57 mice were weighed before modeling. After excluding abnormal mice according to their weight, all mice were randomly divided into two groups, a vehicle group and a drug-treated group, with 10 mice in each group. All mice were anesthetized and located in the granule cell layer of the hippocampus according to the mouse stereotaxic atlas (based on the coordinates of the bregma: AP-2.0mm, ML ⁇ 1.5mm, DV2.0mm). Each mouse was slowly microinjected bilaterally at an injection rate of 0.5 ⁇ L/min and an injection volume of 3 ⁇ L. The model group mice were injected with A ⁇ 1-42 oligomer solution to establish the Alzheimer's model [7], and the model control group mice were injected with PBS solution.
  • mice in the vehicle group and the drug group began to be administered, which was recorded as the first day.
  • the mice in the drug group were injected with plasminogen by tail vein at 1 mg/0.1 ml/mouse/day, and the mice in the vehicle group were injected with 0.1 ml/mouse/day vehicle (4% arginine + 2% glycine solution) by tail vein, and the drug was administered continuously for 28 days.
  • mice On the 29th day, the mice were killed and the brains were fixed in 10% formaldehyde for 24-48 hours.
  • the fixed tissues were dehydrated by gradient alcohol and transparentized by xylene before paraffin embedding. Plasminogen, LAMP2 and DAPI were co-stained as described in Example 3. The sections were observed and photographed under a 400x optical microscope.
  • mice Ten 14-week-old B6/JGpt-Tg(hHTT-CAG130)90/Gpt mice (hHTT130 transgenic mice for short) were selected as the model group, and five C57 mice were selected as the normal control group.
  • hHTT130 mice were randomly divided into two groups, the vehicle group and the drug group, with five mice in each group. After the grouping was completed, all mice began to be administered.
  • the mice in the drug group were injected with plasminogen at a tail vein of 50 mg/kg, and the mice in the normal control group and the vehicle group were injected with the same volume of the vehicle in the tail vein for 28 consecutive days. After the end of the drug administration, the mice were killed and the heart was taken.
  • the heart tissue homogenate was used for huntingtin (HTT) weastern blot detection.
  • a 10% gel was prepared according to the SDS-PAGE gel preparation instructions. After each group of samples were mixed with 4 ⁇ loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5min, centrifuged for 2min after cooling, and then 20 ⁇ L was taken for loading.
  • the electrophoresis conditions were 30V for 45min, and then 100V for electrophoresis to the bottom of the gel. After the electrophoresis, the gel was stripped and transferred to an activated PVDF membrane (GE, A29433753) at 15V for 2.5h.
  • the transferred PVDF membrane was immersed in a blocking solution (5% skim emulsion) and blocked overnight in a refrigerator at 4°C. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), rabbit anti-HTT antibody (Abcam, ab109115) and actin antibody were added and incubated at room temperature for 1.5h. After washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1h. After washing 4 times with TBST, the PVDF membrane was placed on a clean imaging plate, and Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development. The membrane was photographed under a biomolecular imaging instrument and quantitatively analyzed with Image J.
  • MILLIPORE Immobilon Western HRP Substrate
  • Example 12 Plasminogen promotes TDP-43 degradation in the cytoplasm and nucleus of NSC34 cells treated with okadaic acid
  • NSC34 cells (Otwo Biotech, HTX1846) were inoculated in a 9 cm 2 culture dish and cultured in DMEM medium (Gibco, 11965092) containing 10% fetal bovine serum (EVERY GREEN, 11011-8611), and placed in a carbon dioxide incubator for culture at 37.0°C and 5% CO2. After the cells grew for 48 hours and the cell abundance was about 80%-90%, the medium was changed and subsequent experiments were performed. The cells were divided into 4 groups: blank control group, solvent group, drug group and drug + EACA group.
  • okadaic acid (OA) (Shanghai yuanye Bio-Technology, S30686-25ug) at a concentration of 2.5ng/ ⁇ L.
  • OA okadaic acid
  • the medium 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 group, and plasminogen (final concentration was 0.5 mg/mL) and aminocaproic acid (20 mM) were added to the cell culture medium of the drug + EACA group. After adding plasminogen for 24 hours, the cells were harvested.
  • the culture supernatant was aspirated, washed with 1 ⁇ PBS, digested with 0.25 pancreatin 1mL for 2-3 minutes, and the cells were obviously detached. The digestion was terminated with 5-6mL of DMEM complete medium, the cells were slowly blown, the suspension was collected into a centrifuge tube, centrifuged at 1500rpm for 5min to remove the supernatant, resuspended with pre-cooled 1 ⁇ PBS, and the cells were counted. 200 ⁇ L of plasma protein extraction reagent (the volume of 2 ⁇ 10 6 cell pellets is about 20 ⁇ L or 40mg) (Solarbio, R0050) was added to each 20 ⁇ L cell pellet.
  • EACA Aminocaproic acid
  • Example 13 Plasminogen promotes TDP-43 degradation in the cytoplasm and nucleus of NSC34 cells treated with okadaic acid
  • NSC34 cells (Otwo Biotech, HTX1846) were inoculated in a 9 cm 2 culture dish and cultured in DMEM medium (Gibco, 11965092) containing 10% fetal bovine serum (EVERY GREEN, 11011-8611), and placed in a carbon dioxide incubator for culture at 37.0°C and 5% CO2. After the cells grew for 48 hours and the cell abundance was about 80%-90%, the medium was changed and subsequent experiments were performed. The cells were divided into 4 groups: blank control group, solvent group, drug group and drug + EACA group.
  • okadaic acid (OA) (Shanghai yuanye Bio-Technology, S30686-25ug) at a concentration of 2.5ng/ ⁇ L.
  • OA okadaic acid
  • the medium 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 group, and plasminogen (final concentration of 0.5 mg/mL) and aminocaproic acid (20 mM) were added to the cell culture medium of the drug + EACA group. After adding plasminogen for 24 hours, the cells were harvested.
  • the culture supernatant was aspirated, washed with 1 ⁇ PBS, digested with 0.25 pancreatin 1mL for 2-3 minutes, and the cells were obviously detached. The digestion was terminated with 5-6 mL of DMEM complete medium, the cells were slowly blown, the suspension was collected into a centrifuge tube, centrifuged at 1500rpm for 5min to remove the supernatant, resuspended with pre-cooled 1 ⁇ PBS, and the cells were counted.
  • Cell lysis buffer (Solarbio, R0010-100mL) was added to lyse the cells and extract cell protein. The extracted cell proteins were subjected to western blot detection of phosphorylated Tau protein (Abcam, ab151559) and total Tau protein (Proteintech, 10842-1-AP).
  • Plasminogen promotes the increase of plasminogen level and plasmin activity level in the cytoplasm and nucleus of NSC34 cells treated with okadaic acid
  • NSC34 cells (Otwo Biotech, HTX1846) were inoculated in a 9 cm 2 culture dish and cultured in DMEM medium (Gibco, 11965092) containing 10% fetal bovine serum (EVERY GREEN, 11011-8611), and placed in a carbon dioxide incubator for culture at 37.0°C and 5% CO 2 . After the cells grew for 48 hours and reached about 80%-90% abundance, the medium was changed and subsequent experiments were performed. The cells were divided into three groups: vehicle group, drug group, and drug + EACA group.
  • okadaic acid (OA) (Shanghai yuanye Bio-Technology, S30686-25ug) at a concentration of 2.5ng/ ⁇ L.
  • OA okadaic acid
  • the medium 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 group, and plasminogen (final concentration was 0.5 mg/mL) and aminocaproic acid (20 mM) were added to the cell culture medium of the drug + EACA group. After adding plasminogen for 24 hours, the cells were harvested.
  • the culture supernatant was aspirated, washed with 1 ⁇ PBS, and digested with 0.25 pancreatin 1mL for 2-3 minutes. When the cells were obviously detached, the digestion was terminated with 5-6mL of DMEM complete medium, the cells were slowly blown, and the suspension was collected into a centrifuge tube. The supernatant was centrifuged at 1500rpm for 5min to remove the supernatant. The cells were resuspended in pre-cooled 1 ⁇ PBS and the cells were counted. 200 ⁇ L of plasma protein extraction reagent (the volume of 2 ⁇ 106 cell pellets is about 20 ⁇ L or 40mg) (Solarbio, R0050) was added to each 20 ⁇ L cell pellet.
  • the activity of plasmin was detected by enzyme substrate kinetic method. 85 ⁇ L/well of standard solution at seven different concentrations, blank, and sample were added to the ELISA plate in turn, and then 15 ⁇ L 20 mM S-2251 solution (Chromogenix, 82033239) was added to each well and incubated at 37°C. Starting from 0 min of reaction, the A405 absorbance value was read in a multifunctional microplate reader every 5 min until the reaction was 90 min. All reactions were fitted with a straight line using time and absorbance values, and the slope of the straight line was obtained as the reaction rate of the standard/sample ( ⁇ A405/min). Finally, the potency value of the standard and ⁇ A405/min were used as a standard curve to calculate the potency of the measured sample.
  • mice Ten 14-week-old B6/JGpt-Tg(hHTT-CAG130)90/Gpt mice (hHTT130 transgenic mice for short) 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, the vehicle group and the drug group, with five mice in each group. After the grouping was completed, all mice began to be administered.
  • the mice in the drug group were injected with plasminogen at a tail vein of 50 mg/kg, and the mice in the normal control group and the vehicle group were injected with the same volume of the vehicle in the tail vein.
  • the drug was administered for 28 consecutive days. After the end of the drug administration, the mice were killed and the kidneys were taken.
  • the huntingtin (HTT) weastern blot was detected in the kidney tissue homogenate.
  • a 10% gel was prepared according to the SDS-PAGE gel preparation instructions.
  • the samples of each group were mixed with 4 ⁇ loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 minutes, centrifuged for 2 minutes after cooling, and then 20 ⁇ L was taken for loading.
  • the electrophoresis conditions were 30V for 45min, and then 100V for electrophoresis to the bottom of the gel. After the electrophoresis, the gel was stripped and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15V for 2.5h.
  • the transferred PVDF membrane was immersed in a blocking solution (5% skim emulsion) and blocked overnight in a refrigerator at 4°C. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), rabbit anti-HTT antibody (Abcam, ab109115) and actin antibody were added and incubated at room temperature for 1.5h. After washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) was added as a secondary antibody and incubated at room temperature for 1h. After washing 4 times with TBST, the PVDF membrane was placed on a clean imaging plate, and Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development. The membrane was photographed under a biomolecular imaging instrument and quantitatively analyzed using Image J.
  • Example 16 Plasminogen promotes the degradation of huntingtin protein in brain tissue of Huntington model mice
  • the brain slices and culture medium were aspirated, centrifuged at 1200RPM for 5 minutes, the supernatant was removed, and then resuspended with 1 ⁇ PBS, and centrifuged again to remove PBS. The brain tissue precipitate was preserved. After homogenization, huntingtin (HTT) was detected by weastern blot. A 10% gel was prepared according to the SDS-PAGE gel preparation instructions. After each group of samples was mixed with 4 ⁇ loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 minutes, cooled and centrifuged for 2 minutes, and then 20 ⁇ L was taken for sample loading.
  • 4 ⁇ loading buffer (TaKaRa, e2139) at a volume ratio of 3:1
  • the electrophoresis conditions were 30V for 45 minutes, and then 100V electrophoresis to the bottom of the gel.
  • the gel was stripped and transferred to an activated PVDF membrane (GE, A29433753) under the electrophoresis conditions of 15V for 2.5h.
  • the transferred PVDF membrane was immersed in a blocking solution (5% skim milk) and blocked overnight in a 4°C refrigerator. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), rabbit anti-HTT antibody (Abcam, ab109115) and actin antibody were added and incubated at room temperature for 1.5h.
