WO2024094217A1 - 一种促进病理性tdp-43蛋白降解的方法和药物 - Google Patents

一种促进病理性tdp-43蛋白降解的方法和药物 Download PDF

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WO2024094217A1
WO2024094217A1 PCT/CN2023/130019 CN2023130019W WO2024094217A1 WO 2024094217 A1 WO2024094217 A1 WO 2024094217A1 CN 2023130019 W CN2023130019 W CN 2023130019W WO 2024094217 A1 WO2024094217 A1 WO 2024094217A1
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plasminogen
plasmin
mice
tdp
group
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PCT/CN2023/130019
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English (en)
French (fr)
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李季男
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泰伦基国际有限公司
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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
    • 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
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular 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
    • A61P25/28—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 a method for promoting the degradation of pathological TDP-43 protein and treating pathological TDP-43 protein-related diseases, comprising administering an effective amount of a plasminogen activation pathway-related compound such as plasminogen or plasmin to a subject.
  • a plasminogen activation pathway-related compound such as plasminogen or plasmin
  • the present application also relates to a pharmaceutical composition containing a plasminogen activation pathway-related compound such as plasminogen or plasmin for this purpose.
  • TDP-43 Transactive response DNA-binding protein 43
  • Transactive response DNA-binding protein 43 is a protein widely present in cells. It can bind to DNA and RNA, and plays an important role in RNA transcription, alternative splicing, and regulation of mRNA stability in cells.
  • Normal TDP-43 is located in various subcellular structures, including mitochondria, mitochondrial-associated membranes, RNA granules, and stress granules, to regulate endoplasmic reticulum-mitochondria binding, mitochondrial protein translation, and mRNA transport and translation. Therefore, the normal physiological function of TDP-43 is particularly important for cell survival.
  • TDP-43 can also bind proteins to each other to form homodimers and polymers. There is a glutamine-rich region at the C-terminus of TDP-43, which is responsible for most of the aggregation. Recent studies have found that several factors affect the aggregation process of TDP-43, which either change the protein structure itself or change the proteins in the surrounding environment, thereby causing TDP-43 aggregation.
  • TDP-43 aggregates have been identified in an increasing number of neurodegenerative disorders (Lagier-Tourenne et al., Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 R46-R64), including but not limited to: frontotemporal dementia (sporadic or familial, with or without motor neuron disease (MND), with progranulin (GRN) mutation, with TARDBP mutation, with valosine-containing protein (VCP) mutation, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive inclusions, Argyrophilic grain disease, Pick's disease, etc.), amyotrophic lateral sclerosis (sporadic ALS, with TARDBP mutation, with angiogenin (ANG) mutation), Alzheimer's disease (AD, sporadic and familial), Down syndrome (Down syndrome), and other neurodegenerative diseases.
  • frontotemporal dementia sporadic or familial, with or without motor neuron disease (MND), with progran
  • Familial British dementia Familial British dementia
  • polyglutamine diseases Hauntington's disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph Disease)
  • hippocampal sclerosis dementia and myopathies (sporadic inclusion body myositis; inclusion body myopathy with mutations in valosin-containing protein (VCP); and Paget disease of bone and frontotemporal dementia); oculopharyngeal muscular dystrophy with rimmed vacuoles; and myofibrillar myopathy with mutations in the sarcomeric protein (MYOT) gene or mutations in the gene encoding desmin (DES).
  • MYOT sarcomeric protein
  • DES gene encoding desmin
  • Aggregated TDP-43 from patient brains shows a number of abnormal modifications, including hyperphosphorylation, ubiquitination, acetylation, and C-terminal fragmentation cleaved by proteolysis (Arai et al., Biochemical and Biophysical Research Communications 351 (2006) 602-611; Neumann et al., Science 314, (2006), 130-133; Neumann et al., Acta Neuropathol. (2009) 117: 137-149; Hasegawa et al., (2008) Annals of Neurology Vol 64 No 1, 60-70; Cohen et al., Nat Commun. 6: 5845, 2015).
  • TDP-43 pathological conditions Another characteristic feature of TDP-43 pathological conditions is the redistribution and accumulation of TDP-43 from the nucleus to the cytoplasm.
  • the hallmark lesions of FTLD-TDP are neuronal cytoplasmic inclusions and glial cytoplasmic inclusions (NCI (neuronal cytoplasmic inclusion) and GCI (glial cytoplasmic inclusion) respectively) and dystrophic neurites (DN).
  • NCI neuronal cytoplasmic inclusions and glial cytoplasmic inclusions
  • GCI glial cytoplasmic inclusion
  • DN dystrophic neurites
  • Frontotemporal dementia is a clinical term that encompasses a broad spectrum of disorders characterized by degeneration of the frontal and temporal lobes, a pathology known as frontotemporal lobar degeneration (FTLD).
  • FTD is the second most common cause of early degenerative dementia in the age group under 65 years (Le Ber, Revue Neurodoubtedly 169 (2013) 811-819).
  • FTD manifests as several syndromes, including bvFTD characterized by personality and behavioral changes; semantic dementia (SD) and progressive nonfluent aphasia (PNFA) characterized by changes in language function; and corticobasal syndrome (CBS), progressive supranuclear palsy syndrome, and motor neurone disease (FTD-MND) characterized by motor dysfunction.
  • SD semantic dementia
  • PNFA progressive nonfluent aphasia
  • CBS corticobasal syndrome
  • FTD-MND motor neurone disease
  • ALS Amyotrophic lateral sclerosis
  • ALS is a neurodegenerative disease characterized by the premature loss of upper and lower motor neurons. The progression of ALS is marked by fatal paralysis and respiratory failure, with a course of 1 to 5 years from diagnosis to death.
  • the neuropathology is characterized by abnormal cytoplasmic accumulation of TDP-43 in neurons and glial cells of the primary motor cortex, brainstem motor nuclei, spinal cord, and associated white matter tracts.
  • ALS with dementia involves accumulation of TDP-43 in the extramotor neocortex and hippocampus.
  • the role of TDP-43 phosphorylation in ALS patients has been studied with the aid of antibodies. Exploration has been conducted (Hasegawa et al., Ann Neurol 2008; 64: 60-70; Neumannet al., Acta Neuropathol (2009) 117: 137-149).
  • TDP-43 pathology occurs in the brains of up to 57% of patients with Alzheimer's disease (Josephs KA et al., Acta Neuropathol. 2014; 127(6): 811-824; Josephs KA et al., Acta Neuropathol. 2014; 127(3): 441-450; McAleese et al., Brain Pathol. 2017 Jul; 27(4): 472-479).
  • TDP-43 aggregation correlates with patient age and is associated with cognitive decline, memory loss, and medial temporal atrophy in AD.
  • TDP-43-positive patients are 10 times more likely to die with cognitive impairment than TDP-43-negative subjects.
  • TDP-43 follows a general progressive deposition pattern, with TDP-43 first deposited in the amygdala (stage I), then in the hippocampus, limbic, temporal, and ultimately frontostriatum (stage V) (Josephs KA et al., Acta Neuropathol. 2014; 127(6): 811-824; Josephs KA et al., Acta Neuropathol. 2014; 127(3): 441-450).
  • TDP-43 aggregation and pathological spread are the main hallmarks of ALS and FTD, currently incurable and fatal diseases. Mutations in TDP-43 are associated with familial cases of ALS and FTD, providing a causal link between TDP-43 misfolding and disease progression. Therefore, there is a need to find methods that promote the degradation of pathological TDP-43 protein or reduce TDP-43 aggregates to treat diseases associated with pathological TDP-43 protein.
  • plasminogen can promote the degradation of pathological TDP-43 protein in nerve and muscle tissues to some extent, and treat diseases associated with pathological TDP-43 protein aggregation, such as ALS and frontotemporal dementia (also known as frontotemporal dementia).
  • this application relates to the following:
  • a method for promoting degradation of pathological TDP-43 protein comprising administering to a subject a therapeutically 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.
  • the compound has one or more of the following activities: promoting the degradation of pathological TDP-43 proteins in neural tissue, promoting the degradation of pathological TDP-43 proteins in muscle tissue. In some embodiments, the compound promotes the degradation of pathological TDP-43 proteins in and/or outside muscle tissue cells. In some embodiments, the compound promotes the degradation of pathological TDP-43 proteins in and/or outside neural tissue cells (e.g., nerve cells or glial cells).
  • a method for treating a pathological TDP-43 protein-related disease in a subject comprising administering to the subject a therapeutically effective amount of one or more compounds selected from the following: components of the plasminogen activation pathway, proteins that can directly activate plasminogen or activate plasminogen activator
  • the invention relates to a compound that indirectly activates plasminogen by activating an upstream component of the plasminogen pathway, a compound that simulates the activity of plasminogen or plasmin, a compound that can upregulate the expression of plasminogen or plasminogen activator, a plasminogen analog, a plasmin analog, a tPA or uPA analog, and an antagonist of a fibrinolytic inhibitor, wherein the pathological TDP-43 protein-related disease is one or more selected from the following: amyotrophic lateral sclerosis (ALS), bulbar amyotrophic lateral sclerosis, Fus gene mutation amyotrophic lateral sclerosis, Alzheimer's disease, arg
  • 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.