  • Sequence 1 (natural plasminogen (Glu-PLG, Glu-plasminogen) nucleic acid sequence without signal peptide):
  • Sequence 2 (natural plasminogen (Glu-PLG, Glu-plasminogen) nucleic acid sequence without signal peptide):
  • Sequence 3 (nucleic acid sequence of natural plasminogen (derived from Swiss prot) containing a signal peptide):
  • Sequence 4 amino acid sequence of natural plasminogen (derived from Swiss prot) containing a signal peptide:
  • Sequence 7 (delta-plg (delta-plasminogen) nucleic acid sequence):
  • Sequence 13 (nucleic acid sequence of serine protease (structure) domain):
  • SEQ ID NO: 14 amino acid sequence of the serine protease (structure) domain

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Abstract

一种调节以优化泛素蛋白酶体系统和/或自噬溶酶体系统，从而促进细胞内外病理性蛋白降解的方法，以及促进细胞内外病理性蛋白降解的药物及其用途。

Description

一种促进泛素蛋白酶体系统和自噬溶酶体系统对病理性蛋白清除的方法和药物 技术领域
本申请涉及纤维蛋白溶酶原激活途径相关化合物例如纤溶酶原或纤溶酶用于促进泛素蛋白酶体系统和自噬溶酶体系统对病理性蛋白清除的用途。
背景技术
慢性神经退行性疾病是大脑和脊髓细胞神经元丧失的一种疾病状态，包括阿兹海默病、亨廷顿氏病、帕金森氏病、肌萎缩侧索硬化症、脊髓性肌萎缩症等。研究发现，其是由于泛素依赖过程产生的病变，包括26S蛋白酶体的泛素化降解与通过溶酶体途径发生的自噬的异常，这些都会对神经的发育，体内平衡和疾病的产生具有严重的影响。所以，泛素化降解与自噬对神经活动来说是必不可少的，参与了突触发生，细胞-细胞的相互作用。在成人中枢和周围神经系统，基于未分裂的细胞来说，蛋白质的泛素化与去泛素化作用对神经元的生存是至关重要的，多数老年性慢性神经退行性疾病归因于蛋白质总量的积累，而且经常作为核内容物而存在。
泛素蛋白酶体系统(ubiquitin proteasome system，UPS)和自噬溶酶体系统是细胞内的蛋白降解系统，它们的相互作用可促进病理性致病蛋白的降解。人们期望寻找到能够促进泛素蛋白酶体系统(ubiquitin proteasome system，UPS)和自噬溶酶体系统相互作用的物质，从而促进细胞内致病的病理性蛋白的清除。
发明概述
本申请研究发现纤溶酶原能够某种程度上促进泛素蛋白酶体系统(ubiquitin proteasome system，UPS)和自噬溶酶体系统的相互作用，促进细胞内外病理性蛋白的清除。具体地，本申请涉及以下各项：
1.一种促进泛素蛋白酶体系统和自噬溶酶体系统对病理性蛋白清除的 方法，包括给药受试者有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
2.项1所述的方法，其中所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro-plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。
3.项1的方法，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抑制剂，例如抗体。
4.项1-3任一项的方法，其中所述化合物具有以下一项或多项活性：直接在细胞外和/或直接进入细胞内和/或细胞核内清除不同种类的病理性蛋白、促进泛素蛋白酶体系统对病理性蛋白的清除、促进自噬溶酶体系统对病理性蛋白的清除、调节以优化泛素系统成员的表达和/或活性、调节以优化LC3的表达和/或活性、调节以优化自噬溶酶体系统成员的表达和/或活性、调节以优化(特别是促进)LAMP2的表达。
5.项1-4任一项的方法，其中所述化合物为纤溶酶原或纤溶酶。
6.项1-5任一项的方法，其中所述纤溶酶原为Glu-纤溶酶原、Lys-纤溶酶原或其保守取代变体。
7.项1-6任一项的方法，其中所述纤溶酶原与序列2具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且具有纤溶酶原的赖氨酸结合活性和/或蛋白水解活性。
8.项1-7任一项的方法，所述纤溶酶原包含选自如下的一项或多项：
1)具有序列14所示的丝氨酸蛋白酶结构域；
2)与序列14具有至少80％、90％、95％、96％、97％、98％、99％同一性并保留蛋白水解活性的丝氨酸蛋白酶结构域；
3)选自Kringle 1、Kringle 2、Kringle 3、Kringle 4和Kringle 5中一个或多个的Kringle结构域；和
4)与选自Kringle 1、Kringle 2、Kringle 3、Kringle 4和Kringle 5中一个或多个具有至少80％、90％、95％、96％、97％、98％、99％同一性并保留赖氨酸结合活性的Kringle结构域。
9.项1-8任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原的蛋白水解活性的变体。
10.项1-9任一项的方法，所述纤溶酶原包含序列2、6、8、10、12所示的氨基酸序列或包含序列2、6、8、10、12所示氨基酸序列的保守取代变体。
11.项1-10任一项的方法，其中所述纤溶酶原与一种或多种其它治疗方法或药物联合使用。
12.项11的方法，其中所述其它治疗方法包括细胞治疗(包括干细胞治疗)、基因治疗、支持疗法和物理治疗。
13.项1-12任一项的方法，其中所述纤溶酶原通过鼻腔吸入、雾化吸入、滴鼻液、滴眼液、滴耳液、静脉内、腹膜内、皮下、颅内、鞘内、动脉内(例如经由颈动脉)或肌肉内给药。
另一方面，本申请还涉及：
1.一种预防或治疗由于病理性蛋白聚集或者由于泛素/溶酶体功能障碍导致的疾病的方法，包括给药受试者有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
2.项1所述的方法，其中所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多 个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro-plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。
3.项1的方法，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抑制剂，例如抗体。
4.项1-3任一项的方法，其中所述化合物具有以下一项或多项活性：直接在细胞外和/或直接进入细胞内和/或细胞核内清除病理性蛋白、促进泛素蛋白酶体系统对病理性蛋白的清除、促进自噬溶酶体系统对病理性蛋白的清除、调节以优化泛素系统成员的表达和/或活性、调节以优化LC3的表达和/或活性、调节以优化自噬溶酶体系统成员的表达和/或活性、调节以优化(特别是促进)LAMP2的表达。。
5.项1-4任一项的方法，其中所述化合物为纤溶酶原或纤溶酶。
6.项1-5任一项的方法，其中所述纤溶酶原为Glu-纤溶酶原、Lys-纤溶酶原或其保守取代变体。
7.项1-6任一项的方法，其中所述纤溶酶原与序列2具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且具有纤溶酶原的赖氨酸结合活性和/或蛋白水解活性。
8.项1-7任一项的方法，所述纤溶酶原包含选自如下的一项或多项：
1)具有序列14所示的丝氨酸蛋白酶结构域；
2)与序列14具有至少80％、90％、95％、96％、97％、98％、99％同一性并保留蛋白水解活性的丝氨酸蛋白酶结构域；
3)选自Kringle 1、Kringle 2、Kringle 3、Kringle 4和Kringle 5中一个或多个的Kringle结构域；和
4)与选自Kringle 1、Kringle 2、Kringle 3、Kringle 4和Kringle 5中一个或多个具有至少80％、90％、95％、96％、97％、98％、99％同一性并保留赖氨酸结合活性的Kringle结构域。
9.项1-8任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原的蛋白水解活性的变体。
10.项1-9任一项的方法，所述纤溶酶原包含序列2、6、8、10、12所示的氨基酸序列或包含序列2、6、8、10、12所示氨基酸序列的保守取代变体。
11.项1-10任一项的方法，其中所述纤溶酶原与一种或多种其它治疗方法或药物联合使用。
12.项11的方法，其中所述其它治疗方法包括细胞治疗(包括干细胞治疗)、基因治疗、支持疗法和物理治疗。
13.项1-12任一项的方法，其中所述纤溶酶原通过鼻腔吸入、雾化吸入、滴鼻液、滴眼液、滴耳液、静脉内、腹膜内、皮下、颅内、鞘内、动脉内(例如经由颈动脉)或肌肉内给药。在一些实施方案中，所述12.项11的方法，其中所述其它治疗方法包括细胞治疗(包括干细胞治疗)、基因治疗、支持疗法和物理治疗。
14.项1-12任一项的方法，其中所述纤溶酶原通过鼻腔吸入、雾化吸入、滴鼻液、滴眼液、滴耳液、静脉内、腹膜内、皮下、颅内、鞘内、动脉内(例如经由颈动脉)或肌肉内给药。
在一些实施方案中，上述由于病理性蛋白聚集导致的疾病包括：阿尔茨海默病、帕金森病、肌萎缩侧索硬化、朊病毒样病(克雅氏病)、格斯特曼综合征、发散型或家族型致死性失眠症和库鲁病、亨廷顿病、脊髓小脑性共济失调3型、齿状核红核苍白球路易氏体萎缩症、糖尿病、海绵状脑病、黄斑变性、动脉粥样硬化、家族性英国型痴呆、家族性丹麦型痴呆、唐氏综合征、血液透析相关淀粉沉积症、淀粉样心肌病、系统性淀粉样变性、角膜营养不良、血管球性肾炎、淀粉样小体。
在一些实施方案中，上述由于泛素/溶酶体功能障碍导致的疾病包括：范科尼贫血(Fanconi anemia)、着色性干皮病(Xeroderma pigmentosum)、科凯恩综合征(Cockayne syndrome)、癌症、多发性错构瘤综合征(Cowden syndrome)、帕金森病、基因组失稳(Genomic instability)、代谢综合征、肌肉萎缩、希佩尔-林道综合征(Von Hippel Lindau)、多发性骨髓瘤、遗传性假性醛固酮增多症(RIDDLE syndrome)、亨廷顿病、x连锁淋巴增生性疾病、克罗恩病(Crohn’s disease)、阿尔兹海默症(Alzheimer’s disease)、乳腺癌和卵巢癌、肌肉支链淀粉病(muscular  amylopectinosis)、肌萎缩侧索硬化症(Amyotrophic lateral sclerosis)、脊髓性肌萎缩症、系统性红斑狼疮(Systemic lupus erythematosus)、儿童期共济失调、x-连锁帕金森综合征伴痉挛、多系统紊乱、糖尿病、多发性硬化症，胱氨酸病(Cystinosis)、维西综合症(Vici syndrome)、高雪氏病(Gaucher's disease)、额颞叶痴呆(杂合子)或神经元蜡样脂褐质沉积症(纯合子)、Danon’s心肌病、皮质萎缩、癫痫、常染色体隐性脊髓小脑共济失调、遗传痉挛性截瘫、螺旋蛋白相关性神经变性病(Beta-propeller protein-associated neurodegeneration,BPAN)、自闭症谱系障碍、额颞叶痴呆(frontotemporal dementia,FTD)、炎性肠病、非酒精性脂肪性肝病、肺结核、Rett综合征、骨硬化症、2B型Charcot-Marie-Tooth病(Charcot-Marie-Tooth type 2B disease)、幼年关节炎、斯奈德-罗宾逊综合征(Snyder-Robinson syndrome,SRS)、维斯科特-奥尔德里奇综合征(Wiskott-Aldrich syndrome)、原发性小头畸形(Primary microcephaly)、II型传性感觉和自主神经病变、原发性开角型青光眼(Primary open angle glaucoma,POAG)、佩吉特骨病(Paget’s disease of the bone，PGD)、结肠癌、肺癌、脑癌、常染色体隐性遗传和散发性早发帕金森病、泽尔维格综合征谱系障碍(Zellweger syndrome spectrum disorders)、远端肌病、溃疡性结肠炎、家族性地中海热、共济失调伴痉挛、法布里病、戈谢病、溶酶体酸性脂肪酶缺乏症、黏多糖贮积症、天使综合征。
在一些具体实施方案中，所述纤溶酶原途径激活剂与一种或多种其它药物和/或治疗方法联合施用，优选地，所述治疗方法包括细胞疗法(例如干细胞疗法)和基因疗法，例如反义RNA、小分子剪接修饰剂。
在一些具体实施方案中，所述纤溶酶原途径激活剂为纤维蛋白溶酶原激活途径的组分，例如纤溶酶原。在一些具体实施方案中，所述纤溶酶原包含或具有与序列2、6、8、10或12所示氨基酸序列具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，并且具有纤溶酶原活性和/或赖氨酸结合活性。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且具有纤溶酶原活性和/或赖氨酸结合活性的蛋白质。在一些具体实施方案中，所述纤溶酶原活 性为纤溶酶原的蛋白水解活性。在一些具体实施方案中，所述纤溶酶原为包含纤溶酶原活性片段、并且具有纤溶酶原活性和/或赖氨酸结合活性的蛋白质。在一些具体实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水解活性。在一些具体实施方案中，所述纤溶酶原活性片段包含或具有纤溶酶原丝氨酸蛋白酶结构域或称纤溶酶原蛋白酶结构域。在一些具体实施方案中，所述纤溶酶原活性片段的氨基酸序列如序列14所示。在一些具体实施方案中，所述纤溶酶原选自Glu-纤溶酶原(人全长纤溶酶原)、Lys-纤溶酶原(在第76-77位氨基酸之间切割后的人全长纤溶酶原)、小纤溶酶原(包含Kringle 5(K5)和丝氨酸蛋白酶结构域)、微纤溶酶原(包含丝氨酸蛋白酶结构域)、delta-纤溶酶原(包含Kringle 1和丝氨酸蛋白酶结构域)或它们的保留纤溶酶原活性的变体。在一些具体实施方案中，所述纤溶酶原为人全长纤溶酶原、或其仍然保留纤溶酶原活性和/或赖氨酸结合活性的变体或片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性和/或赖氨酸结合活性的变体或片段。在一些实施方案中，所述纤溶酶原包含如序列2、6、8、10或12所示的氨基酸序列。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。
在一些具体实施方案中，所述纤溶酶原途径激活剂以全身或局部方式给药，例如通过静脉内、肌肉内、鼻腔吸入、雾化吸入、滴鼻液形式给药。在一些实施方案中，所述受试者是人。在一些实施方案中，所述受试者缺乏或缺失纤溶酶原。在一些实施方案中，所述缺乏或缺失是先天的、继发的和/或局部的。在一些实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量，每天、每二天或每三天连续施用。