  • the antagonist of the fibrinolytic inhibitor is an inhibitor of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin, such as an antibody.
  • 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 group consisting of:
  • 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.
  • the plasminogen is administered by nasal inhalation, nebulized inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial or intramuscular administration.
  • the plasminogen pathway activator is administered in combination with one or more other drugs and/or treatment methods, preferably, the treatment method includes cell therapy (e.g., stem cell therapy) and 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 sequence 2, 6, 8, 10 or 12, and has plasminogen activity and/or lysine binding activity.
  • the plasminogen is based on sequence 2, 6, 8, 10 or 12, with additions, deletions and/or substitutions 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 acid, and a protein with plasminogen activity and/or lysine binding activity.
  • 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. In some specific embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In some specific embodiments, the plasminogen active fragment comprises or has a plasminogen serine protease domain or a plasminogen protease domain. In some specific embodiments, 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 a serine protease domain), micro-plasminogen (comprising a serine protease domain), delta-plasminogen (comprising Kringle 1 and a 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 SEQ ID NOs: 2, 6, 8, 10, or 12.
  • the plasminogen is human native 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, drug, preparation comprises a pharmaceutically acceptable carrier and a plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as plasminogen.
  • the kit and article comprise one or more containers, wherein the container comprises the pharmaceutical composition, drug or preparation.
  • the kit or article further comprises a label or instructions for use, which indicates the use of a plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as plasminogen for the above method.
  • the kit or article further comprises one or more additional containers, wherein the container contains 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 plasminogen Activation pathway components, such as plasminogen.
  • the plasminogen comprises or has an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in sequence 2, 6, 8, 10 or 12, and has plasminogen activity and/or lysine binding activity.
  • the plasminogen is a protein that adds, deletes and/or replaces 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, and has plasminogen activity and/or lysine binding activity.
  • 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 a serine protease domain), micro-plasminogen (comprising a serine protease domain), delta-plasminogen (comprising Kringle 1 and a 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 SEQ ID NOs: 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, 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, medicament 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-mentioned purposes.
  • 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 mechanism by which plasminogen promotes pathological protein degradation in the central nervous system.
  • 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, microglia, and nucleus.
  • Plg plasminogen receptor
  • tPA tissue-type plasminogen activator
  • CAP conformationally abnormal proteins
  • Plm plasmin generated protein fragments
  • PDP plasmin degradation products
  • UBI ubiquitin activating enzyme
  • E1 ubiquitin conjugating enzyme
  • Plasminogen can promote the degradation of central nervous system pathological proteins such as TDP-43 and superoxide dismutase-1 (SOD1), and can improve the clinical symptoms of various neurodegenerative diseases including amyotrophic lateral sclerosis.
  • SOD1 superoxide dismutase-1
  • the degraded protein fragments are further phagocytosed by microglia and then degraded by lysosomes;
  • Plasminogen enters the cell or the nucleus through endocytosis, and is activated to form plasmin, which promotes the degradation of central nervous system pathological proteins such as TDP-43, SOD1, TAU, ⁇ -synuclein, etc.;
  • Plasminogen enters the cell to regulate the intracellular protein degradation system - the ubiquitin proteasome system (ubiquitin proteasome 4)
  • Plasminogen enters the cell to regulate the function of the intracellular protein degradation system - the autophagy-lysosome system, and degrades the central nervous system pathological proteins through the autophagy-lysosome system;
  • central nervous system pathological proteins including TDP-43 and SOD1 have similar infectivity to prions, and plasminogen may have the ability to prevent central nervous system pathological proteins from spreading between cells.
  • FIG. 2A-B Effects of plasminogen on TDP-43 protein in normal mouse brain homogenate.
  • A is a Western blot image
  • B is the result of quantitative analysis of the optical density of the TDP-43 protein band.
  • the results showed that the molecular weight of the recombinant TDP-43 protein monomer was about 43kDa, the molecular weight of the high molecular weight TDP-43 protein (HMW) was >55kDa, and the molecular weight of the low molecular weight TDP-43 fragment (LMW) was ⁇ 40kDa.
  • HMW high molecular weight TDP-43 protein
  • LMW low molecular weight TDP-43 fragment
  • the amount of TDP-43 monomer, HMW and LMW in the plasminogen group was significantly lower than that in the vehicle control group, and the difference was extremely significant (*** represents P ⁇ 0.001, * represents P ⁇ 0.05). This suggests that plasminogen can promote the cleavage of TDP-43 in normal mouse brain homogenate.
  • FIG. 3A-B Effects of plasminogen on TDP-43 protein in brain homogenate of ALS model mice.
  • A is a Western blot image
  • B is the result of quantitative analysis of TDP-43 protein band optical density.
  • the results showed that the molecular weight of the recombinant TDP-43 protein monomer was about 43 kDa, the molecular weight of the high molecular weight TDP-43 protein (HMW) was >55 kDa, and the molecular weight of the low molecular weight TDP-43 fragment (LMW) was ⁇ 40 kDa.
  • HMW high molecular weight TDP-43 protein
  • LMW low molecular weight TDP-43 fragment
  • the amount of TDP-43 monomer, HMW and LMW in the plasminogen group was significantly lower than that in the vehicle control group, and the difference was extremely significant (*** represents P ⁇ 0.001, ** represents P ⁇ 0.01). This suggests that plasminogen can promote the cleavage of TDP-43 in the brain homogenate of ALS model mice.
  • FIG. 4A-B Plasminogen promotes TDP-43 protein degradation in spinal cord tissue of ALS model mice.
  • A is a Western blot image
  • B is the result of quantitative analysis of TDP-43 protein band optical density. The results showed that the amount of TDP-43 monomer and low molecular weight TDP-43 in the spinal cord tissue of mice in the drug-treated group was significantly lower than that in the vehicle group, and the statistical difference was significant (* represents P ⁇ 0.05). This indicates that plasminogen can promote the degradation of TDP-43 in the spinal cord tissue of ALS model mice.
  • FIG. 5A-B Plasminogen promotes the degradation of TDP-43 protein in the brain tissue of ALS model mice injected with pathological TDP-43 protein.
  • A is a Western blot image
  • B is the result of quantitative analysis of the optical density of TDP-43 protein bands.
  • the results showed that the amount of TDP-43 monomer and low molecular weight TDP-43 in the brain tissue of mice in the drug-treated group was significantly lower than that in the vehicle group, and the statistical difference was significant (* represents P ⁇ 0.05). This shows that plasminogen can promote the degradation of TDP-43 in the brain tissue of ALS model mice.
  • Figure 6A-I Representative images of immunofluorescence co-localization staining of plasminogen and TDP-43 in the spinal cord tissue of mice after administration of plasminogen to ALS model mice.
  • AC normal control group
  • DF vehicle group
  • GI drug administration group.
  • the results showed that the positive staining of plasminogen (green fluorescence) in the spinal cord tissue of the drug administration group was significantly more than that of the vehicle group, indicating that the administered plasminogen can enter the spinal cord tissue and be enriched in the spinal cord tissue.
  • plasminogen is present in the cytoplasm (as shown in ⁇ ) and the nucleus (as shown in Plasminogen co-localizes with TDP-43 (red fluorescence) in the cytoplasm ( ⁇ ) and in the nucleus (
  • TDP-43 level in the spinal cord tissue of the drug-treated group was lower than that of the vehicle group, and the co-localization of plasminogen and TDP-43 in the drug-treated group was more than that in the vehicle group. This indicates that plasminogen can enter the spinal cord tissue and cells in the ALS model mice, co-localize with TDP-43, and degrade TDP-43.
  • Figure 7A-I Representative images of immunofluorescence co-localization staining of plasminogen and TDP-43 in muscle tissue of mice after plasminogen was administered to ALS model mice.
  • AC normal control group
  • DF vehicle group
  • GI drug administration group.
  • the results showed that the positive staining of plasminogen (green fluorescence) in muscle tissue of the drug administration group was significantly more than that of the vehicle group, indicating that plasminogen can be enriched in muscle tissue.
  • plasminogen is present in the cytoplasm (as shown in ⁇ ) and the nucleus (as shown in Plasminogen co-localizes with TDP-43 (red fluorescence) in the cytoplasm ( ⁇ ) and in the nucleus ( This indicates that plasminogen can be enriched in muscle tissue, enter cells, and co-localize with TDP-43 in ALS model mice.
  • FIG. 8A-B shows the results of WB detection of TDP-43 levels in brain homogenates of mice with dementia induced by okadaic acid after administration of plasminogen.
  • A is a Western blot image
  • B is the result of quantitative analysis of the optical density of TDP-43 protein bands.
  • the results showed that the levels of TDP-43 monomers and low molecular weight TDP-43 in the brain tissue of mice in the drug-treated group were significantly lower than those in the vehicle group. This indicates that plasminogen can promote the degradation of TDP-43 in the brain tissue of mice with dementia induced by okadaic acid.