一方面，本申请还涉及用于上述方法的药物组合物、药物、制剂、试剂盒、制品，包含以上所述的纤溶酶原途径激活剂，例如以上所述的纤溶酶原。
在一些实施方案中，所述药物组合物、药物、制剂包含药学上可接受的载体和纤溶酶原途径激活剂，例如纤溶酶原激活途径的组分，例如纤溶 酶原。在一些实施方案中，所述试剂盒和制品包含一个或多个容器，所述容器中包含所述药物组合物、药物或制剂。在一些实施方案中，所述试剂盒或制品还包含标签或使用说明书，该标签或使用说明书指示使用纤溶酶原途径激活剂，例如纤溶酶原激活途径的组分，例如纤溶酶原用于上述方法。在一些实施方案中，所述试剂盒或制品还包含另外的一个或多个容器，该容器中含有一种或多种其他药物。
一方面本申请还涉及用于以上所述用途的纤溶酶原途径激活剂，例如以上所述的纤溶酶原。
一方面，本申请还涉及治疗有效量的上述纤溶酶原途径激活剂在制备用于上述方法的药物组合物、药物、制剂、试剂盒、制品中的用途。
在一些实施方案中，所述纤溶酶原途径激活剂选自如下的一种或多种：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
在一些具体实施方案中，所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro-plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。在一些具体实施方案中，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的拮抗剂，例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抗体。
在一些具体实施方案中，所述纤溶酶原途径激活剂与一种或多种其它药物和/或治疗方法联合施用，优选地，所述治疗方法包括细胞疗法(例如干细胞疗法)和基因疗法，例如反义RNA、小分子剪接修饰剂。
在一些具体实施方案中，所述纤溶酶原途径激活剂为纤维蛋白溶酶原激活途径的组分，例如纤溶酶原。在一些具体实施方案中，所述纤溶酶原包含或具有与序列2、6、8、10或12所示氨基酸序列具有至少75％、 80％、85％、90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，并且具有纤溶酶原活性和/或赖氨酸结合活性。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且具有纤溶酶原活性和/或赖氨酸结合活性的蛋白质。在一些具体实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水解活性。在一些具体实施方案中，所述纤溶酶原为包含纤溶酶原活性片段、并且具有纤溶酶原活性和/或赖氨酸结合活性的蛋白质。在一些具体实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水解活性。在一些具体实施方案中，所述纤溶酶原活性片段包含或具有纤溶酶原丝氨酸蛋白酶结构域或称纤溶酶原蛋白酶结构域。在一些具体实施方案中，所述纤溶酶原活性片段的氨基酸序列如序列14所示。在一些具体实施方案中，所述纤溶酶原选自Glu-纤溶酶原(人全长纤溶酶原)、Lys-纤溶酶原(在第76-77位氨基酸之间切割后的人全长纤溶酶原)、小纤溶酶原(包含Kringle 5(K5)和丝氨酸蛋白酶结构域)、微纤溶酶原(包含丝氨酸蛋白酶结构域)、delta-纤溶酶原(包含Kringle 1和丝氨酸蛋白酶结构域)或它们的保留纤溶酶原活性的变体。在一些具体实施方案中，所述纤溶酶原为人全长纤溶酶原、或其仍然保留纤溶酶原活性和/或赖氨酸结合活性的变体或片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性和/或赖氨酸结合活性的变体或片段。在一些实施方案中，所述纤溶酶原包含如序列2、6、8、10或12所示的氨基酸序列。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。
在一些实施方案中，所述纤溶酶原途径激活剂，例如纤溶酶原激活途径的组分，例如纤溶酶原与一种或多种其它药物和/或治疗方法联合施用。在一些实施方案中，所述纤溶酶原途径激活剂，例如纤溶酶原激活途径的组分，例如纤溶酶原通过静脉内、肌肉内、鞘内、鼻腔吸入、雾化吸入、滴鼻液或滴眼液形式给药。
在一些实施方案中，所述药物组合物、药物、制剂包含药学上可接受的载体和纤溶酶原途径激活剂，例如纤溶酶原激活途径的组分，例如纤溶酶原。在一些实施方案中，所述试剂盒和制品包含一个或多个容器，所述容器中包含所述药物组合物、药物或制剂。在一些实施方案中，所述试剂 盒或制品还包含标签或使用说明书，该标签或使用说明书指示使用纤溶酶原途径激活剂，例如纤溶酶原激活途径的组分，例如纤溶酶原用于上述用途。
在一些实施方案中，所述试剂盒或制品还包含另外的一个或多个容器，该容器中含有一种或多种其他药物。
本发明明确涵盖了属于本发明实施方案之间的技术特征的所有组合，并且这些组合后的技术方案在本申请中已经明确公开，就像上述技术方案已经单独且明确公开一样。另外，本发明还明确涵盖各个实施方案及其要素的之间的组合，该组合后的技术方案在本文中明确公开。
附图简述
图1纤溶酶原与泛素蛋白酶体系统和自噬溶酶体系统相互作用，促进细胞内中枢神经病理性蛋白降解示意图。血脑屏障(Blood-brain barrier)，基底膜(Basement membrane)，内皮细胞(Endothelial)，纤溶酶原(Plasminogen，Plg)，纤溶酶原受体(Plasminogen receptor,PlgR)，组织型纤溶酶原活化因子(tPA)，构象异常蛋白(conformationally abnormal proteins，CAP)，纤溶酶(Plm)，纤溶酶产生的蛋白片段(plasmin generated protein fragments，PGPFs)，纤溶酶降解产物(plasmin degradation products，PDP)，溶酶体(Lysosome)，泛素(ubiquitin，UBI)，泛素激活酶(E1)，泛素结合酶(E2)，泛蛋白连接酶(E3)，蛋白酶体(Proteasome)，细胞核(Nucleus)。现有结果显示，纤溶酶原可进入细胞内与细胞内的蛋白降解系统—泛素蛋白酶体系统(ubiquitin proteasome system，UPS)和自噬溶酶体系统相互作用。纤溶酶原由此可促进细胞内异常沉积中枢神经系统病理性蛋白降解，改善中枢神经系统退行性疾病。
图2纤溶酶原在肌萎缩侧索硬化症模型小鼠脊髓组织内定位情况以及纤溶酶原与泛素的共定位情况。结果显示，给药组纤溶酶原(绿色荧光)在脊髓组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入脊髓组织，并在脊髓组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与泛素(红色荧光)在胞质内共定位(如Δ所示)(图2)，且给药组脊髓组织泛素表达水平似乎少于溶媒组。说明在肌萎缩侧索硬化症模型小 鼠中纤溶酶原能够进入脊髓组织，进入细胞内，与泛素共定位，且降低泛素的表达。提示纤溶酶原可能与泛素发生相互作用。
图3纤溶酶原在肌萎缩侧索硬化症模型小鼠脊髓组织内定位情况以及纤溶酶原与LC3B的共定位情况。结果显示，给药组纤溶酶原(绿色荧光)在脊髓组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入脊髓组织，并在脊髓组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LC3B(红色荧光)在胞质内共定位(如Δ所示)，且给药组脊髓组织LC3B表达水平似乎少于溶媒组。说明在肌萎缩侧索硬化症模型小鼠中纤溶酶原能够进入脊髓组织，进入细胞内，与LC3B共定位，提示纤溶酶原与LC3B之间存在相互作用。
图4纤溶酶原在肌萎缩侧索硬化症模型小鼠脊髓组织内定位情况以及纤溶酶原与LAMP2的共定位情况。结果显示，给药组纤溶酶原(绿色荧光)在脊髓组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入脊髓组织，并在脊髓组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LAMP2(红色荧光)在胞质内共定位(如Δ所示)，且给药组脊髓组织LAMP2表达水平似乎多于溶媒组，进入细胞内，与LAMP2共定位，且促进LAMP2的表达，促进溶酶体作用。
图5给予纤溶酶原促进肌萎缩侧索硬化症模型小鼠脊髓组织LC3的表达。结果显示，给药组LC3的表达显著高于溶媒组，且统计差异显著(*表示P<0.05)。表明纤溶酶原能够促进肌萎缩硬化症模型小鼠LC3基因的转录，从而参与自噬过程的调控。
图6纤溶酶原在帕金森模型小鼠黑质和纹状体组织中的定为情况及纤溶酶原与泛素的共定位情况。结果显示，给药组纤溶酶原(绿色荧光)在黑质和纹状体组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入黑质和纹状体组织，并在黑质和纹状体组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与泛素(红色荧光)在胞质内共定位(如Δ所示)。说明在帕金森模型小鼠中纤溶酶原能够进入黑质和纹状体组织，进入细胞内，与泛素共定位。提示纤溶酶原可能与泛素发生相互作用。
图7纤溶酶原在帕金森模型小鼠黑质和纹状体组织中的定为情况及纤溶酶原与LC3B的共定位情况。结果显示，给药组纤溶酶原(绿色荧光)在黑质和纹状体组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进 入黑质和纹状体组织，并在黑质和纹状体组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LC3B(红色荧光)在胞质内共定位(如Δ所示)。说明在帕金森模型小鼠中纤溶酶原能够进入黑质和纹状体组织，进入细胞内，与LC3B共定位。提示纤溶酶原可能与LC3发生相互作用。
图8纤溶酶原在帕金森模型小鼠黑质和纹状体组织中的定为情况及纤溶酶原与LAMP2的共定位情况。结果显示，给药组纤溶酶原(绿色荧光)在黑质和纹状体组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入黑质和纹状体组织，并在黑质和纹状体组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LAMP2(红色荧光)在胞质内共定位(如所示)。说明在帕金森模型小鼠中纤溶酶原能够进入黑质和纹状体组织，进入细胞内，与LAMP2共定位。提示纤溶酶原可能与LAMP2发生相互作用。
图9纤溶酶原在阿尔兹海默模型小鼠海马组织中的定位情况及纤溶酶原与泛素的共定位情况。结果显示，给药组纤溶酶原(绿色荧光)在海马组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入海马组织，并在海马组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与泛素(红色荧光)在胞质内共定位(如Δ所示)。说明在阿尔兹海默模型小鼠中纤溶酶原能够进入海马组织，进入细胞内。提示纤溶酶原可能与泛素发生相互作用。
图10纤溶酶原在阿尔兹海默模型小鼠海马组织中的定位情况及纤溶酶原与LC3B的共定位情况。结果显示，给药组纤溶酶原(绿色荧光)在海马组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入海马组织，并在海马组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LC3B(红色荧光)在胞质内共定位(如Δ所示)。说明在阿尔兹海默模型小鼠中纤溶酶原能够进入海马组织，进入细胞内。提示纤溶酶原可能与LC3B发生相互作用。
图11纤溶酶原在阿尔兹海默模型小鼠海马组织中的定位情况及纤溶酶原与LAMP2的共定位情况。结果显示，给药组纤溶酶原(绿色荧光)在海马组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入海马组织，并在海马组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶 原与LAMP2(红色荧光)在胞质内共定位(如Δ所示)。说明在阿尔兹海默模型小鼠中纤溶酶原能够进入海马组织，进入细胞内。提示纤溶酶原可能与LAMP2发生相互作用。
图12A-B给予纤溶酶原28天后亨廷顿模型小鼠心脏组织中亨廷顿蛋白western blot定量分析结果。A为Western blot图片，B为亨廷顿蛋白条带光密度定量分析结果。结果显示，溶媒组小鼠心脏HTT水平明显高于正常对照组小鼠，给药组小鼠心脏HTT水平明显低于溶媒组小鼠，且统计差异显著(*代表P<0.05)。提示纤溶酶原能够促进亨廷顿模型小鼠心脏组织中亨廷顿蛋白降解。
图13给予纤溶酶原于冈田酸处理NSC34细胞浆(A-B)和细胞核(C-D)中TDP-43水平WB检测结果。A和C为Western blot图片，C和D为TDP-43条带定量分析结果。结果显示，给药组细胞浆和细胞核中TDP-43的水平显著低于溶媒组细胞核，而加入EACA能够完全的抑制纤溶酶原对TDP-43的效应。*代表P<0.05，**代表P<0.01，***代表P<0.001。提示纤溶酶原能够促进细胞浆和细胞核中TDP-43降解，而纤溶酶原的这种作用与其结构上的赖氨酸结合位点密切相关。
图14给予纤溶酶原于冈田酸处理NSC34细胞中磷酸化Tau蛋白(A-B)、总Tau蛋白和磷酸化Tau蛋白与总Tau蛋白比值。A和C为Western blot图片，B、D和E分别为磷酸化Tau蛋白、总Tau蛋白和磷酸化Tau蛋白与总Tau蛋白比值结果。结果结果显示，给药组磷酸化Tau蛋白的水平显著低于溶媒组，而加入EACA能够完全的抑制纤溶酶原对磷酸化Tau蛋白的效应；给药组总Tau蛋白水平与溶媒组未见显著差异；给药组磷酸化Tau蛋白与总Tau蛋白的比值显著低于溶媒组，而加入EACA能够完全的抑制纤溶酶原的这种效应。*代表P<0.05，***代表P<0.001。提示纤溶酶原能够促进细胞中磷酸化Tau蛋白降解，而纤溶酶原的这种作用与其结构上的赖氨酸结合位点密切相关。
图15A-D给予纤溶酶原冈田酸处理NSC34细胞浆和细胞核中纤溶酶原和纤溶酶活性水平检测结果。A为细胞浆纤溶酶原水平ELISA法检测结果，B为细胞核中纤溶酶原水平ELISA法检测结果，C为细胞浆纤溶酶活性水平酶底物动力学法检测结果，D为细胞核纤溶酶活性水平酶底物动力学法检测结果。结果显示，给药组细胞浆和细胞核中人纤溶酶原水平和纤 溶酶活性水平显著高于溶媒组，且统计差异显著；加入EACA能够完全的抑制纤溶酶原这些的效应(**代表P<0.01，***代表P<0.001)。提示纤溶酶原能够进入细胞，甚至细胞核中，增加纤溶酶活性，而纤溶酶原进入细胞和细胞核与其赖氨酸结合活性密切相关。
图16A-B给予纤溶酶原28天后亨廷顿模型小鼠肾脏组织中亨廷顿蛋白western blot定量分析结果。A为Western blot图片，B为亨廷顿蛋白条带光密度定量分析结果。结果显示，溶媒组小鼠肾脏HTT水平明显高于正常对照组小鼠，给药组小鼠肾脏HTT水平明显低于溶媒组小鼠(图16A-B)，且统计差异显著(*代表P<0.05，***代表P<0.001)。提示纤溶酶原能够促进亨廷顿模型小鼠肾脏组织中亨廷顿蛋白降解。