  • Figure 9A-B shows the results of WB detection of TDP-43 levels in the nuclei of renal cells in mice with amyotrophic sclerosis treated with plasminogen.
  • A is a Western blot image
  • B is the result of quantitative analysis of the optical density of TDP-43 protein bands.
  • the results showed that the level of TDP-43 in the nuclei of renal cells in the drug-treated group was significantly lower than that in the vehicle group (* represents P ⁇ 0.05). This suggests that plasminogen can promote the degradation of TDP-43 in the nuclei of renal cells.
  • Figure 10A-D is the WB test results of TDP-43 levels in the cytoplasm and nucleus of NSC34 treated with plasminogen okadaic acid.
  • A is the Western blot image of the cytoplasm
  • B is the quantitative analysis result of the optical density of the TDP-43 protein band in the cytoplasm
  • C is the Western blot image of the nucleus
  • D is the quantitative analysis result of the optical density of the TDP-43 protein band in the nucleus.
  • Figure 11A-D is the result of the detection of plasminogen and plasmin activity levels in the cytoplasm and nucleus of NSC34 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.
  • FIG. 12 ELISA test results of plasma plasminogen levels at different time points after tail vein injection of plasminogen in SOD1-G93A mice.
  • the results of plasma ELISA test results of SOD1-G93A mice showed that the plasma plasminogen levels of SOD1-G93A mice increased significantly after tail vein injection of 50 mg/kg and 6 mg/kg plasminogen, and the plasminogen level in the 50 mg/kg group was significantly higher than that in the 6 mg/kg group.
  • the plasminogen level gradually decreased 2 hours after administration and was basically metabolized completely within 12 to 24 hours.
  • * represents P ⁇ 0.05, ** represents P ⁇ 0.01, and *** represents P ⁇ 0.001.
  • the level of plasminogen in plasma has a dose-dependent effect, and the higher the concentration of plasminogen administered, the more it aggregates; (2) the level of plasminogen in plasma has a time-dependent effect, first increasing and then gradually decreasing within 2 to 12 hours.
  • Figure 13A-B shows the results of ELISA test for brain tissue plasminogen level and the ratio of plasminogen in brain tissue to plasminogen in plasma at different time points after intravenous injection of plasminogen in SOD1-G93A mice.
  • A is the result of ELISA test for brain tissue plasminogen level
  • B is the ratio of plasminogen in brain tissue to plasminogen in plasma.
  • the results of ELISA level test for SOD1-G93A mouse brain showed that the level of plasminogen in SOD1-G93A mouse brain tissue increased significantly after tail vein injection of 50mg/kg and 6mg/kg plasminogen, and the level of plasminogen in the 50mg/kg administration group was significantly higher than that in the 6mg/kg administration group.
  • the level of plasminogen gradually decreased 2 hours after administration and was basically metabolized within 12 to 24 hours.
  • the ratio of plasminogen level in brain tissue to plasminogen level in blood was 3.47%, 4.94% and 6.79% respectively 2, 6 and 12 hours after administration of plasminogen.
  • FIG 14 ELISA test results of spinal cord tissue plasminogen levels at different time points after intravenous injection of plasminogen in SOD1-G93A mice.
  • the results of ELISA test of SOD1-G93A mouse spinal cord plasminogen levels showed that the level of SOD1-G93A mouse spinal cord plasminogen increased significantly after tail vein injection of 50mg/kg and 6mg/kg plasminogen, and the plasminogen level in the 50mg/kg group was significantly higher than that in the 6mg/kg group. Plasminogen levels gradually decreased 2 hours after administration and were basically metabolized within 12 to 24 hours. * represents P ⁇ 0.05, ** represents P ⁇ 0.01.
  • Figure 15A-B shows the results of plasminogen level and plasmin activity detection in brain tissue homogenate after single intravenous injection of plasminogen in SOD1-G93A mice.
  • A is the result of ELISA test for plasminogen level
  • B is the result of enzyme substrate kinetic test for plasmin activity level.
  • the results showed that the plasminogen level and plasmin activity level in brain tissue homogenate of mice in the drug administration group were significantly higher than those in the vehicle group, and the statistical difference was significant (* represents P ⁇ 0.05, *** represents P ⁇ 0.001). It is suggested that intravenous administration of plasminogen can promote the increase of plasminogen level and plasmin activity in brain tissue.
  • Figure 16 shows the results of the detection of plasminogen levels in the nuclei of brain tissue, spinal cord tissue and kidney tissue after continuous intravenous injection of plasminogen in SOD1-G93A mice for 7 days.
  • the results showed that after 7 days of administration of plasminogen, the levels of human plasminogen in the nuclei of brain tissue, spinal cord tissue and kidney tissue of SOD1-G93A mice in the administration group were significantly higher than those in the vehicle group, and the statistical difference was extremely significant (*** represents P ⁇ 0.001). This suggests that intravenous administration of plasminogen can promote the increase of human plasminogen levels in the nuclei of brain tissue, spinal cord tissue and kidney tissue.
  • FIG 17 ELISA test results of blood plasminogen levels at different time points after tail vein injection of plasminogen in Parkinson's model mice. The results showed that the blood plasminogen levels in the drug-treated mice were Significantly higher than the vehicle group, the level of plasminogen gradually decreased 2 hours after administration and was basically metabolized completely between 12 and 24 hours. *** represents P ⁇ 0.001.
  • FIG 18 ELISA test results of blood plasminogen levels at different time points after tail vein injection of plasminogen in Parkinson's model mice.
  • the results showed that the plasminogen level in the brain tissue of the mice in the drug group was significantly higher than that in the vehicle group.
  • the plasminogen level gradually decreased 2 hours after administration and was basically metabolized completely within 12 to 24 hours.
  • the results showed that the plasminogen injected into the tail vein can cross the blood-brain barrier and promote the increase of plasminogen level in the brain tissue of Parkinson's model mice. *** represents P ⁇ 0.001.
  • FIG 19 ELISA test results of spinal cord tissue plasminogen levels at different time points after intravenous injection of plasminogen in Parkinson's model mice.
  • the results showed that the level of plasminogen in the spinal cord tissue of the drug-treated mice was significantly higher than that of the vehicle-treated mice.
  • the level of plasminogen gradually decreased 2 hours after administration and was basically metabolized completely within 12 to 24 hours.
  • the results showed that plasminogen injected into the tail vein can cross the blood-brain barrier and promote the increase of plasminogen levels in the spinal cord tissue of Parkinson's model mice. ** represents P ⁇ 0.01, *** represents P ⁇ 0.001.
  • Figure 20 The ratio of the level of plasminogen in the spinal cord or brain tissue to the level of plasminogen in the blood at different time points after the tail vein injection of plasminogen in Parkinson's model mice.
  • the results showed that the ratio of the level of plasminogen in the spinal cord tissue to the level of plasminogen in the blood was 1.24%, 1.16% and 1.46% 2, 6 and 12 hours after the administration of plasminogen, respectively, and the ratio of the level of plasminogen in the brain tissue to the level of plasminogen in the blood was 3.47%, 4.18% and 8.51% 2, 6 and 12 hours after the administration of plasminogen, respectively.
  • the results show that the plasminogen injected by the tail vein can cross the blood-brain barrier and promote the increase of plasminogen levels in the brain and spinal cord tissues of Parkinson's model mice.
  • Figure 21 Results of the detection of plasmin activity in brain tissue of Parkinson's model mice after tail vein injection of plasminogen. The results showed that the plasmin activity level in the brain tissue of the mice in the drug group was significantly higher than that in the vehicle group, and the statistical difference was significant. * represents P ⁇ 0.05. The results showed that plasminogen injected into the tail vein can cross the blood-brain barrier and promote the increase of plasmin activity level in the brain tissue of Parkinson's model mice.
  • Figure 22 Results of ELISA test on plasma plasminogen levels at different time points after tail vein injection of plasminogen in FAD mice.
  • the results of plasma ELISA test on FAD mice showed that the plasma plasminogen levels of FAD mice increased significantly after tail vein injection of 50mg/kg and 6mg/kg plasminogen, and the plasminogen levels in the 50mg/kg group were significantly higher than those in the 6mg/kg group. Plasminogen levels gradually decreased 2 hours after administration and were basically metabolized within 12 to 24 hours.
  • Figure 23A-B Results of ELISA test of brain tissue plasminogen levels at different time points after tail vein injection of plasminogen in FAD mice (A), and ratio of brain tissue plasminogen levels to blood plasminogen levels at different time points (B).
  • the results of ELISA test of FAD mouse brain levels showed that the plasminogen levels in FAD mouse brain tissues increased significantly after tail vein injection of 50 mg/kg and 6 mg/kg plasminogen, and the plasminogen levels in the 50 mg/kg group were significantly higher than those in the 6 mg/kg group. Plasminogen levels gradually decreased 2 hours after administration and were basically metabolized completely within 12 to 24 hours.