图17A-B给予纤溶酶原2天后体外培养的亨廷顿模型小鼠脑组织中亨廷顿蛋白western blot定量分析结果。A为Western blot图片，B为亨廷顿蛋白条带光密度定量分析结果。结果显示，给药组小鼠的脑组织HTT水平明显低于溶媒组小鼠，且统计差异显著(*代表P<0.05)。提示纤溶酶原能够促进亨廷顿模型小鼠脑组织中亨廷顿蛋白降解。
发明详述
纤维蛋白溶解系统(Fibrinolytic system)也称纤溶系统，为参与纤维蛋白溶解(纤溶)过程的一系列化学物质组成的系统，主要包括纤维蛋白溶解酶原(纤溶酶原)、纤溶酶、纤溶酶原激活物、纤溶抑制剂。纤溶酶原激活物包括组织型纤溶酶原激活物(t-PA)和尿激酶型纤溶酶原激活物(u-PA)。t-PA是一种丝氨酸蛋白酶，由血管内皮细胞合成。t-PA激活纤溶酶原，此过程主要在纤维蛋白上进行；尿激酶型纤溶酶原激活物(u-PA)由肾小管上皮细胞和血管内皮细胞产生，可以直接激活纤溶酶原而不需要纤维蛋白作为辅因子。纤溶酶原(PLG)由肝脏合成，当血液凝固时，PLG 大量吸附在纤维蛋白网上，在t-PA或u-PA的作用下，被激活为纤溶酶，促使纤维蛋白溶解。纤溶酶(PL)是一种丝氨酸蛋白酶，作用如下：降解纤维蛋白和纤维蛋白原；水解多种凝血因子Ⅴ、Ⅷ、Ⅹ、Ⅶ、Ⅺ、Ⅱ等；使纤溶酶原转变为纤溶酶；水解补体等。纤溶抑制物：包括纤溶酶原激活物抑制剂(PAI)和α2抗纤溶酶(α2-AP)。PAI主要有PAI-1和PAI-2两种形式，能特异性与t-PA以1：1比例结合，从而使其失活，同时激活PLG。α2-AP由肝脏合成，与PL以1：1比例结合形成复合物，抑制PL活性；FⅩⅢ使α2-AP以共价键与纤维蛋白结合，减弱了纤维蛋白对PL作用的敏感性。体内抑制纤溶系统活性的物质：PAI-1，补体C1抑制物；α2抗纤溶酶；α2巨球蛋白。
本发明“纤维蛋白溶酶原途径激活剂”或“纤溶酶原途径激活剂”术语涵盖纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
本发明的术语“纤维蛋白溶酶原激活途径的组分”或“纤溶酶原激活途径的组分”涵盖：
1.纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、微纤溶酶原(micro-plasminogen)、delta-纤溶酶原；它们的变体或类似物；
2.纤维蛋白溶酶以及它们的变体或类似物；和
3.纤维蛋白溶酶原激活剂，例如tPA和uPA以及包含一个或多个tPA或uPA的结构域(如一个或多个kringle结构域和蛋白水解结构域)的tPA或uPA变体和类似物。
所述的“纤溶抑制剂的拮抗剂”术语涵盖PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的拮抗剂，例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抗体。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式，以及通过添加、删除和/或取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸、仍然具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA活性的蛋白 质。例如，纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”包括通过例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个保守性氨基酸取代获得的这些蛋白质的突变变体。
本发明的“纤溶酶原变体”涵盖包含或具有与序列2、6、8、10或12所示氨基酸序列具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且具有纤溶酶原活性和/或赖氨酸结合活性的蛋白质。例如本发明的“纤溶酶原变体”可以是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性和/或赖氨酸结合活性的蛋白质。具体地，本发明纤溶酶原变体包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式，以及通过保守性氨基酸取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸获得的这些蛋白质的突变变体。
本发明的纤溶酶原可以为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性和/或赖氨酸结合活性的变体，例如序列2、6、8、10或12所示的纤溶酶原，例如序列2所示的人天然纤溶酶原。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“类似物”包括分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用的化合物。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和“类似物”涵盖包含一个或多个结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和“类似物”。例如，纤维蛋白溶酶原的“变体”和“类似物”涵盖包含一个或多个纤溶酶原结构域(例如一个或多个kringle(k)结构域和蛋白水解结构域(或称丝氨酸蛋白酶结构域，或称纤溶酶原蛋白酶结构域)的纤维蛋白溶酶原变体和类似物，例如小纤维蛋白溶酶原(mini-plasminogen)。纤维蛋白溶酶的“变体”和“类似物”涵盖包含一个或多个纤维蛋白溶酶结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶“变体”和“类似物”，例如小纤维蛋白溶酶(mini-plasmin)和δ-纤维蛋白溶酶(delta-plasmin)。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的“变体”或“类似物”是否分别具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的活性，或者是否分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用可以通过本领域已知方法进行检测，例如，通过基于酶谱法(enzymography)、ELISA(酶联免疫吸附测定)和FACS(荧光激活细胞分选方法)通过激活的纤维蛋白溶酶活性水平来衡量，例如可以参照选自如下文献中记载的方法测量：Ny，A.，Leonardsson，G.，Hagglund，A.C，Hagglof，P.，Ploplis，V.A.，Carmeliet，P.and Ny，T.(1999).Ovulation inplasminogen-deficient mice.Endocrinology 140，5030-5035；Silverstein RL,Leung LL,Harpel PC,Nachman RL(November 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(February2008)."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.
在本发明的一些实施方案中，本发明的“纤维蛋白溶酶原激活途径的组分”为纤溶酶原，选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性和/或赖氨酸结合活性的保守突变变体或其片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性和/或赖氨酸结合活性的保守突变变体或其片段。在一些实施方案中，所述纤溶酶原的氨基酸序列包含或具有如序列2、6、8、10或12所示的氨基酸序列。在一些实施方案中，所述纤溶酶原是人全长纤溶酶原。在一些实施方案中，所述纤溶酶原是如序列2所示的人全长纤溶酶原。
“能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物”指能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶 原的任何化合物，例如tPA、uPA、链激酶、沙芦普酶、阿替普酶、瑞替普酶、替奈普酶、阿尼普酶、孟替普酶、拉诺替普酶、帕米普酶、葡激酶。
本发明“纤溶抑制剂的拮抗剂”为拮抗、减弱、封闭、阻止纤溶抑制剂作用的化合物。所述纤溶抑制剂例如PAI-1、补体C1抑制物、α2抗纤溶酶和α2巨球蛋白。所述拮抗剂例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抗体，或阻断或下调例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白表达的反义RNA或小RNA，或占据PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的结合位点但无PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白功能的化合物”，或封闭PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的结合结构域和/或活性结构域的化合物。
纤溶酶是纤溶酶原激活系统(PA系统)的关键组分。它是一种广谱的蛋白酶，能够水解细胞外基质(ECM)的几个组分，包括纤维蛋白、明胶、纤连蛋白、层粘连蛋白和蛋白聚糖。此外，纤溶酶能将一些金属蛋白酶前体(pro-MMPs)激活形成具有活性的金属蛋白酶(MMPs)。因此纤溶酶被认为是胞外蛋白水解作用的一个重要的上游调节物。纤溶酶是由纤溶酶原通过两种生理性的PAs：组织型纤溶酶原激活剂(tPA)或尿激酶型纤溶酶原激活剂(uPA)蛋白水解形成的。由于纤溶酶原在血浆和其他体液中相对水平较高，传统上认为PA系统的调节主要通过PAs的合成和活性水平实现。PA系统组分的合成受不同因素严格调节，如激素、生长因子和细胞因子。此外，还存在纤溶酶和PAs的特定生理抑制剂。纤溶酶的主要抑制剂是α2-抗纤溶酶(α2-antiplasmin)。PAs的活性同时被uPA和tPA的纤溶酶原激活剂抑制剂-1(PAI-1)抑制以及主要抑制uPA的溶酶原激活剂抑制剂-2(PAI-2)调节。某些细胞表面具有直接水解活性的uPA特异性细胞表面受体(uPAR)。
纤溶酶原是一个单链糖蛋白，由791个氨基酸组成，分子量约为92kDa。纤溶酶原主要在肝脏合成，大量存在于胞外液中。血浆中纤溶酶原含量约为2μM。因此纤溶酶原是组织和体液中蛋白质水解活性的一个巨大的潜在来源。纤溶酶原存在两种分子形式：谷氨酸-纤溶酶原(Glu-plasminogen)和赖氨酸-纤溶酶原(Lys-plasminogen)。天然分泌和未裂解形式的纤溶酶原具有一个氨基末端(N-末端)谷氨酸，因此被称为谷氨酸-纤溶酶原。然而，在纤溶酶存在时，谷氨酸-纤溶酶原在Lys76-Lys77处水解成为赖氨酸-纤溶酶原。与谷氨酸-纤溶酶原相比，赖氨酸-纤溶酶原与纤维 蛋白具有更高的亲和力，并可以更高的速率被PAs激活。这两种形式的纤溶酶原的Arg560-Val561肽键可被uPA或tPA切割，导致二硫键连接的双链蛋白酶纤溶酶的形成。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringles，羧基末端部分包含蛋白酶结构域。一些kringles含有介导纤溶酶原与纤维蛋白及其抑制剂α2-AP特异性相互作用的赖氨酸结合位点。最新发现一个纤溶酶原为38kDa的片段，其中包括kringles1-4，是血管生成的有效抑制剂。这个片段被命名为血管抑素，可通过几个蛋白酶水解纤溶酶原产生。
纤溶酶的主要底物是纤维蛋白，纤维蛋白的溶解是预防病理性血栓形成的关键。纤溶酶还具有对ECM几个组分的底物特异性，包括层粘连蛋白、纤连蛋白、蛋白聚糖和明胶，表明纤溶酶在ECM重建中也起着重要作用。间接地，纤溶酶还可以通过转化某些蛋白酶前体为活性蛋白酶来降解ECM的其他组分，包括MMP-1，MMP-2，MMP-3和MMP-9。因此，有人提出，纤溶酶可能是细胞外蛋白水解的一个重要的上游调节器。此外，纤溶酶具有激活某些潜在形式的生长因子的能力。在体外，纤溶酶还能水解补体系统的组分并释放趋化补体片段。
“纤溶酶”是存在于血液中的一种非常重要的酶，能将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体。
“纤溶酶原”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源纤溶酶原氨基酸序列(序列4)计算由810个氨基酸组成，分子量约为90kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的纤溶酶原包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基Cys103-Cys181，Kringle2包括残基Glu184-Cys262，Kringle3包括残基Cys275-Cys352，Kringle4包括残基Cys377-Cys454，Kringle5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位 氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。Delta-纤溶酶原(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶(结构)域(也可称为蛋白水解结构域，或称纤溶酶原蛋白酶结构域)，有文献报道了delta-纤溶酶原的氨基酸序列(序列8)，编码该氨基酸序列的cDNA序列如序列7。小纤溶酶原(Mini-plasminogen)由Kringle5和丝氨酸蛋白酶域组成，有文献报道其包括残基Val443-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，其氨基酸序列如序列10所示，编码该氨基酸序列的cDNA序列如序列9所示。而微纤溶酶原(Micro-plasminogen)仅含有丝氨酸蛋白酶结构域，有文献报道其氨基酸序列包括残基Ala543-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，也有专利文献CN102154253A报道其序列包括残基Lys531-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，本专利序列参考专利文献CN102154253A，其氨基酸序列如序列12所示，编码该氨基酸序列的cDNA序列如序列11所示。
本发明的“纤溶酶”与“纤维蛋白溶酶”、“纤维蛋白溶解酶”可互换使用，含义相同；“纤溶酶原”与“纤维蛋白溶酶”、“纤维蛋白溶解酶原”可互换使用，含义相同。
在本申请中，所述纤溶酶原“缺乏”的含义或活性为受试者体内纤溶酶原的含量比正常人低，低至足以影响所述受试者的正常生理功能；所述纤溶酶原“缺失”的含义或活性为受试者体内纤溶酶原的含量显著低于正常人，甚至活性或表达极微，只有通过外源提供才能维持正常生理功能。