  • the ratio of plasminogen in brain tissue to that in blood of mice in the 6 mg/kg plasminogen group was 3.59% and 4.23% 2 and 6 hours after plasminogen injection, respectively; the ratio of plasminogen in brain tissue to that in blood of mice in the 50 mg/kg plasminogen group was 2.49%, 2.31% and 3.32% 2, 6 and 12 hours after plasminogen injection, respectively.
  • plasminogen administration under physiological and pathological conditions can promote plasminogen to cross the blood-brain barrier and accumulate in brain tissue;
  • intravenous injection of plasminogen into mice significantly increased the level of plasminogen in brain tissue;
  • the accumulation of plasminogen in brain tissue has a time-dependent effect, which increases first, then gradually decreases from 2 to 12 hours, and is almost completely metabolized from 12 to 24 hours;
  • the accumulation of plasminogen in brain tissue has a dose-dependent effect, and the higher the dose, the higher the level of plasminogen in brain tissue.
  • Figure 24 Results of the enzyme substrate kinetic method to detect the activity of plasmin in brain homogenate 2 hours after tail vein injection of plasminogen in FAD mice.
  • the results showed that the activity level of plasmin in brain tissue of FAD mice increased significantly after tail vein injection of 50mg/kg and 6mg/kg plasminogen, and the plasminogen level in the 50mg/kg group was significantly higher than that in the 6mg/kg group.
  • the results show that the level of plasmin in brain tissue increased significantly after plasminogen was injected into mice.
  • the activity of plasmin in brain tissue has a dose-dependent effect. The higher the dose, the higher the level of plasminogen in brain tissue.
  • Figure 25A-B Results of ELISA test of spinal cord tissue plasminogen levels at different time points after tail vein injection of plasminogen in FAD mice (A), and ratio of spinal cord tissue plasminogen levels to blood plasminogen levels at different time points (B).
  • the results of ELISA test of FAD mouse spinal cord levels showed that the level of plasminogen in the spinal cord tissue of FAD mice increased significantly after tail vein injection of 50mg/kg and 6mg/kg plasminogen, and the level of plasminogen in the 50mg/kg group was significantly higher than that in the 6mg/kg group. Plasminogen levels gradually decreased 2 hours after administration and were basically metabolized within 12 to 24 hours.
  • the ratio of plasminogen in the spinal cord tissue to that in the blood of mice in the 6 mg/kg plasminogen group was 0.93% and 1.62% 2 and 6 hours after plasminogen injection, respectively; the ratio of plasminogen in the spinal cord tissue to that in the blood of mice in the 50 mg/kg plasminogen group was 0.33%, 0.40% and 1.56% 2, 6 and 12 hours after plasminogen injection, respectively.
  • plasminogen administration under physiological and pathological conditions can promote plasminogen to cross the blood-brain barrier and accumulate in the spinal cord tissue;
  • intravenous injection of plasminogen into mice significantly increased the level of plasminogen in the spinal cord tissue;
  • the enrichment of plasminogen in the spinal cord tissue has a time-dependent effect, which increases first, then gradually decreases from 2 to 12 hours, and is almost completely metabolized from 12 to 24 hours;
  • the enrichment of plasminogen in the spinal cord tissue has a dose-dependent effect, and the higher the dose, the higher the level of plasminogen in the spinal cord tissue.
  • Figure 26A-C shows the clinical phenotype changes of ALS patients before and after plasminogen treatment.
  • A ALSFRS-R scores of 9 ALS patients before and after plasminogen treatment
  • B Maximum number of steps walked by patient 5 during the second course of treatment
  • C ALSFRS-R scores of 9 ALS patients treated with plasminogen for 0.5 months (indicated by solid lines) and ALSFRS-R scores of ALS patients treated with Riluzole or Edaravone for 6 months (indicated by dotted lines).
  • the fibrinolytic system also known as the fibrinolytic system, is a system composed of a series of chemical substances involved in the fibrinolysis process, mainly including plasminogen (plasminogen), plasmin, plasminogen activator, and fibrinolysis inhibitor.
  • 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 is a serine protease synthesized by vascular endothelial cells.
  • t-PA activates plasminogen, and this process mainly occurs on fibrin;
  • urokinase plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells, and can directly activate plasminogen without fibrin as a cofactor.
  • Plasminogen (PLG) is synthesized by the liver. When blood coagulates, PLG is adsorbed on the fibrin network in large quantities. Under the action of t-PA or u-PA, it is activated into plasmin, promoting fibrinolysis.
  • Plasmin (PL) is a serine protease that has the following functions: degrade fibrin and fibrinogen; hydrolyze various coagulation factors V, VIII, X, VII, XI, II, etc.; convert plasminogen into plasmin; hydrolyze complement, etc.
  • Fibrinolytic 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 it at the same time.
  • PAI plasminogen activator inhibitor
  • PAI-2 ⁇ 2 antiplasmin
  • ⁇ 2-AP is synthesized by the liver and combines with 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 still 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, e.g., 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 those comprising or having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12. or 99% sequence identity, and has plasminogen activity and/or lysine binding activity.
  • the "plasminogen variant" of the present invention can be a protein that adds, deletes and/or replaces 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, and still has 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, reference can be made to the following literature The method described in: Ny, A., Leonardsson, G., Hagglund, AC, Hagglof, P., Ploplis, VA, Carmeliet, P.
  • 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.
  • a compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway refers to any compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway, such as tPA, uPA, streptokinase, saruplase,reteplase, reteplase, tenecteplase, anistreplase, monteplase, lanoteplase, pamiplase, staphylokinase.
  • the "antagonist of fibrinolytic inhibitor” of the present invention is a compound that antagonizes, weakens, blocks, or prevents the action of fibrinolytic inhibitor.
  • 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 PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, or ⁇ 2 macroglobulin, or occupies PAI-1, complement C1 inhibitor, or "Compounds that block the binding sites of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin but do not have the functions of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin", or compounds that block the binding domain and/or active domain 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 92 kDa. 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.
  • the naturally secreted and uncleaved form of plasminogen has an amino-terminal (N-terminal) glutamic acid, so it is called glutamate-plasminogen.
  • lysine-plasminogen has a higher affinity for fibrin 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.
  • 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, according to the natural human plasminogen amino acid sequence containing a signal peptide (Sequence 4), it is composed of 810 amino acids and has a molecular weight of about 90kD. It is a glycoprotein that is 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 the natural full-length plasminogen, consisting of 791 amino acids (excluding the 19-amino acid signal peptide).
  • the cDNA sequence encoding this sequence is shown in Sequence 1, and its amino acid sequence is shown in Sequence 2.
  • Sequence 6 In vivo, there is also a Lys-plasminogen formed by hydrolysis of the 76th-77th amino acids of Glu-plasminogen, as shown in Sequence 6, and the cDNA sequence encoding this amino acid sequence is shown in Sequence 5.
  • Delta-plasminogen is a fragment of the full-length plasminogen that lacks the Kringle2-Kringle5 structure and only contains Kringle1 and a serine protease (structure) domain (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 this amino acid sequence is shown in Sequence 7.
  • Mini-plasminogen Mini-plasminogen consists of Kringle5 and serine protease (structure) domains.
  • the serine protease domain is composed of a serine protease domain, and it is reported in the literature 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, and it is reported in the literature that its amino acid sequence includes residues Ala543-Asn791 (with the Glu residue of the Glu-plasminogen sequence without a signal peptide as the starting amino acid), and there is also a patent document CN102154253A reporting that its sequence includes residues Lys531-Asn791 (with the Glu residue of the Glu-plasminogen sequence without a signal peptide as the starting amino acid).
  • the sequence of this patent refers to the patent document CN102154253A, and its amino acid sequence is shown in Sequence 12, and the cDNA sequence encoding the amino acid sequence is shown in Sequence 11.
  • 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 the fibrin clot into fibrin degradation products and D-dimers, thereby dissolving the thrombus.
  • 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 RB, Mukhametova LI. Structure and function of plasminogen/plasmin system [J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6): 590-605); 2) an active fragment that performs a proteolytic function in the plasminogen protein.
  • the plasminogen is a protein comprising a plasminogen activity fragment shown in sequence 14. In some embodiments of the present application, the plasminogen is a protein comprising a lysine binding fragment of Kringle 1, Kringle 2, Kringle 3, Kringle 4 and/or Kringle 5.
  • the plasminogen activity 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 activity fragment and still retaining 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 to 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. They specifically refer to proteins or genes evolved from the same ancestral gene in different species.
  • the plasminogen of the present invention includes human natural plasminogen, and also includes orthologs or orthologs of plasminogen derived from different species and having plasminogen activity.
  • Constant substitution variants refer to variants in which a given amino acid residue is changed but does not change the overall conformation and function of the protein or enzyme, including but not limited to replacing an amino acid in the amino acid sequence of the parent protein with an amino acid of similar properties (such as acidity, basicity, hydrophobicity, etc.).
  • the properties of amino acids are well known. For example, arginine, histidine and lysine are hydrophilic basic amino acids and can be interchanged. Similarly, 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.
  • Constants also include polypeptides or enzymes with more than 60% amino acid identity determined by BLAST or FASTA algorithms, and better if it can reach more than 75%, preferably more than 85%, and even more than 90% is the best, and compared with the natural or parent protein or enzyme, it has the same or substantially similar properties or functions.
  • Phathological TDP-43 protein is a term relative to "physiologically functional TDP-43 protein". Physiologically functional TDP-43 protein refers to a TDP-43 protein that is in a state where it can exhibit its desired function in the in vivo cellular environment. In contrast, “pathological TDP-43 protein” refers to a TDP-43 protein that cannot exhibit its desired function in the in vivo cellular environment.
  • TDP-43 proteins include, but are not limited to: TDP-43 proteins that have mutated and lost their physiological functions (e.g., lost more than 50%, 60%, 70%, 80%, 90% of their relevant physiological functions), TDP-43 proteins that form protein aggregates, misfolded TDP-43 proteins, abnormally modified TDP-43 proteins (including hyperphosphorylation, ubiquitination, acetylation, and C-terminal fragments cleaved by proteolysis), and TDP-43 proteins that have undergone protein denaturation.
  • TDP-43 proteins that have mutated and lost their physiological functions e.g., lost more than 50%, 60%, 70%, 80%, 90% of their relevant physiological functions
  • TDP-43 proteins that form protein aggregates e.g., misfolded TDP-43 proteins
  • abnormally modified TDP-43 proteins including hyperphosphorylation, ubiquitination, acetylation, and C-terminal fragments cleaved by proteolysis
  • TDP-43 pathological conditions Another characteristic feature of TDP-43 pathological conditions is the redistribution and accumulation of TDP-43 from the nucleus to the cytoplasm, and such proteins are also included in the scope of pathological TDP-43 proteins of the present application.
  • plasminogen promoting TDP-43 (protein) degradation
  • plasminogen promotes the degradation of pathological TDP-43 (protein).
  • 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 orpeptide
  • protein is used interchangeably herein to refer 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. Included are 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.
  • Percentage (%) of amino acid sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after introducing gaps, if necessary, to achieve maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Contrast for the purpose of determining percentage amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences. However, for purposes of the present invention, percentage values of amino acid sequence identity 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 and purified from nature for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When synthesizing polypeptides by chemical synthesis, they can be synthesized via liquid or solid phase. Solid phase peptide synthesis (SPPS) (wherein the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence) is a method suitable for chemical synthesis of plasminogen. Various forms of SPPS, such as Fmoc and Boc can be used to synthesize plasminogen. Techniques for solid phase synthesis are 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 plasminogen encoding polynucleotides.
  • 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 generally contain expression control sequences (e.g., replication origins) that are compatible with the host cells.
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from bacteriophage lambda.
  • the promoter will generally 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.
  • Plasminogen having the desired purity can be mixed with an optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980))
  • the therapeutic formulation is prepared by forming a lyophilized preparation or an aqueous solution.
  • Acceptable carriers, excipients, stabilizers are non-toxic 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; chloride), benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; 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, hist
  • 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. Pat. No.
  • Polymers such as ethylene-vinyl acetate and lactic acid-co-glycolic acid can sustain the release of molecules 100 Some hydrogels release proteins for more than 10 days, while some release proteins for a shorter time. Rational strategies for protein stabilization can be designed based on the relevant mechanisms. For example, if the mechanism of aggregation is found to be the formation of intermolecular SS bonds through the interchange of thiodisulfide bonds, stabilization can be achieved by modifying sulfhydryl 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 an article of manufacture or kit comprising the plasminogen or plasmin of the present invention which can be used to treat cardiovascular disease and related conditions caused by diabetes.
  • the article of manufacture 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, the composition The composition 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 a 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 caused by diabetes and related conditions 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 other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.
  • the product includes 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.
  • muscle atrophy refers to the reduction in the amount of muscle tissue, structural abnormality, reduction or loss and/or functional abnormality, weakening or loss caused by various reasons.
  • the causes of muscle atrophy are mainly various muscle diseases or trauma, including syringomyelia, myelitis, radiculoarthropathy, basal arachnoiditis, brainstem lesions, and brain and spinal neuropathy.
  • the human plasminogen used in all the following examples is 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.
  • Example 1 Plasminogen promotes the cleavage of pathological TDP-43 protein in normal mouse brain homogenate
  • mice Four C57BL/6J male mice aged 11 to 12 weeks and weighing 18 to 25 g were sacrificed and the whole brain was removed and weighed. 1 ⁇ PBS (Thermo Fisher, pH 7.4; 10010-031, homogenize at 4°C (1 min, 3-4 times), centrifuge at 4°C (12000 rpm, 20 min) after homogenization, take the supernatant, i.e. brain homogenate, and place it in a new EP tube.
  • PBS Thermo Fisher, pH 7.4; 10010-031, homogenize at 4°C (1 min, 3-4 times), centrifuge at 4°C (12000 rpm, 20 min) after homogenization, take the supernatant, i.e. brain homogenate, and place it in a new EP tube.
  • Eppendorf (EP) tubes were set as 1 blank group, 2 blank control group, 3 vehicle control group, and 4 plasminogen group, with 5 parallels in each group.
  • the blank group was added with 21.5 ⁇ L of normal saline, 4.6 ⁇ L of vehicle solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 ⁇ L of mouse brain homogenate;
  • the blank control group was added with 21.5 ⁇ L of normal saline, 2.3 ⁇ L of plasminogen solution (2 mg/mL), and 23.9 ⁇ L of mouse brain homogenate;
  • the vehicle control group was added with 20.5 ⁇ L of TDP-43 (Nanjing GeneScript Biotechnology Co., Ltd., custom-expressed human TDP-43, C134WHE160-2/P5HF001, 1.05 mg/mL), 4.6 ⁇ L of vehicle solution, and 23.9 ⁇ L of mouse brain homogenate;
  • the molecular weight of the recombinant TDP-43 protein monomer was about 43kDa
  • the molecular weight of the high molecular weight TDP-43 protein (HMW) was >55kDa
  • the molecular weight of the low molecular weight TDP-43 fragment (LMW) was ⁇ 40kDa.
  • the amount of TDP-43 monomer, HMW and LMW in the plasminogen group was significantly lower than that in the vehicle control group, and the difference was extremely significant (*** represents P ⁇ 0.001, * represents P ⁇ 0.05) ( Figure 2). This suggests that plasminogen can promote the cleavage of TDP-43 in normal mouse brain homogenate.
  • Example 2 Plasminogen promotes the cleavage of pathological TDP-43 protein in the brain homogenate of amyotrophic sclerosis model mice
  • SOD1-G93A transgenic mice Four B6.Cg-Tg(SOD1-G93A)1Gur/J transgenic male mice (referred to as SOD1-G93A transgenic mice) were killed, the whole brain was taken out and weighed, 1 ⁇ PBS (Thermo Fisher, pH7.4; 10010-031) was added at 150 mg tissue/mL PBS, and homogenized at 4°C (1 min, 3-4 times), and then centrifuged at 4°C (12000 rpm, 20 min). The supernatant, i.e., the brain homogenate, was placed in a new EP tube.
  • 1 ⁇ PBS Thermo Fisher, pH7.4; 10010-031
  • Eppendorf (EP) tubes were set as 1 blank group, 2 blank control group, 3 vehicle control group, and 4 plasminogen group, with 5 parallels in each group.
  • the blank group was added with 21.5 ⁇ L of normal saline, 4.6 ⁇ L of vehicle solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 ⁇ L of mouse brain homogenate;
  • the blank control group was added with 21.5 ⁇ L of normal saline, 2.3 ⁇ L of plasminogen solution (2 mg/mL), and 23.9 ⁇ L of mouse brain homogenate;
  • the vehicle control group was added with 20.5 ⁇ L of TDP-43 (Nanjing GeneScript Biotechnology Co., Ltd., custom-expressed human TDP-43, C134WHE160-2/P5HF001, 1.05 mg/mL), 4.6 ⁇ L of vehicle solution, and 23.9 ⁇ L of mouse brain homogenate;
  • 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 through the tail vein, and the drug administration group mice were injected with plasminogen (10 mg/ml) at 50 mg/kg body weight through the tail vein.
  • the mice were killed 24 hours after administration and the spinal cord was collected. After homogenization, TDP-43 western blot was performed.
  • mice Nine 6-7 week old C57BL/6J female mice were selected and weighed before modeling. After weighing, all mice were randomly divided into two groups: blank control group (3 mice) and model group (6 mice). After grouping, mice in sham operation group and model group were anesthetized by intraperitoneal injection of tribromoethanol, with an anesthetic dose of 20mL/kg. Model mice were located in the hippocampus according to the mouse stereotaxic atlas (according to the coordinates of the bregma: AP-2.54mm, ML ⁇ 2mm, DV-2.4mm), and each mouse was slowly microinjected bilaterally. The sham operation group mice were only drilled at the coordinate positioning point and no injection was performed [4].