本领域技术人员可以理解，本发明纤溶酶原的所有技术方案适用于纤溶酶，因此，本发明描述的技术方案涵盖了纤溶酶原和纤溶酶。在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时，在纤溶酶原激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性纤溶酶。具有活性的纤溶酶可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中纤溶酶原的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为纤溶酶原激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”在本申请中包括1)在纤溶酶原蛋白中，能够与底物中的靶序列结合的活性片段，也称为赖氨酸结合片段，例如包含Kringle 1、Kringle 2、Kringle 3、Kringle 4和/或Kringle 5的片段(所述纤溶酶原结构参见Aisina R B,Mukhametova L I.Structure and function of plasminogen/plasmin system[J].Russian Journal of Bioorganic Chemistry,2014,40(6):590-605所述)；2)在纤溶酶原蛋白中发挥蛋白水解功能的活性片段，例如包含序列14所示的纤溶酶原活性(蛋白水解功能)的片段；3)在纤溶酶原蛋白中，既具有与底物中的靶序列结合活性(赖氨酸结合活性)又具有纤溶酶原活性(蛋白水解功能)的片段。在本申请的一些实施方案中，所述纤溶酶原为包含序列14所示的纤溶酶原活性片段的蛋白质。在本申请的一些实施方案中，所述纤溶酶原为包含Kringle 1、Kringle 2、Kringle 3、Kringle 4和/或Kringle 5的赖氨酸结合片段的蛋白质。在一些实施方案中，本申请的纤溶酶原活性片段包含序列14、与序列14具有至少80％、90％、95％、96％、97％、98％、99％同源性的氨基酸序列的蛋白质。因此，本发明所述的纤溶酶原包括含有该纤溶酶原活性片段、并且仍然保持该纤溶酶原活性的蛋白。在一些实施方案中，本申请的纤溶酶原包含Kringle 1、Kringle 2、Kringle 3、Kringle 4和/或Kringle 5、或与Kringle 1、Kringle 2、Kringle 3、Kringle 4或Kringle 5具有至少80％、90％、95％、96％、97％、98％、99％同源性并且仍然具有赖氨酸结合活性的蛋白质。
目前，对于血液中纤溶酶原及其活性测定方法包括：对组织纤溶酶原激活剂活性的检测(t-PAA)、血浆组织纤溶酶原激活剂抗原的检测(t-PAAg)、对血浆组织纤溶酶原活性的检测(plgA)、血浆组织纤溶酶原抗原的检测(plgAg)、血浆组织纤溶酶原激活剂抑制物活性的检测、血浆组织纤溶酶原激活剂抑制物抗原的检测、血浆纤维蛋白溶酶-抗纤维蛋白溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤溶酶原活性成正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其 具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤溶酶原包括人的天然纤溶酶原，还包括来源于不同物种的、具有纤溶酶原活性的纤溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性，碱性，疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸，蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤溶酶原是指从其天然环境分离和/或回收的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原会纯化(1)至大于90％、大于95％、或大于98％的纯度(按重量计)，如通过Lowry法所确定的，例如超过99％(按重量计)，(2)至足以通过使用旋转杯序列分析仪获得N端或内部氨基酸序列的至少15个残基的程度，或(3)至同质性，该同质性是通过使用考马斯蓝或银染在还原性或非还原性条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定的。分离的纤溶酶原也包括通过生物工程技术从重组细胞制备，并通过至少一个纯化步骤分离的纤溶酶原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指任何长度的氨基酸的聚合形式，其可以包括遗传编码的和非遗传编码的氨基酸，化学或生物化学修饰的或衍生化的氨基酸，和具有经修饰的肽主链的多肽。该术语包括融合蛋白，包括但不限于具有异源氨基酸序列的融合蛋白，具有异源和同源前导序列(具有或没有N端甲硫氨酸残基)的融合物；等等。
关于参照多肽序列的“氨基酸序列同一性百分数(％)”定义为在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式实现，例如使用公众可得到的计算机软件，诸 如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员能决定用于比对序列的适宜参数，包括对所比较序列全长实现最大对比需要的任何算法。然而，为了本发明的目的，氨基酸序列同一性百分数值是使用序列比较计算机程序ALIGN-2产生的。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：
分数X/Y乘100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基的数目，且其中Y是B中的氨基酸残基的总数。应当领会，在氨基酸序列A的长度与氨基酸序列B的长度不相等的情况下，A相对于B的％氨基酸序列同一性会不等于B相对于A的％氨基酸序列同一性。除非另有明确说明，本文中使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
术语“个体”、“受试者”和“患者”在本文中可互换使用，指哺乳动物，包括但不限于鼠(大鼠、小鼠)、非人灵长类、人、犬、猫、有蹄动物(例如马、牛、绵羊、猪、山羊)等。
“治疗有效量”或“有效量”指在对哺乳动物或其它受试者施用以治疗疾病时足以实现对疾病的所述预防和/或治疗的纤溶酶原的量。“治疗有效量”会根据所使用的纤溶酶原、要治疗的受试者的疾病和/或其症状的严重程度以及年龄、体重等而变化。
术语疾病状态的“治疗”包括抑制或阻止所述疾病状态或其临床症状的发展，或减轻所述疾病状态或症状，使得所述疾病状态或其临床症状暂时或永久性的退去。
本发明纤溶酶原的制备
纤溶酶原可以从自然界分离并纯化用于进一步的治疗用途，也可以通过标准的化学肽合成技术来合成。当通过化学合成多肽时，可以经液相或固相进行合成。固相多肽合成(SPPS)(其中将序列的C末端氨基酸附接于不溶性支持物，接着序贯添加序列中剩余的氨基酸)是适合纤溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤溶酶原。用于 固相合成的技术描述于Barany和Solid-Phase Peptide Synthesis；第3-284页于The Peptides:Analysis,Synthesis,Biology.第2卷：Special Methods in Peptide Synthesis,Part A.,Merrifield,等J.Am.Chem.Soc.,85:2149-2156(1963)；Stewart等,Solid Phase Peptide Synthesis,2nd ed.Pierce Chem.Co.,Rockford,Ill.(1984)；和Ganesan A.2006Mini Rev.Med Chem.6:3-10和Camarero JA等2005Protein Pept Lett.12:723-8中。简言之，用其上构建有肽链的功能性单元处理小的不溶性多孔珠。在偶联/去保护的重复循环后，将附接的固相游离N末端胺与单个受N保护的氨基酸单元偶联。然后，将此单元去保护，露出可以与别的氨基酸附接的新的N末端胺。肽保持固定在固相上，之后将其切掉。
可以使用标准重组方法来生产本发明的纤溶酶原。例如，将编码纤溶酶原的核酸插入表达载体中，使其与表达载体中的调控序列可操作连接。表达调控序列包括但不限于启动子(例如天然关联的或异源的启动子)、信号序列、增强子元件、和转录终止序列。表达调控可以是载体中的真核启动子系统，所述载体能够转化或转染真核宿主细胞(例如COS或CHO细胞)。一旦将载体掺入合适的宿主中，在适合于核苷酸序列的高水平表达及纤溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆纤溶酶原编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(enterobacteriaceae)，诸如沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物种。在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子系统，色氨酸(trp)启动子系统，beta-内酰胺酶启动子系统，或来自噬菌体λ的启动子系统。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
在微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达并生成本发明的纤溶酶原(例如编码纤溶酶原的多核苷酸)。参见Winnacker,From Genes to Clones,VCH Publishers,N.Y.,N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起点，启动子和增强子(Queen等,Immunol.Rev.89:49(1986))，以及必需的加工信息位点，诸如核糖体结合位点，RNA剪接位点，多聚腺苷酸化位点，和转录终止子序列。合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛乳头瘤病毒、巨细胞病毒等衍生的启动子。参见Co等,J.Immunol.148:1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀，亲和柱，柱层析，高效液相层析(HPLC)，凝胶电泳等来纯化本发明所述的纤溶酶原。该纤溶酶原是基本上纯的，例如至少约80％至85％纯的，至少约85％至90％纯的，至少约90％至95％纯的，或98％至99％纯的或更纯的，例如不含污染物，所述污染物如细胞碎片，除纤溶酶原以外的大分子，等等。
药物配制剂
可以通过将具有所需纯度的纤溶酶原与可选的药用载体，赋形剂，或稳定剂(Remington's Pharmaceutical Sciences,16版，Osol,A.ed.(1980))混合形成冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽 (少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。例如，抗高血压的药物，抗心律失常的药物，治疗糖尿病的药物等。
本发明的纤溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送系统(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980)。
用于体内给药的本发明的纤溶酶原必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤溶酶原可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J.Biomed.Mater.Res.,15:167-277(1981)；Langer,Chem.Tech.,12:98-105(1982))或聚(乙烯醇)，聚交酯(美国专利3773919,EP 58,481)，L-谷氨酸与γ乙基-L-谷氨酸的共聚物(Sidman，等，Biopolymers 22:547(1983))，不可降解的乙烯－乙烯乙酸酯(ethylene-vinyl acetate)(Langer，等，出处同上)，或可降解的乳酸-羟基乙酸共聚物如Lupron DepotTM(由乳酸-羟基乙酸共聚物和亮氨酰脯氨酸(leuprolide)乙酸酯组成的可注射的微球体)，以及聚D-(-)-3-羟丁酸。聚合物如乙烯-乙酸乙烯酯和乳酸-羟基乙酸能持续释放分子100天以上，而一些水凝胶释放蛋白的时间却较短。可以根据相关机理来设计使蛋白稳定的合理策略。例如，如果发现凝聚的机理是通过硫代二硫键互换而形成分子间S-S键，则可通过修饰巯基残基、从酸性溶液中冻干、控制湿度、采用合适的添加剂、和开发特定的聚合物基质组合物来实现稳定。
给药和剂量
可以通过不同方式，例如通过静脉内，腹膜内，皮下，颅内，鞘内，动脉内(例如经由颈动脉)，肌内来实现本发明药物组合物的施用。
用于胃肠外施用的制备物包括无菌水性或非水性溶液、悬浮液和乳剂。非水性溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油，和可注射有机酯，如油酸乙酯。水性载体包括水、醇性/水性溶液、乳剂或悬浮液，包括盐水和缓冲介质。胃肠外媒介物包含氯化钠溶液、林格氏右旋糖、右旋糖和氯化钠、或固定油。静脉内媒介物包含液体和营养补充物、电解质补充物，等等。也可以存在防腐剂和其他添加剂，诸如例如，抗微生物剂、抗氧化剂、螯合剂、和惰性气体，等等。
医务人员会基于各种临床因素确定剂量方案。如医学领域中公知的，任一患者的剂量取决于多种因素，包括患者的体型、体表面积、年龄、要施用的具体化合物、性别、施用次数和路径、总体健康、和同时施用的其它药物。本发明包含纤溶酶原的药物组合物的剂量范围可以为每天约0.0001至2000mg/kg，或约0.001至500mg/kg(例如0.02mg/kg，0.25mg/kg，0.5mg/kg，0.75mg/kg，10mg/kg，50mg/kg等等)受试者体重。例如，剂量可以是1mg/kg体重或50mg/kg体重或在1-50mg/kg的范围，或至少1mg/kg。高于或低于此例示性范围的剂量也涵盖在内，特别是考虑到上述的因素。上述范围中的中间剂量也包含在本发明的范围内。受试者可以每天、隔天、每周或根据通过经验分析确定的任何其它日程表施用此类剂量。例示性的剂量日程表包括连续几天1-10mg/kg。在本发明的药物施用过程中需要实时评估治疗效果和安全性。
制品或药盒
本发明的一个实施方案涉及一种制品或药盒，其包含可用于治疗由糖尿病引起的心血管病及其相关病症的本发明纤溶酶原或纤溶酶。所述制品优选包括一个容器，标签或包装插页。适当的容器有瓶子，小瓶，注射器等。容器可由各种材料如玻璃或塑料制成。所述容器含有组合物，所述组合物可有效治疗本发明的疾病或病症并具有无菌入口(例如所述容器可为静脉内溶液包或小瓶，其含有可被皮下注射针穿透的塞子的)。所述组合物中至少一种活性剂为纤溶酶原/纤溶酶。所述容器上或所附的标签说明所述组合物用于治疗本发明所述由糖尿病引起的心血管病及其相关病症。所述制品可进一步包含含有可药用缓冲液的第二容器，诸如磷酸盐缓冲的盐水，林格氏溶液以及葡萄糖溶液。其可进一步包含从商业和使用者角度来看所 需的其它物质，包括其它缓冲液，稀释剂，过滤物，针和注射器。此外，所述制品包含带有使用说明的包装插页，包括例如指示所述组合物的使用者将纤溶酶原组合物以及治疗伴随的疾病的其它药物给药患者。