  • the model group mice were injected with TDP-43 solution at an injection rate of 0.5 ⁇ L/min and an injection volume of 3 ⁇ L/side. After the injection, the syringe stayed for 5min and then slowly withdrew. Three days after the brain localization injection, all mice were weighed, and the model group mice were intraperitoneally injected with 5 mg/kg LPS solution according to their body weight. Then the model group mice were randomly divided into two groups, 3 in the drug group and 3 in the vehicle group. 24 hours after LPS injection, the sham operation group mice and the vehicle group mice were injected with 5 mL/kg of the vehicle in the tail vein, and the drug group mice were injected with 50 mg/kg of plasminogen in the tail vein. The drugs were administered continuously for 3 days. Two hours after the third administration, the mice were killed and brain tissues were obtained. TDP-43 western blot detection was performed after homogenization.
  • Plasminogen is enriched in the spinal cord tissue of amyotrophic lateral sclerosis model mice and co-localizes with TDP-43 in cells
  • 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.
  • Plasminogen was present in the cytoplasm (as shown in ⁇ ) and the nucleus (as shown in Plasminogen co-localizes with TDP-43 (red fluorescence) in the cytoplasm ( ⁇ ) and in the nucleus (
  • the TDP-43 level in the spinal cord tissue of the drug-treated group was lower than that of the vehicle group, and the co-localization of plasminogen and TDP-43 in the drug-treated group was more than that in the vehicle group ( Figure 6). Plasminogen can enter the spinal cord tissue, enter the cells, co-localize with TDP-43, and degrade TDP-43.
  • Plasminogen is enriched in the muscle tissue of amyotrophic lateral sclerosis model mice and co-localizes with TDP-43 in cells
  • 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 gluteal muscle tissue was fixed in formalin fixative. The fixed tissue was dehydrated by alcohol gradient and transparentized by 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.
  • plasminogen green fluorescence
  • plasminogen is present in the cytoplasm (as shown in ⁇ ) and the nucleus (as shown in Plasminogen and TDP-43 (red fluorescence) light) in the cytoplasm (as shown in ⁇ ) and in the nucleus (as shown in This indicates that plasminogen can be enriched in muscle tissue, enter cells, and co-localize with TDP-43 in ALS model mice.
  • Example 7 Plasminogen promotes TDP-43 degradation in brain tissue of okadaic acid-induced dementia model mice
  • mice aged 30-32 weeks purchased from Jackson lab, stock number: 034840
  • Five C57 female mice aged 6-7 weeks were selected as the blank group.
  • the mice in the blank group and the model group were anesthetized by intraperitoneal injection of tribromoethanol, with an anesthetic dose of 20 mL/kg.
  • mice were located in the basolateral amygdala according to the mouse stereotaxic atlas (according to the coordinates of the bregma: AP-1.94 mm, ML ⁇ 3.15 mm, DV-4.5 mm), and each mouse was slowly microinjected bilaterally.
  • the mice in the blank group were only drilled at the coordinate location point and no injection was performed [5].
  • mice in the model group were injected with 50ng/ ⁇ L okadaic acid (manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., catalog number S30686-25ug:) solution, the injection rate was 0.5 ⁇ L/min, the injection volume was 2 ⁇ L, and after the injection, the syringe stayed for 6min and then slowly withdrew.
  • okadaic acid manufactured by Shanghai Yuanye Biotechnology Co., Ltd., catalog number S30686-25ug:
  • mice and the vehicle group mice were injected with 5mL/kg of the vehicle through the tail vein, and the drug group mice were injected with 50mg/k of plasminogen through the tail vein.
  • the mice were killed and brain tissue was obtained. TDP-43 western blot detection was performed after homogenization.
  • Example 8 Plasminogen promotes TDP-43 degradation in the nuclei of renal cells in amyotrophic lateral sclerosis model mice
  • SOD1-G93A mice (Jackson Laboratory, Stock Number: 004435) and C57BL/6J mice aged 9-10 weeks were taken.
  • SOD1-G93A mice were randomly divided into two groups, a vehicle group and a drug-treated group, and C57BL/6J mice were used as a normal control group, with 3 mice in each group.
  • the mice in the vehicle group were injected with 5 mL/kg of vehicle (10 mM citric acid sodium citrate solution, pH 7.4) through the tail vein every day, and the mice in the drug-treated group were injected with 50 mg/kg of plasminogen through the tail vein every day. Normal control mice were not treated with drugs.
  • the mice were killed after 7 days, and the kidney tissue was collected.
  • the collected kidney tissue was placed in pre-cooled RPMI-1640 (Sigma-Aldrich) culture medium on ice. After rinsing with PBS, the kidney tissue was cut into small pieces and then incubated Digest with 0.25% pancreatin at 37°C for 10 minutes, shaking every 2 minutes. Add DMEM medium containing 10% fetal newborn calf serum to terminate digestion. After centrifugation (1500rpm, 5 minutes), remove the supernatant and obtain a single cell pellet. Add 200 ⁇ L plasma protein extraction reagent (the volume of 2 ⁇ 10 6 cell pellets is about 20 ⁇ L or 40mg) (Solarbio, R0050) to each 20 ⁇ L cell pellet.
  • RPMI-1640 Sigma-Aldrich
  • Example 9 Plasminogen promotes TDP-43 degradation in the cytoplasm and nucleus of NSC34 cells treated with okadaic acid
  • NSC34 cells (Otwo Biotech, HTX1846) were seeded 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 4 groups: blank control group, vehicle group, drug group and drug + EACA group.
  • the cells in the blank control group were not treated after the medium was changed; the cells in the vehicle group, drug group and drug + EACA group were exposed to okadaic acid (OA) (Shanghai yuanye Bio-Technology, S30686-25ug) at a concentration of 2.5ng/ ⁇ L.
  • OA okadaic acid
  • the vehicle was added to the cell culture medium of the vehicle group, plasminogen (0.5 mg/mL) was added to the cell culture medium of the drug 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 another 24 hours, the cells were harvested.
  • the culture supernatant was aspirated, washed with 1 ⁇ PBS, and digested with 0.25 pancreatic enzyme 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, 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 (2 ⁇ 10 6 cell pellets) was added to each 20 ⁇ L cell pellet. Volume is about 20 ⁇ L or 40mg)(Solarbio, R0050).
  • EACA Aminocaproic acid
  • 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 vehicle was added to the cell culture medium of the vehicle group, plasminogen (0.5 mg/mL) was added to the cell culture medium of the drug 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 another 24 hours, the cells were harvested.
  • the culture supernatant was aspirated, washed with 1 ⁇ PBS, and digested with 0.25 pancreatic enzyme 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 blown slowly, and the suspension was collected into a centrifuge tube. The supernatant was centrifuged at 1500rpm for 5 minutes to remove the supernatant. Resuspend in pre-cooled 1 ⁇ PBS and count the cells. Add 200 ⁇ L of plasma protein extraction reagent to every 20 ⁇ L of cell pellet. (The volume of 2 ⁇ 106 cell pellets is about 20 ⁇ L or 40mg) (Solarbio, R0050).
  • the test was performed according to the instructions of Human Plasminogen ELISA Kit (Manufacturer: AssayMax, Catalog No.: EP1200-1).
  • the concentration of each sample was calibrated using the human plasminogen working standard in the kit as the internal standard.
  • the calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per unit of total protein in each sample and perform statistical analysis.
  • the activity of plasmin was detected by enzyme substrate kinetic method. 85 ⁇ L/well of seven different concentrations of standard solution, 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 Twenty-seven 10-15 week old B6.Cg-Tg(SOD1-G93A)1Gur/J(SOD1-G93A) mice (pedigree number: 004435) (referred to as SOD1-G93A mice) (breeding mice purchased from Jackson Laboratory, USA) were randomly divided into three groups: a vehicle control group of 3 mice, a 6 mg/kg plasminogen group of 12 mice, and a 50 mg/kg plasminogen group of 12 mice.
  • mice in the vehicle control group were treated with 5 ml/kg tail
  • the vehicle was injected intravenously, and the mice in the 6mg/kg plasminogen group were injected with plasminogen (1.2mg/ml) at 6mg/kg body weight through the tail vein, and the mice in the 50mg/kg plasminogen group were injected with plasminogen (10mg/ml) at 6mg/kg body weight through the tail vein.
  • the mice in the vehicle control group were killed 2 hours after administration, and blood was collected.
  • Three mice in the 6mg/kg plasminogen group and the 50mg/kg plasminogen group were killed at 2, 6, 12 and 24 hours after administration, and blood was collected.
  • the results of plasma ELISA test of SOD1-G93A mice showed that the plasma plasminogen level of SOD1-G93A mice increased significantly after tail vein injection of 50mg/kg and 6mg/kg plasminogen, and the plasminogen level of the 50mg/kg group was significantly higher than that of the 6mg/kg group.
  • the plasminogen level gradually decreased 2 hours after administration and was basically metabolized within 12 to 24 hours (Figure 12).