在一些实施方案中，上述由于病理性蛋白聚集导致的疾病包括：阿尔茨海默病、帕金森病、肌萎缩侧索硬化、朊病毒样病(克雅氏病)、格斯特曼综合征、发散型或家族型致死性失眠症和库鲁病、亨廷顿病、脊髓小脑性共济失调3型、齿状核红核苍白球路易氏体萎缩症。
在一些实施方案中，上述由于由于泛素/溶酶体功能障碍导致的疾病包括：范科尼贫血(Fanconi anemia)、着色性干皮病(Xeroderma pigmentosum)、科凯恩综合征(Cockayne syndrome)、癌症、多发性错构瘤综合征(Cowden syndrome)、帕金森病、基因组失稳(Genomic instability)、代谢综合征、肌肉萎缩、希佩尔-林道综合征(Von Hippel Lindau)、多发性骨髓瘤、遗传性假性醛固酮增多症(RIDDLE syndrome)、亨廷顿病、x连锁淋巴增生性疾病、克罗恩病(Crohn’s disease)、阿尔兹海默症(Alzheimer’s disease)、乳腺癌和卵巢癌、肌肉支链淀粉病(muscular amylopectinosis)、肌萎缩侧索硬化症(Amyotrophic lateral sclerosis)、脊髓性肌萎缩症、系统性红斑狼疮(Systemic lupus erythematosus)、儿童期共济失调、x-连锁帕金森综合征伴痉挛、多系统紊乱、糖尿病、多发性硬化症，胱氨酸病(Cystinosis)、维西综合症(Vici syndrome)、高雪氏病(Gaucher's disease)、额颞叶痴呆(杂合子)或神经元蜡样脂褐质沉积症(纯合子)、Danon’s心肌病、皮质萎缩、癫痫、常染色体隐性脊髓小脑共济失调、遗传痉挛性截瘫、螺旋蛋白相关性神经变性病(Beta-propeller protein-associated neurodegeneration,BPAN)、自闭症谱系障碍、额颞叶痴呆(frontotemporal dementia,FTD)、炎性肠病、非酒精性脂肪性肝病、肺结核、Rett综合征、骨硬化症、2B型Charcot-Marie-Tooth病(Charcot-Marie-Tooth type 2B disease)、幼年关节炎、斯奈德-罗宾逊综合征(Snyder-Robinson syndrome,SRS)、维斯科特-奥尔德里奇综合征(Wiskott-Aldrich syndrome)、原发性小头畸形(Primary microcephaly)、II型传性感觉和自主神经病变、原发性开角型青光眼(Primary open angle glaucoma,POAG)、佩吉特骨病(Paget’s disease of the bone，PGD)、结肠癌、肺癌、脑癌、常染色体隐性遗传和散发性早发帕金森病、泽尔维格综合征谱系障碍(Zellweger syndrome spectrum disorders)、远端肌病、溃 疡性结肠炎、家族性地中海热、共济失调伴痉挛、法布里病、戈谢病、溶酶体酸性脂肪酶缺乏症、黏多糖贮积症、天使综合征。
实施例
以下所有实施例中使用的人纤溶酶原来自捐赠者血浆，基于文献(KennethC 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.1976Jun25；251(12):3693-9；HAGAN JJ,ABLONDI FB,DE RENZO EC.Purification and biochemical properties of human plasminogen.J Biol Chem.1960Apr；235:1005-10)[1-3]所描述的方法并进行工艺优化，从人捐赠者血浆纯化获得，其中人Lys-纤维蛋白溶酶原(Lys-纤溶酶原)和Glu-纤维蛋白溶酶原(Glu-纤溶酶原)>98％。
实施例
实施例1纤溶酶原在ALS模型小鼠脊髓组织中富集且与泛素共定位
转基因突变SOD1具有在散发性和家族性肌萎缩性侧索硬化症(Amyotrophic Lateral Sclerosis,ALS)临床中观察到的组织病理学特征。本案ALS模型小鼠为B6.Cg-Tg(SOD1-G93A)1Gur/J转基因小鼠(简称SOD1-G93A)，购自Jackson实验室，谱系号：004435，在SPF级环境下进行动物相关试验。SOD1-G93A模型小鼠目前已被广泛应用于ALS的机制研究及新药研发的临床前试验研究当中。
取周龄相近野生型雄性小鼠5只和雄性SOD1-G93A小鼠9只。野生型小鼠作为空白对照组，SOD1-G93A小鼠从第14周发病后腿颤抖时开始观察记录，记录每只小鼠发病时间，发病14天后开始给药，所有小鼠根据发病情况随机分成溶媒组和给药组，其中溶媒组小鼠5只，每天尾静脉注射0.1ml/只溶媒(柠檬酸钠缓冲液)；给药组4只，每天尾静脉注射1mg/0.1ml/只纤溶酶原，SPF环境下连续给药，濒死取材，最长给药61天。取材脊髓组织于福尔马林固定液中固定。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。将切片浸入 抗原修复工作液(0.01M柠檬酸钠缓冲液)中微波修复，预热5分钟，高火2分钟，低火15分钟。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories,Inc.,USA)封闭30分钟；时间到后，弃除羊血清液，滴加抗纤溶酶原抗体(自产)，4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按照XTSA520IHC试剂盒(Alpha X Biotech,AXT6202500)说明书操作进行对应的抗纤溶酶原二抗的绿色荧光显色。PBS洗3次，每次5分钟。重复上述的抗原修复和封闭操作，然后进行抗泛素(ubiquitin)抗体(Abcam，ab7780)染色，37℃孵育1小时。PBS洗3次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按照XTSA620IHC试剂盒(Alpha X Biotech,AXT6502500)说明书操作进行对应的抗泛素二抗的红色荧光显色。PBS洗3次，每次5分钟。进行DAPI(BOSTER，11K16B77)核染色。PBS洗3次，每次5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察拍照。
泛素(ubiquitin，Ub)是一类分子量约为8500且高度保守的小分子量蛋白质。它由76个氨基酸组成，并广泛存在于真核生物细胞中。泛素的主要功能是参与真核细胞中大部分蛋白质的降解，而蛋白酶体就是泛素介导蛋白质降解的场所。泛素-蛋白酶体通路是“消化”细胞内蛋白质的重要途径之一。
结果显示，给药组纤溶酶原(绿色荧光)在脊髓组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入脊髓组织，并在脊髓组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与泛素(红色荧光)在胞质内共定位(如Δ所示)(图2)，且给药组脊髓组织泛素表达水平似乎少于溶媒组。说明在肌萎缩侧索硬化症模型小鼠中纤溶酶原能够进入脊髓组织，进入细胞内，与泛素共定位，且降低泛素的表达。提示纤溶酶原可能与泛素发生相互作用。
实施例2纤溶酶原在ALS模型小鼠脊髓组织中富集且与LC3B共定位
取周龄相近野生型雄性小鼠5只和雄性SOD1-G93A小鼠9只。野生型小鼠作为空白对照组，SOD1-G93A小鼠从第14周发病后腿颤抖时开始观察记录，记录每只小鼠发病时间，发病14天后开始给药，所有小鼠根据发病情况随机分成溶媒组和给药组，其中溶媒组小鼠5只，每天尾静脉注射0.1ml/只溶媒(柠檬酸钠缓冲液)；给药组4只，每天尾静脉注射1mg/0.1ml/只纤溶酶原，SPF环境下连续给药，濒死取材，最长给药61天。取材脊髓组织于福尔马林固定液中固定。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。将切片浸入抗原修复工作液(0.01M柠檬酸钠缓冲液)中微波修复，预热5分钟，高火2分钟，低火15分钟。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories,Inc.,USA)封闭30分钟；时间到后，弃除羊血清液，滴加抗纤溶酶原抗体(自产)，4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按照XTSA520IHC试剂盒(Alpha X Biotech,AXT6202500)说明书操作进行对应的抗纤溶酶原二抗的绿色荧光显色。PBS洗3次，每次5分钟。重复上述的抗原修复和封闭操作，然后进行抗LC3B抗体(Proteintech，18725-1-AP)染色，37℃孵育1小时。PBS洗3次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按照XTSA620 IHC试剂盒(Alpha X Biotech,AXT6502500)说明书操作进行对应的抗LC3B二抗的红色荧光显色。PBS洗3次，每次5分钟。进行DAPI(BOSTER，11K16B77)核染色。PBS洗3次，每次5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察拍照。
自噬是一种通过溶酶体在细胞内部降解功能失调的细胞组分的过程。自噬可以降解和消化受损、变性的细胞器、蛋白质与核酸等生物大分子。为细胞的再生和修复提供原料，实现细胞内物质的循环利用。LC3指蛋白轻链3，是自噬过程的标志物。它的功能主要参与了自噬小体的形成，LC3前体分子被裂解去C端的5肽，裂解形成胞质形式LC3-I。然后被APG7L/ATG7激活，转移至ATG3并偶联脂酰乙醇胺(PE)以形成膜结合形式LC3- II(LC3B)，它可以附着到自噬体(autophagosome)的膜上，是自噬体的结构蛋白。
结果显示，给药组纤溶酶原(绿色荧光)在脊髓组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入脊髓组织，并在脊髓组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LC3B(红色荧光)在胞质内共定位(如Δ所示)，且给药组脊髓组织LC3B表达水平似乎少于溶媒组。说明在肌萎缩侧索硬化症模型小鼠中纤溶酶原能够进入脊髓组织，进入细胞内，与LC3B共定位，提示纤溶酶原与LC3B之间存在相互作用。
实施例3纤溶酶原在ALS模型小鼠脊髓组织中富集且与LAMP2共定位
取周龄相近野生型雄性小鼠5只和雄性SOD1-G93A小鼠9只。野生型小鼠作为空白对照组，SOD1-G93A小鼠从第14周发病后腿颤抖时开始观察记录，记录每只小鼠发病时间，发病14天后开始给药，所有小鼠根据发病情况随机分成溶媒组和给药组，其中溶媒组小鼠5只，每天尾静脉注射0.1ml/只溶媒(柠檬酸钠缓冲液)；给药组4只，每天尾静脉注射1mg/0.1ml/只纤溶酶原，SPF环境下连续给药，濒死取材，最长给药61天。取材脊髓组织于福尔马林固定液中固定。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。将切片浸入抗原修复工作液(0.01M柠檬酸钠缓冲液)中微波修复，预热5分钟，高火2分钟，低火15分钟。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories,Inc.,USA)封闭30分钟；时间到后，弃除羊血清液，滴加抗纤溶酶原抗体(自产)，4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按照XTSA520IHC试剂盒(Alpha X Biotech,AXT6202500)说明书操作进行对应的抗纤溶酶原二抗的绿色荧光显色。PBS洗3次，每次5分钟。重复上述的抗原修复和封闭操作，然后进行抗LAMP2抗体(BOSTER,BM4357)染色，37℃孵育1小时。PBS洗3次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体 (Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按照XTSA620IHC试剂盒(Alpha X Biotech,AXT6502500)说明书操作进行对应的抗LAMP2二抗的红色荧光显色。PBS洗3次，每次5分钟。进行DAPI(BOSTER，11K16B77)核染色。PBS洗3次，每次5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察拍照。
溶酶体相关膜蛋白-2(LAMP2)是一种相当丰富的溶酶体糖蛋白，它是直接导入溶酶体的蛋白的受体，也是自噬体/吞噬体成熟的中介[4]。
结果显示，给药组纤溶酶原(绿色荧光)在脊髓组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入脊髓组织，并在脊髓组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LAMP2(红色荧光)在胞质内共定位(如Δ所示)，且给药组脊髓组织LAMP2表达水平似乎多于溶媒组(图4)。说明在肌萎缩侧索硬化症模型小鼠中纤溶酶原能够进入脊髓组织，进入细胞内，与LAMP2共定位，且促进LAMP2的表达，促进溶酶体作用。
实施例4纤溶酶原促进肌萎缩侧索硬化症模型小鼠脊髓组织LC3的表达
取10-15周龄SOD1-G93A小鼠6只，随机分为2组，溶媒对照组3只，给药组3只。溶媒对照组小鼠按照5ml/kg尾静脉注射施予溶媒，给药组小鼠按照50mg/kg体重尾静脉注射施予纤溶酶原(10mg/ml)。给药24小时后处死小鼠，取材脊髓。匀浆后行LC3基因转录的RT-PCR检测，记录CT值。计算100ng总RNA中LC3基因转录mRNA的CT值。
结果显示，给药组LC3的表达显著高于溶媒组，且统计差异显著(*表示P<0.05)(图5)。表明纤溶酶原能够促进肌萎缩硬化症模型小鼠LC3基因的转录，从而参与自噬过程的调控。
实施例5纤溶酶原在帕金森模型小鼠黑质和纹状体组织中富集且与泛素共定位
取9周龄C57雄性小鼠12只，造模前1天称重，小鼠按照30mg/kg体重每天腹腔注射5mg/ml MPTP溶液，连续注射5天，建立帕金森模型[5，6]MPTP溶液配制：用注射器吸取10ml去离子水，加入到100mg MPTP粉末(sigma，M0896)中，配制成10mg/ml的母液，然后吸取1ml母液于安瓶中，再加入1ml去离子水，最终浓度为5mg/ml。造模完成后小鼠随机分为 两组，给溶媒PBS对照组和给纤溶酶原组各6只小鼠，并开始给药，记为第1天，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原溶液，给溶媒PBS对照组尾静脉给予相同体积PBS，持续给药14天。于给药第15天处死小鼠，取材小鼠黑质和纹状体于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。如实施例1所述方法进行纤溶酶原、泛素和DAPI共染色。切片在400倍光学显微镜下观察拍照。
结果显示，给药组纤溶酶原(绿色荧光)在黑质和纹状体组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入黑质和纹状体组织，并在黑质和纹状体组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与泛素(红色荧光)在胞质内共定位(如Δ所示)(图6)。说明在帕金森模型小鼠中纤溶酶原能够进入黑质和纹状体组织，进入细胞内，与泛素共定位。提示纤溶酶原可能与泛素发生相互作用。
实施例6溶酶原在帕金森模型小鼠黑质和纹状体组织中富集且与LC3B共定位
取9周龄C57雄性小鼠12只，造模前1天称重，小鼠按照30mg/kg体重每天腹腔注射5mg/ml MPTP溶液，连续注射5天，建立帕金森模型[5，6]。MPTP溶液配制：用注射器吸取10ml去离子水，加入到100mg MPTP粉末(sigma，M0896)中，配制成10mg/ml的母液，然后吸取1ml母液于安瓶中，再加入1ml去离子水，最终浓度为5mg/ml。造模完成后小鼠随机分为两组，给溶媒PBS对照组和给纤溶酶原组各6只小鼠，并开始给药，记为第1天，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原溶液，给溶媒PBS对照组尾静脉给予相同体积PBS，持续给药14天。于给药第15天处死小鼠，取材小鼠黑质和纹状体于4％多聚甲醛固定24-48小时。后续如实施2所述方法进行纤溶酶原、LC3B和DAPI共染色。切片在400倍光学显微镜下观察拍照。
结果显示，给药组纤溶酶原(绿色荧光)在黑质和纹状体组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入黑质和纹状体组织，并在黑质和纹状体组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LC3B(红色荧光)在胞质内共定位(如Δ所示)(图7)。说明 在帕金森模型小鼠中纤溶酶原能够进入黑质和纹状体组织，进入细胞内，与LC3B共定位。提示纤溶酶原可能与LC3发生相互作用。
实施例7纤溶酶原在帕金森模型小鼠黑质和纹状体组织中富集且与LAMP2共定位
取9周龄C57雄性小鼠12只，造模前1天称重，小鼠按照30mg/kg体重每天腹腔注射5mg/ml MPTP溶液，连续注射5天，建立帕金森模型[5，6]。MPTP溶液配制：用注射器吸取10ml去离子水，加入到100mg MPTP粉末(sigma，M0896)中，配制成10mg/ml的母液，然后吸取1ml母液于安瓶中，再加入1ml去离子水，最终浓度为5mg/ml。造模完成后小鼠随机分为两组，给溶媒PBS对照组和给纤溶酶原组各6只小鼠，并开始给药，记为第1天，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原溶液，给溶媒PBS对照组尾静脉给予相同体积PBS，持续给药14天。于给药第15天处死小鼠，取材小鼠黑质和纹状体于4％多聚甲醛固定24-48小时。后续如实施3所述方法进行纤溶酶原、LAMP2和DAPI共染色。切片在400倍光学显微镜下观察拍照。
结果显示，给药组纤溶酶原(绿色荧光)在黑质和纹状体组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入黑质和纹状体组织，并在黑质和纹状体组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LAMP2(红色荧光)在胞质内共定位(如Δ所示)(图8)。说明在帕金森模型小鼠中纤溶酶原能够进入黑质和纹状体组织，进入细胞内，与LAMP2共定位。提示纤溶酶原可能与LAMP2发生相互作用。
实施例8纤溶酶原在阿尔兹海默模型小鼠海马组织中富集且与泛素共定位
取8周龄的雄性C57小鼠20只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为两组，溶媒组和给药组，每组各10只。所有小鼠麻醉，根据小鼠立体定位图谱，定位于海马的粒细胞层(根据前卤点的坐标定位：AP-2.0mm，ML±1.5mm，DV2.0mm)，每只小鼠双侧缓慢微量注射，注射速率为0.5μL/min，注射体积3μL，模型组小鼠注射Aβ1-42寡聚体溶液，以建立阿尔兹海默模型[7]，模型对照组小鼠注射PBS溶液。Aβ1-42寡聚体溶液制备(10μM)：取β-Amyloid(1-42)(上海强耀生物科技有限公 司，04010011521)，加入冷的六氟异丙醇，配制浓度为1mg/ml，室温放置3天，分装45μL/管，即10nmol/mL，放置通风橱中过夜，置于25℃干燥箱中干燥1小时，-80℃保存。使用时每管加入二甲基亚砜溶液10μl复溶，注射时加入无菌PBS溶液990μL，4℃放置24h后使用。脑定位注射21天后，溶媒组及给药组小鼠开始给药，记为第1天，给药组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原，溶媒组小鼠尾静脉注射0.1ml/只/天溶媒(4％精氨酸+2％甘氨酸溶液)，连续给药28天。第29天处死小鼠取材大脑于10％甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。如实施例1所述方法进行纤溶酶原、泛素和DAPI共染色。切片在400倍光学显微镜下观察拍照。
结果显示，给药组纤溶酶原(绿色荧光)在海马组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入海马组织，并在海马组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与泛素(红色荧光)在胞质内共定位(如Δ所示)(图9)。说明在阿尔兹海默模型小鼠中纤溶酶原能够进入海马组织，进入细胞内。提示纤溶酶原可能与泛素发生相互作用。
实施例9纤溶酶原在阿尔兹海默模型小鼠海马组织中富集且与LC3B共定位
取8周龄的雄性C57小鼠20只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为两组，溶媒组和给药组，每组各10只。所有小鼠麻醉，根据小鼠立体定位图谱，定位于海马的粒细胞层(根据前卤点的坐标定位：AP-2.0mm，ML±1.5mm，DV2.0mm)，每只小鼠双侧缓慢微量注射，注射速率为0.5μL/min，注射体积3μL，模型组小鼠注射Aβ1-42寡聚体溶液，以建立阿尔兹海默模型[7]，模型对照组小鼠注射PBS溶液。Aβ1-42寡聚体溶液制备(10μM)：取β-Amyloid(1-42)(上海强耀生物科技有限公司，04010011521)，加入冷的六氟异丙醇，配制浓度为1mg/ml，室温放置3天，分装45μL/管，即10nmol/mL，放置通风橱中过夜，置于25℃干燥箱中干燥1小时，-80℃保存。使用时每管加入二甲基亚砜溶液10μl复溶，注射时加入无菌PBS溶液990μL，4℃放置24h后使用。脑定位注射21天后，溶媒组及给药组小鼠开始给药，记为第1天，给药组小鼠按照1mg/0.1ml/只/天尾静 脉注射纤溶酶原，溶媒组小鼠尾静脉注射0.1ml/只/天溶媒(4％精氨酸+2％甘氨酸溶液)，连续给药28天。第29天处死小鼠取材大脑于10％甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。如实施例2所述方法进行纤溶酶原、LC3B和DAPI共染色。切片在400倍光学显微镜下观察拍照。
结果显示，给药组纤溶酶原(绿色荧光)在海马组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入海马组织，并在海马组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LC3B(红色荧光)在胞质内共定位(如Δ所示)(图10)。说明在阿尔兹海默模型小鼠中纤溶酶原能够进入海马组织，进入细胞内。提示纤溶酶原可能与LC3B发生相互作用。
实施例10纤溶酶原在阿尔兹海默模型小鼠海马组织中富集且与LAMP2共定位
取8周龄的雄性C57小鼠20只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为两组，溶媒组和给药组，每组各10只。所有小鼠麻醉，根据小鼠立体定位图谱，定位于海马的粒细胞层(根据前卤点的坐标定位：AP-2.0mm，ML±1.5mm，DV2.0mm)，每只小鼠双侧缓慢微量注射，注射速率为0.5μL/min，注射体积3μL，模型组小鼠注射Aβ1-42寡聚体溶液，以建立阿尔兹海默模型[7]，模型对照组小鼠注射PBS溶液。Aβ1-42寡聚体溶液制备(10μM)：取β-Amyloid(1-42)(上海强耀生物科技有限公司，04010011521)，加入冷的六氟异丙醇，配制浓度为1mg/ml，室温放置3天，分装45μL/管，即10nmol/mL，放置通风橱中过夜，置于25℃干燥箱中干燥1小时，-80℃保存。使用时每管加入二甲基亚砜溶液10μl复溶，注射时加入无菌PBS溶液990μL，4℃放置24h后使用。脑定位注射21天后，溶媒组及给药组小鼠开始给药，记为第1天，给药组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原，溶媒组小鼠尾静脉注射0.1ml/只/天溶媒(4％精氨酸+2％甘氨酸溶液)，连续给药28天。第29天处死小鼠取材大脑于10％甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。如实施例3所述方法进行纤溶酶原、LAMP2和DAPI共染色。切片在400倍光学显微镜下观察拍照。
结果显示，给药组纤溶酶原(绿色荧光)在海马组织中的阳性着色明显多于溶媒组，说明给药纤溶酶原能够进入海马组织，并在海马组织富集。此外，纤溶酶原存在于胞质和细胞核内。纤溶酶原与LAMP2(红色荧光)在胞质内共定位(如Δ所示)(图11)。说明在阿尔兹海默模型小鼠中纤溶酶原能够进入海马组织，进入细胞内。提示纤溶酶原可能与LAMP2发生相互作用。
实施例11纤溶酶原促进亨廷顿模型小鼠心脏亨廷顿蛋白(huntingtin)降解
选取14周龄B6/JGpt-Tg(hHTT-CAG130)90/Gpt小鼠(简称hHTT130转基因小鼠)10只作为模型组，C57小鼠5只作为正常对照组。hHTT130小鼠随机分为两组，溶媒组和给药组，各5只。分组完成后，所有小鼠开始给药，给药组小鼠按照50mg/kg尾静脉注射纤溶酶原，正常对照组和溶媒组小鼠尾静脉注射相同体积溶媒，连续给药28天。给药结束后，处死小鼠取材心脏。心脏组织匀浆行亨廷顿蛋白(huntingtin，HTT)weastern blot检测。根据SDS-PAGE配胶说明制备10％凝胶。各组样品分别与4×上样缓冲液(TaKaRa，e2139)以体积比为3:1混匀后，100℃加热5min，冷却后离心2min，然后取20μL上样。电泳条件为30V跑45min，然后100V电泳至胶底。电泳结束后剥取凝胶转移到活化的PVDF膜(GE，A29433753)上，电泳条件为15V，2.5h。转移后的PVDF膜浸泡在封闭液(5％脱脂乳液)中于4℃冰箱中封闭过夜，TBST(0.01M Tris-NaCl，pH7.6缓冲液)洗4次后，加入兔抗HTT抗体(Abcam,ab109115)和actin抗体室温孵育1.5h，TBST洗4次后，加入山羊抗兔IgG(HRP)抗体(Abcam，ab6721)二抗室温孵育1h，TBST洗4次后，将PVDF膜放于干净成像板上，加入Immobilon Western HRP Substrate(MILLIPORE，WBKLS0100)显色，在生物分子成像仪下拍照并用Image J定量分析。
结果显示，溶媒组小鼠心脏HTT水平明显高于正常对照组小鼠，给药组小鼠心脏HTT水平明显低于溶媒组小鼠(图12A-B)，且统计差异显著(*代表P<0.05)。提示纤溶酶原能够促进亨廷顿模型小鼠心脏组织中亨廷顿蛋白降解。
实施例12纤溶酶原促进冈田酸处理的NSC34细胞浆和细胞核中TDP-43降解
将106个NSC34细胞(Otwo Biotech，HTX1846)接种于9cm2的培养皿中，用含有10％胎牛血清(EVERY GREEN,11011-8611)的DMEM培养基(Gibco,11965092)中培养，并置于二氧化碳培养箱进行培养，培养温度37.0℃，5％CO2，待细胞生长48小时，细胞生长约80％-90％丰度时，换液后，进行后续实验。细胞被分为4组：空白对照组、溶媒组、给药组和给药+EACA组。空白对照组细胞换液后不做处理；溶媒组、给药组和给药+EACA组细胞进行冈田酸(okadaic acid，OA)(Shanghai yuanye Bio-Technology,S30686-25ug)暴露处理，浓度为2.5ng/μL。冈田酸刺激24小时后，溶媒组细胞培养液中加入溶媒，给药组细胞培养液中加入纤溶酶原(0.5mg/mL)，给药+EACA组细胞培养液中加入纤溶酶原(最终浓度为0.5mg/mL)和氨基己酸(Aminocaproic Acid，EACA)(20mM)。再加入纤溶酶原作用24小时后，收获细胞。吸去培养上清液，用1×PBS进行清洗，使用0.25胰酶1mL进行消化2-3分钟，看细胞明显脱落，用5-6mL的DMEM完全培养基终止消化，缓慢吹打细胞，收集悬液到离心管，1500rpm，5min进行离心去上清，用预冷的1×PBS进行重悬，对细胞进行计数。每20μL细胞沉淀加入200μL浆蛋白抽提试剂(2×106个细胞沉淀的体积约20μL或40mg)(Solarbio,R0050)。用移液器吹打或高速涡旋15秒，必须使细胞沉淀完全分散开成单细胞悬液。冰浴10分钟。最高转速剧烈涡旋10秒，4℃12000～16000g离心10分钟。上清即为抽提得到的细胞浆蛋白，立即吸取上清至预冷的样品管中备用。沉淀即为细胞核，要完全吸尽残余的上清(避免细胞浆蛋白的污染)，加入50-100μL核蛋白抽提试剂。用移液器吹打或高速涡旋15秒(可适当延长)至沉淀完全分散，冰浴10分钟。最高转速剧烈涡旋10秒，4℃12000～16000g离心10分钟。立即吸取上清至预冷的样品管中，此即为抽提得到的细胞核蛋白。提取的核蛋白行TDP-43 western blot检测。
氨基己酸(Aminocaproic Acid，EACA)是一种赖氨酸类似物，阻断纤溶酶原上的高亲和力赖氨酸结合位点[8]。
结果显示，给药组细胞浆和细胞核中TDP-43的水平显著低于溶媒组细胞核，而加入EACA能够完全的抑制纤溶酶原对TDP-43的效应(图13A-D)。提示纤溶酶原能够促进细胞浆和细胞核中TDP-43降解，而纤溶酶原的这种作用与其结构上的赖氨酸结合位点密切相关。
实施例13纤溶酶原促进冈田酸处理的NSC34细胞浆和细胞核中TDP-43降解
将106个NSC34细胞(Otwo Biotech，HTX1846)接种于9cm2的培养皿中，用含有10％胎牛血清(EVERY GREEN,11011-8611)的DMEM培养基(Gibco,11965092)中培养，并置于二氧化碳培养箱进行培养，培养温度37.0℃，5％CO2，待细胞生长48小时，细胞生长约80％-90％丰度时，换液后，进行后续实验。细胞被分为4组：空白对照组、溶媒组、给药组和给药+EACA组。空白对照组细胞换液后不做处理；溶媒组、给药组和给药+EACA组细胞进行冈田酸(okadaic acid，OA)(Shanghai yuanye Bio-Technology,S30686-25ug)暴露处理，浓度为2.5ng/μL。冈田酸刺激24小时后，溶媒组细胞培养液中加入溶媒，给药组细胞培养液中加入纤溶酶原(0.5mg/mL)，给药+EACA组细胞培养液中加入纤溶酶原(最终浓度为0.5mg/mL)和氨基己酸(Aminocaproic Acid，EACA)(20mM)。再加入纤溶酶原作用24小时后，收获细胞。吸去培养上清液，用1×PBS进行清洗，使用0.25胰酶1mL进行消化2-3分钟，看细胞明显脱落，用5-6mL的DMEM完全培养基终止消化，缓慢吹打细胞，收集悬液到离心管，1500rpm，5min进行离心去上清，用预冷的1×PBS进行重悬，对细胞进行计数。加入细胞裂解液(Solarbio，R0010-100mL)裂解细胞，提取细胞蛋白。提取的细胞蛋白行磷酸化Tau蛋白(Abcam，ab151559)和总Tau蛋白(Proteintech，10842-1-AP)western blot检测。