  • Example 12 Administration of plasminogen promotes increase in plasminogen levels in brain tissue of SOD1-G93A mice
  • Brain tissue was obtained from the mice killed in Example 11, homogenized, and tested according to the instructions of Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using the human plasminogen working standard in the kit as the internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per unit of total protein in each sample and perform statistical analysis.
  • the results of ELISA level detection in the brain of SOD1-G93A mice showed that the level of plasminogen in the brain tissue of SOD1-G93A mice increased significantly after tail vein injection of 50 mg/kg and 6 mg/kg plasminogen, and the level of plasminogen in the 50 mg/kg group was significantly higher than that in the 6 mg/kg group.
  • the level of plasminogen gradually decreased 2 hours after administration and was basically metabolized completely within 12 to 24 hours (Figure 13A).
  • the ratio of the level of plasminogen in brain tissue to the level of plasminogen in the blood was 3.47%, 4.94% and 6.79% 2, 6 and 12 hours after administration of plasminogen, respectively ( Figure 13B).
  • Plasminogen is enriched in brain tissue;
  • Example 13 Administration of plasminogen promotes increase in plasminogen levels in spinal cord tissue of SOD1-G93A mice
  • the results of the ELISA test of the spinal cord of SOD1-G93A mice showed that the level of plasminogen in the spinal cord of SOD1-G93A mice increased significantly after tail vein injection of 50mg/kg and 6mg/kg plasminogen, and the level of plasminogen in the 50mg/kg group was significantly higher than that in the 6mg/kg group.
  • the level of plasminogen gradually decreased 2 hours after administration and was basically metabolized completely within 12 to 24 hours (Figure 14).
  • Example 14 Administration of plasminogen promotes the increase of plasminogen level and plasmin activity level in brain tissue of SOD1-G93A mice
  • mice Eight 22-week-old SOD1-G93A mice were randomly divided into two groups, a vehicle group and a drug-treated group, with 4 mice in each group.
  • 2.5 mg/mL bacterial lipopolysaccharide (LPS) (Beijing Solebow Technology Co., Ltd., L8880) was administered to the vehicle group and the drug-treated group mice by tracheal instillation, with a modeling dose of 5 mg/kg.
  • LPS bacterial lipopolysaccharide
  • Four C57 mice of the same age were taken as normal control mice.
  • Drug administration began 3 days after LPS treatment.
  • the blank group mice and the vehicle group mice were injected with 5 mL/kg of the vehicle through the tail vein, and the drug-treated group mice were injected with 50 mg/k of plasminogen through the tail vein.
  • Six hours after a single dose the mice were killed and brain tissue was obtained. After homogenization, the plasminogen level ELISA test and the plasmin activity enzyme substrate method
  • Example 15 Administration of plasminogen promotes the increase of plasminogen levels in the nuclei of brain tissue, spinal cord tissue and kidney tissue of SOD1-G93A mice
  • mice Ten 9-week-old SOD1-G93A mice were randomly divided into two groups, a vehicle group and a drug group, with 5 mice in each group. Five C57 mice of the same age were taken as normal control mice. The normal group mice and the vehicle group mice were injected with 5 mL/kg of the vehicle through the tail vein, and the drug group mice were injected with 50 mg/kg of plasminogen through the tail vein every day for 7 consecutive days. After 7 days, the mice were killed to obtain brain tissue, spinal cord tissue and kidney tissue. The tissue was cut into small pieces, digested with 0.25% trypsin (Beyotime Biotechnology, C0201-500mL), and filtered with a 200-mesh cell sieve to obtain a single cell suspension.
  • trypsin Beyotime Biotechnology, C0201-500mL
  • a pipette to blow or high-speed vortex for 15 seconds to make the cell pellet completely dispersed into a single cell suspension. Ice bath for 10 minutes. Vortex vigorously at the highest speed for 10 seconds, and centrifuge at 12000-16000g for 10 minutes at 4°C. The supernatant is the extracted cytoplasmic protein, and the supernatant should be immediately aspirated into a pre-cooled sample tube for later use.
  • the precipitate is the cell nucleus, and the remaining supernatant should be completely aspirated (to avoid contamination by cytoplasmic proteins), and 50-100 ⁇ L of nuclear protein extraction reagent should be added.
  • the extracted nuclear protein is tested for human plasminogen level by ELISA.
  • Example 16 Administration of plasminogen promotes increase in blood plasminogen levels in Parkinson's disease model mice
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • Preparation of MPTP solution 45 mg of MPTP (Sigma, M0896) was dissolved in 9 ml of saline solution to a final concentration of 5 mg/ml.
  • the mice were randomly divided into two groups according to their body weight, a vehicle group of 6 mice and a drug group of 12 mice. Drug administration began on day 1. The mice in the drug group were injected intravenously into the tail vein.
  • Plasminogen solution was administered by tail vein injection at 50 mg/kg body weight, and the vehicle group was injected with 5 mL/kg body weight of vehicle solution (10 mM citric acid-sodium citrate solution, pH 7.4) by tail vein injection. Three mice in the vehicle group were killed at 2 and 24 hours after administration, and blood was collected. Mice in the drug group were killed at 2, 6, 12, and 24 hours after administration, and blood was collected. After blood centrifugation (3500 rpm, 10 min, 4 ° C), the supernatant was taken and tested according to the instructions of Human Plasminogen ELISA Kit (Manufacturer: AssayMax, Catalog No.: EP1200-1). The concentration of each sample was calibrated with human plasminogen working standard as the internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen in each sample unit total protein and perform statistical analysis.
  • vehicle solution 10 mM citric acid-sodium citrate solution, pH 7.4
  • Example 17 Administration of plasminogen promotes increase in plasminogen level in brain tissue of Parkinson's model mice
  • Brain tissue was obtained from the mice killed in Example 16, homogenized, and tested according to the instructions of Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using the human plasminogen working standard as the internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per unit of total protein in each sample and perform statistical analysis.
  • Example 18 Administration of plasminogen promotes increase in plasminogen levels in spinal cord tissue of Parkinson's disease model mice
  • Example 19 Administration of plasminogen promotes increase in plasminogen levels in brain and spinal cord tissues of Parkinson's disease model mice
  • Brain and spinal cord tissues were collected from the mice killed in Example 16, homogenized, and tested according to the instructions of Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1).
  • the concentration of each sample was calibrated using the human plasminogen working standard as the internal standard.
  • the calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per unit of total protein in each sample and perform statistical analysis.
  • Plasminogen promotes the increase of plasmin activity in brain tissue of Parkinson's model mice
  • mice Fifteen female mice were taken and weighed before modeling. They were randomly divided into two groups according to their body weight, a blank control group of 5 mice and a model group of 10 mice. All mice in the model group were intraperitoneally injected with 5 mg/mL MPTP solution at 35 mg/kg/mouse, and no mice in the blank control group were intraperitoneally injected with 7 mL/kg normal saline. The modeling time was set at 9 am every day for 5 consecutive days. 24 hours after the last injection of MPTP, all mice were weighed and intraperitoneally injected with 5 mg/kg LPS solution.
  • the model group mice were randomly divided into two groups according to their body weight, a drug administration group of 5 mice and a vehicle group of 5 mice.
  • the vehicle group mice were injected with the vehicle through the tail vein, and the drug administration group mice were injected with 50 mg/kg plasminogen through the tail vein.
  • the drugs were administered once and the samples were dissected 2 hours after administration.
  • the activity of plasmin was detected by enzyme substrate kinetics in brain tissue homogenate.
  • the activity of plasmin was detected by enzyme substrate kinetics.
  • Example 21 Administration of plasminogen promotes increase in blood plasminogen levels in Alzheimer's model mice
  • mice were injected with vehicle at 5ml/kg through the tail vein, the 6mg/kg plasminogen group mice were injected with plasminogen (1.2mg/ml) at 6mg/kg body weight through the tail vein, and the 50mg/kg plasminogen group mice were injected with plasminogen (10mg/ml) at 6mg/kg body weight through the tail vein.
  • the mice in the vehicle control group were killed 2 hours after administration, and blood was collected.
  • Three mice in the 6 mg/kg plasminogen group and the 50 mg/kg plasminogen group were killed at 2, 6, 12 and 24 hours after administration, and blood was collected.
  • the results of plasma ELISA test of FAD mice showed that the plasma plasminogen level of FAD mice increased significantly after tail vein injection of 50mg/kg and 6mg/kg plasminogen, and the plasminogen level of the 50mg/kg group was significantly higher than that of the 6mg/kg group.
  • the plasminogen level gradually decreased 2 hours after administration and was basically metabolized completely within 12 to 24 hours (Figure 22).
  • Example 22 Administration of plasminogen promotes increase in plasminogen levels in brain tissue of Alzheimer's model mice
  • Brain tissue was obtained from the mice killed in Example 21, homogenized, and tested according to the instructions of Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using the human plasminogen working standard as the internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per unit of total protein in each sample and perform statistical analysis.