结果显示，给药组磷酸化Tau蛋白的水平显著低于溶媒组，而加入EACA能够完全的抑制纤溶酶原对磷酸化Tau蛋白的效应(图14A-B)；给药组总Tau蛋白水平与溶媒组未见显著差异；给药组磷酸化Tau蛋白与总Tau蛋白的比值显著低于溶媒组，而加入EACA能够完全的抑制纤溶酶原的这种效应(图14C-E)，*代表P<0.05，***代表P<0.001。提示纤溶酶原能够促 进细胞中磷酸化Tau蛋白降解，而纤溶酶原的这种作用与其结构上的赖氨酸结合位点密切相关。
实施例14纤溶酶原促进冈田酸处理的NSC34细胞浆和细胞核中纤溶酶原水平和纤溶酶活性水平增加
将106个NSC34细胞(Otwo Biotech，HTX1846)接种于9cm2的培养皿中，用含有10％胎牛血清(EVERY GREEN,11011-8611)的DMEM培养基(Gibco,11965092)中培养，并置于二氧化碳培养箱进行培养，培养温度37.0℃，5％CO2，待细胞生长48小时，细胞生长约80％-90％丰度时，换液后，进行后续实验。细胞被分为3组：溶媒组、给药组和给药+EACA组。溶媒组、给药组和给药+EACA组细胞进行冈田酸(okadaic acid，OA)(Shanghai yuanye Bio-Technology,S30686-25ug)暴露处理，浓度为2.5ng/μL。冈田酸刺激24小时后，溶媒组细胞培养液中加入溶媒，给药组细胞培养液中加入纤溶酶原(0.5mg/mL)，给药+EACA组细胞培养液中加入纤溶酶原(最终浓度为0.5mg/mL)和氨基己酸(Aminocaproic Acid，EACA)(20mM)。再加入纤溶酶原作用24小时后，收获细胞。吸去培养上清液，用1×PBS进行清洗，使用0.25胰酶1mL进行消化2-3分钟，看细胞明显脱落，用5-6mL的DMEM完全培养基终止消化，缓慢吹打细胞，收集悬液到离心管，1500rpm，5min进行离心去上清。预冷的1×PBS进行重悬，对细胞进行计数。每20μL细胞沉淀加入200μL浆蛋白抽提试剂(2×106个细胞沉淀的体积约20μL或40mg)(Solarbio,R0050)。用移液器吹打或高速涡旋15秒，必须使细胞沉淀完全分散开成单细胞悬液。冰浴10分钟。最高转速剧烈涡旋10秒，4℃12000～16000g离心10分钟。上清即为抽提得到的细胞浆蛋白，立即吸取上清至预冷的样品管中备用。沉淀即为细胞核，要完全吸尽残余的上清(避免细胞浆蛋白的污染)，加入50-100μL核蛋白抽提试剂。用移液器吹打或高速涡旋15秒(可适当延长)至沉淀完全分散，冰浴10分钟。最高转速剧烈涡旋10秒，4℃12000～16000g离心10分钟。立即吸取上清至预冷的样品管中，此即为抽提得到的细胞核蛋白。
细胞裂解后，按照Human Plasminogen ELISA Kit(厂家：AssayMax，货号：EP1200-1)说明书操作进行检测。以试剂盒中人纤溶酶原工作标准品作为内标，对每个样本的浓度进行浓度标定，标定后的浓度除以总蛋白浓度计算出每个样本单位总蛋白量中纤溶酶原的量并进行统计分析。
用酶底物动力学法检测纤溶酶活性。依次向酶标板中加入85μL/孔七个不同浓度点的标准品溶液、空白、样本，然后每孔加入15μL 20mM S-2251溶液(Chromogenix，82033239)，于37℃温育。从反应0min开始，每隔5min在多功能酶标仪中读取A405吸光值，至反应90min止。所有的反应以时间为和吸光值进行直线拟合，得出直线的斜率为标准品/样本的反应速率(△A405/min)。最后以标准品的效价值和△A405/min作标准曲线进行计算所测样本效价。
结果显示，给药组细胞浆和细胞核中人纤溶酶原水平和纤溶酶活性水平显著高于溶媒组，且统计差异显著；加入EACA能够完全的抑制纤溶酶原这些的效应(图15A-D)。提示纤溶酶原能够进入细胞，甚至细胞核中，增加纤溶酶活性，而纤溶酶原进入细胞和细胞核与其赖氨酸结合活性密切相关。
实施例15纤溶酶原促进亨廷顿模型小鼠肾脏脏亨廷顿蛋白(huntingtin)降解
选取14周龄B6/JGpt-Tg(hHTT-CAG130)90/Gpt小鼠(简称hHTT130转基因小鼠)10只作为模型组，C57小鼠5只作为正常对照组。hHTT130小鼠随机分为两组，溶媒组和给药组，各5只。分组完成后，所有小鼠开始给药，给药组小鼠按照50mg/kg尾静脉注射纤溶酶原，正常对照组和溶媒组小鼠尾静脉注射相同体积溶媒，连续给药28天。给药结束后，处死小鼠取材肾脏。肾脏组织匀浆行亨廷顿蛋白(huntingtin，HTT)weastern blot检测。根据SDS-PAGE配胶说明制备10％凝胶。各组样品分别与4×上样缓冲液(TaKaRa，e2139)以体积比为3:1混匀后，100℃加热5min，冷却后离心2min，然后取20μL上样。电泳条件为30V跑45min，然后100V电泳至胶底。电泳结束后剥取凝胶转移到活化的PVDF膜(GE，A29433753)上，电泳条件为15V，2.5h。转移后的PVDF膜浸泡在封闭液(5％脱脂乳液)中于4℃冰箱中封闭过夜，TBST(0.01M Tris-NaCl，pH7.6缓冲液)洗4次后，加入兔抗HTT抗体(Abcam,ab109115)和actin抗体室温孵育1.5h，TBST洗4次后，加入山羊抗兔IgG(HRP)抗体(Abcam，ab6721)二抗室温孵育1h，TBST洗4次后，将PVDF膜放于干净成像板上，加入Immobilon Western HRP Substrate(MILLIPORE，WBKLS0100)显色，在生物分子成像仪下拍照并用Image J定量分析。
结果显示，溶媒组小鼠肾脏HTT水平明显高于正常对照组小鼠，给药组小鼠肾脏HTT水平明显低于溶媒组小鼠(图16A-B)，且统计差异显著(*代表P<0.05)。提示纤溶酶原能够促进亨廷顿模型小鼠肾脏组织中亨廷顿蛋白降解。
实施例16纤溶酶原促进亨廷顿模型小鼠脑组织亨廷顿蛋白(huntingtin)降解
选取2周龄B6/JGpt-Tg(hHTT-CAG130)90/Gpt小鼠，处死后取材脑组织，置于预冷的1640培养基(厂家：Gibco，货号：31800-105)中。在6孔板底部放置0.8μm孔径的醋酸纤维滤膜，滤膜上加入1mL B27培养基(厂家：Gibco，货号：A3653401)，进行湿润滤膜。把脑组织置于含预冷的PBS的平皿中，用1×PBS冲洗3遍，清洗组织表面的血，使用刀片把脑组织切成薄薄的脑片。脑片平均分6个样品，把脑片平铺在有培养基的滤膜上，保证培养基刚好能浸没脑片，进行脑片培养。6孔板放置于CO2培养箱，37℃，5％CO2培养。第二天更换培养基，同时给药组(3个样品)添加0.4mg/mL的纤溶酶原，溶媒组(3个样品)添加同等体积的生理盐水。每天进行更换培养液，相应的实验组添加与上述相同量的纤溶酶原或生理盐水。培养3天后(纤溶酶原作用了2天时间)，吸取脑片及培养液，1200RPM离心5min，去上清液，再用1×PBS重悬，再离心去PBS。保存脑组织沉淀。匀浆后行亨廷顿蛋白(huntingtin，HTT)weastern blot检测。根据SDS-PAGE配胶说明制备10％凝胶。各组样品分别与4×上样缓冲液(TaKaRa，e2139)以体积比为3:1混匀后，100℃加热5min，冷却后离心2min，然后取20μL上样。电泳条件为30V跑45min，然后100V电泳至胶底。电泳结束后剥取凝胶转移到活化的PVDF膜(GE，A29433753)上，电泳条件为15V，2.5h。转移后的PVDF膜浸泡在封闭液(5％脱脂乳液)中于4℃冰箱中封闭过夜，TBST(0.01M Tris-NaCl，pH7.6缓冲液)洗4次后，加入兔抗HTT抗体(Abcam,ab109115)和actin抗体室温孵育1.5h，TBST洗4次后，加入山羊抗兔IgG(HRP)抗体(Abcam，ab6721)二抗室温孵育1h，TBST洗4次后，将PVDF膜放于干净成像板上，加入Immobilon Western HRP Substrate(MILLIPORE，WBKLS0100)显色，在生物分子成像仪下拍照并用Image J定量分析。
结果显示，给药组小鼠的脑组织HTT水平明显低于溶媒组小鼠，且统计差异显著(*代表P<0.05)(图17A-B)。提示纤溶酶原能够促进亨廷顿模型小鼠脑组织中亨廷顿蛋白降解。
参考文献
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序列表
序列1(不含有信号肽的天然纤溶酶原(Glu-PLG，Glu-纤维蛋白溶酶原)核酸序列)：

序列2(不含有信号肽的天然纤溶酶原(Glu-PLG，Glu-纤维蛋白溶酶原)核酸序列)：
序列3(含有信号肽的天然纤溶酶原(来源于swiss prot)的核酸序列)：

序列4(含有信号肽的天然纤溶酶原(来源于swiss prot)的氨基酸序列)：
序列5(LYS77-PLG(Lys-纤溶酶原)核酸序列)：
序列6(LYS77-PLG(Lys-纤溶酶原)氨基酸序列)：
序列7(delta-plg(delta-纤溶酶原)核酸序列)：

序列8(delta-plg(delta-纤溶酶原)氨基酸序列)：
序列9(Mini-plg(小纤维蛋白溶酶原)核酸序列)：
序列10(Mini-plg(小纤维蛋白溶酶原)氨基酸序列)：
序列11(Micro-plg(微纤维蛋白溶酶原)核酸序列)：
序列12(Micro-plg(微纤维蛋白溶酶原)氨基酸序列)：
序列13(丝氨酸蛋白酶(结构)域的核酸序列)：
序列14(丝氨酸蛋白酶(结构)域的氨基酸序列)：

Claims (15)

  1. 一种促进泛素蛋白酶体系统和自噬溶酶体系统对病理性蛋白清除的方法，包括给药受试者有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
  2. 一种清除细胞外和/或细胞质内和/或细胞核内不同种类的病理性蛋白的方法，包括给药受试者有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
  3. 权利要求1或2所述的方法，其中所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro-plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。
  4. 权利要求1或2的方法，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抑制剂，例如抗体。
  5. 权利要求1-4任一项的方法，其中所述化合物具有以下一项或多项活性：清除细胞外和/或细胞质内和/或细胞核内的病理性蛋白、促进泛素蛋白酶体系统对病理性蛋白的清除、促进自噬溶酶体系统对病理性蛋白的清除作用、调节以优化泛素系统成员的表达和/或活性、调节以优化LC3的表达和/或活性、调节以优化自噬溶酶体系统成员的表达和/或活性、调节以优 化(特别是促进)LAMP2的表达。
  6. 权利要求1-5任一项的方法，其中所述化合物为纤溶酶原或纤溶酶。
  7. 权利要求1-6任一项的方法，其中所述纤溶酶原为Glu-纤溶酶原、Lys-纤溶酶原或其保守取代变体。
  8. 权利要求1-7任一项的方法，其中所述纤溶酶原与序列2具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且具有纤溶酶原的赖氨酸结合活性和/或蛋白水解活性。
  9. 权利要求1-8任一项的方法，所述纤溶酶原包含选自如下的一项或多项：
    1)具有序列14所示的丝氨酸蛋白酶结构域；
    2)与序列14具有至少80％、90％、95％、96％、97％、98％、99％同一性并保留蛋白水解活性的丝氨酸蛋白酶结构域；
    3)选自Kringle 1、Kringle 2、Kringle 3、Kringle 4和Kringle 5中一个或多个的Kringle结构域；和
    4)与选自Kringle 1、Kringle 2、Kringle 3、Kringle 4和Kringle 5中一个或多个具有至少80％、90％、95％、96％、97％、98％、99％同一性并保留赖氨酸结合活性的Kringle结构域。
  10. 权利要求1-9任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原的蛋白水解活性的变体。
  11. 权利要求1-10任一项的方法，所述纤溶酶原包含序列2、6、8、10、12所示的氨基酸序列或包含序列2、6、8、10、12所示氨基酸序列的保守取代变体。
  12. 权利要求1-11任一项的方法，其中所述纤溶酶原与一种或多种其它治疗方法或药物联合使用。
  13. 权利要求12的方法，其中所述其它治疗方法包括细胞治疗(包括干细胞治疗)、支持疗法和物理治疗。
  14. 权利要求1-13任一项的方法，其中所述纤溶酶原通过鼻腔吸入、雾化吸入、滴鼻液、滴眼液、滴耳液、静脉内、腹膜内、皮下、舌下、颅内、鞘内、动脉内(例如经由颈动脉)或肌肉内给药。
  15. 权利要求1-14任一项的方法，其中所述病理性蛋白选自如下的一种或多种：其中所述病理性蛋白选自如下的一项或多项：囊性纤维化跨膜电导调节因子(CFTR，cystic fibrosis transmembrane conductance regulator)、α1-抗胰蛋白酶(α1-antitryspin)、Parkin蛋白、p53肿瘤抑制蛋白、Wilms’Tumor 1蛋白(WT1蛋白)、von Hippel Lindau(VHL)肿瘤抑制蛋白、Merlin蛋白、Src蛋白激酶、晶体蛋白(Crystallin)、甲状腺素运载蛋白(Transthyretin)、短链酰基辅酶A脱氢酶变异体(SCAD variant)、低密度脂蛋白受体(Low density lipoprotein receptor)、亨廷顿蛋白(huntingtin)、神经丝蛋白(Neurofilament protein)、外周蛋白(Peripherin)、1α-中间纤丝蛋白(α-Internexin)、胰岛淀粉样多肽(Islet Amyloid Polypeptide)、β2-微球蛋白(β2-microglobulin)、血清淀粉样蛋白A(Serum amyloid A Protein)、免疫球蛋白轻链(Immunoglobulin light Chains)、人溶菌酶(Human Lysozyme)、α-乳清蛋白(α-Lactalbumin)、胸腺素α原(Prothymosinα)、载脂蛋白E(Apolipoprotein E)、载脂蛋白J(Apolipoprotein J)、β-淀粉样蛋白(amyloidβ-protein,A β)、Tau蛋白、α-突触核蛋白(Alpha-synuclein,α-syn)、TAR DNA结合蛋白(transactive response DNA-binding protein 43,TDP-43)、朊蛋白(prion protein)、铜锌超氧化物歧化酶(Copper-zinc Superoxide Dismutase,SOD1)、血浆铜蓝蛋白、重链IgG、轻链IgG、干扰素诱导蛋白6-16(IF16-6，G1P3)、突变的雄性激素受体、ataxin-3。
PCT/CN2023/130018 2022-11-04 2023-11-06 一种促进泛素蛋白酶体系统和自噬溶酶体系统对病理性蛋白清除的方法和药物 WO2024094216A1 (zh)

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