  • the results of ELISA level test of FAD mouse brain showed that the plasminogen level in FAD mouse brain tissue increased significantly after tail vein injection of 50mg/kg and 6mg/kg plasminogen, and the plasminogen level in the 50mg/kg group was significantly higher than that in the 6mg/kg group.
  • the plasminogen level gradually decreased 2 hours after administration and was basically metabolized completely in 12 to 24 hours (Figure 23A).
  • the ratio of plasminogen in brain tissue to plasminogen in blood of mice in the 6mg/kg plasminogen group was 3.59% and 4.23% respectively after 2 and 6 hours of plasminogen injection; the ratio of plasminogen in brain tissue to plasminogen in blood of mice in the 50mg/kg plasminogen group was 2.49%, 2.31% and 3.32% respectively after 2, 6 and 12 hours of plasminogen injection (Figure 23B).
  • Example 23 Administration of plasminogen promotes increase in plasminogen levels in brain tissue of Alzheimer's model mice
  • Brain tissue was obtained from the mice killed in Example 21, and the enzyme substrate kinetic method of plasmin was performed after homogenization. 85 ⁇ L/well of seven different concentration points of standard solution, blank, and sample were added to the ELISA plate (manufacturer: NUNC, item number: 446469) in turn, and then 15 ⁇ L of 20mM S-2251 solution (manufacturer: Chromogenix, item number: 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.
  • mice significantly increased the level of plasmin in brain tissue.
  • the activity of plasmin in brain tissue had a dose-dependent effect. The higher the dose, the higher the level of plasminogen in brain tissue.
  • Example 24 Administration of plasminogen promotes increase in plasminogen levels in spinal cord tissue of Alzheimer's model mice
  • the results of the ELISA level test on the spinal cord of FAD mice showed that the level of plasminogen in the spinal cord tissue of FAD mice increased significantly after tail vein injection of 50 mg/kg and 6 mg/kg plasminogen, and the level of plasminogen in the 50 mg/kg group was significantly higher than that in the 6 mg/kg group.
  • the level of plasminogen gradually decreased 2 hours after administration and was basically completely metabolized within 12 to 24 hours (Figure 25A).
  • the ratio of plasminogen in the spinal cord tissue to plasminogen in the blood of the mice in the 6 mg/kg plasminogen group was 0.93% and 1.62% 2 and 6 hours after plasminogen injection, respectively; the ratio of plasminogen in the spinal cord tissue of the mice in the 50 mg/kg plasminogen group was 0.93% and 1.62% 2, 6 and 12 hours after plasminogen injection, respectively.
  • the ratios of plasminogen in tissue to plasminogen in blood were 0.33%, 0.40% and 1.56%, respectively ( FIG. 25B ).
  • Tissue enrichment has a time-dependent effect, first increasing, then gradually decreasing from 2 to 12 hours, and almost completely metabolized within 12 to 24 hours; (4) Plasminogen enrichment in spinal cord tissue has a dose-dependent effect, and the higher the dose, the higher the level of plasminogen in the spinal cord tissue.
  • Example 25 Plasminogen improves the condition of patients with amyotrophic lateral sclerosis
  • ALS amyotrophic lateral sclerosis
  • Human plasminogen lyophilized powder was dissolved in sterile water at a concentration of 5 mg/ml and administered to the patient via intravenous injection or nebulizer.
  • the basic information of the patient and the use of plasminogen are shown in Table 1.
  • ALS Functional Rating Scale–Revised (ALSFRS-R, ALS FRS-R) is a widely used and validated assessment tool for monitoring disability progression in ALS patients [8].
  • ALSFRS-R score of the 9 patients before administration was 20.22 ⁇ 10.01, which increased to 23.13 ⁇ 10.82 after 0.5-4 months of plasminogen administration, an increase of 4.11 ⁇ 5.30 points.
  • Patient 8 is a patient with medullary ALS. After only 2 weeks of plasminogen treatment, the patient's ALSFRS-R score increased rapidly from 20 to 36.
  • Patient 6 carries a FUS gene mutation. After 12 days of plasminogen treatment, the patient's ALSFRS-R score increased from 27 to 29.
  • plasminogen The therapeutic effects of plasminogen on ALS were compared with two existing FDA-approved drugs for the treatment of ALS, Riluzole and Edaravone. According to literature reports, after 6 months of treatment with Riluzole, the ALSFRS-R score of ALS patients decreased by -7.0 ⁇ 7.1 points; after 6 months of treatment with Edaravone, the ALSFRS-R score of ALS patients decreased by -5.01 ⁇ 0.64 points [9,10]. After 0.5 months of treatment with plasminogen, the ALSFRS-R scores of 9 ALS patients increased by 4.11 ⁇ 5.30 points, and no side effects were observed during the use of plasminogen (Figure 26C and Table 3).

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Abstract

本申请涉及一种促进病理性TDP-43蛋白降解的方法，包括给药受试者治疗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。本申请还涉及促进病理性TDP-43蛋白降解的药物及其用途。

Description

一种促进病理性TDP-43蛋白降解的方法和药物 技术领域
本申请涉及一种促进病理性TDP-43蛋白降解和治疗病理性TDP-43蛋白相关疾病的方法，包括给药受试者有效量的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。本申请还涉及用于此用途的、包含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的药物组合物。
背景技术
TDP-43全称为反式反应DNA结合蛋白43(Transactive response DNA-binding protein 43)是一种广泛存在于细胞中的蛋白质。它可以与DNA和RNA结合，在细胞内的RNA转录、选择性剪接以及mRNA稳定性调节等过程中发挥重要的作用。正常的TDP-43位于各种亚细胞结构中，包括线粒体、线粒体相关膜、RNA颗粒和应激颗粒，以调节内质网-线粒体结合、线粒体蛋白翻译以及mRNA转运和翻译。因此，TDP-43的正常生理功能对细胞存活尤为重要。
TDP-43还可以使蛋白质-蛋白质相互结合，形成同源二聚体和多聚体。在TDP-43 C末端存在着一个谷氨酰胺的富集区，该区域负责大部分的聚集。最近的研究发现，有几个因素影响了TDP-43的聚集过程，它们要么改变了蛋白质结构本身，要么改变了周围环境的蛋白质，从而造成TDP-43的聚集。
已在越来越多的神经退行性病症中鉴定出TDP-43聚集体(Lagier-Tourenne etal.，Human Molecular Genetics，2010，Vol.19，Review Issue 1 R46-R64)，包括但不限于：额颞叶痴呆(散发性或家族性、伴有或不伴有运动神经元疾病(motor-neuron disease，MND)、有颗粒蛋白前体(GRN)突变、有TARDBP突变、有含缬酪肽蛋白(valosine-containingprotein，VCP)突变、与9p染色体连锁、皮质基底节变性、有泛素阳性包涵体的额颞叶变性、嗜银颗粒病(Argyrophilic grain disease)、皮克病(Pick’s disease)，等)、肌萎缩侧索硬化(散发性ALS、有TARDBP突变、有血管生成蛋白(angiogenin，ANG)突变)、阿尔茨海默病(AD，散发性和家族性)、唐氏综合征(Down  syndrome)、家族性英国型痴呆(FamilialBritish dementia)、多聚谷氨酰胺病(亨廷顿病(Huntington’s disease)和脊髓小脑共济失调3型(SCA3(spinocerebellar ataxia type 3)；也称为马查多-约瑟夫病(MachadoJoseph Disease)))、海马硬化性痴呆和肌病(散发性包涵体肌炎；包涵体肌病，有含缬酪肽蛋白(VCP)突变；以及骨佩吉特病(Paget disease of bone)和额颞痴呆)；有镶边空泡(rimmed vacuole)的眼咽肌营养不良；有肌节蛋白(MYOT)基因突变或编码结蛋白(DES)之基因的突变的肌原纤维肌病)。
来自患者脑的聚集TDP-43显示出大量异常修饰，包括过度磷酸化、泛素化、乙酰化和通过蛋白水解切割的C-末端片段(Arai et al.，Biochemical and BiophysicalResearch Communications 351(2006)602-611；Neumann et al.，Science 314，(2006)，130-133；Neumann et al.，Acta Neuropathol.(2009)117：137-149；Hasegawa et al.，(2008)Annals of Neurology Vol 64 No 1，60-70；Cohen et al.，Nat Commun.6：5845，2015)。TDP-43病理状况的另一个特征性特征是TDP-43从细胞核到胞质的重分布和积累。FTLD-TDP的标志性病变是神经元胞质包涵体和胶质细胞胞质包涵体(分别为NCI(neuronalcytoplasmic inclusion)和GCI(glial cytoplasmic inclusion))和营养不良性神经突(dystrophic neurite，DN)。
额颞痴呆(frontotemporal dementia,FTD)是一个临床术语，其涵盖基于额和颞叶的变性-称为额颞叶变性(FTLD)的病理性特征的广谱障碍。FTD是65岁以下年龄组中早期退行性痴呆的第二大最主要原因(Le Ber，Revue Neurologique 169(2013)811-819)。FTD表现为数种综合征，包括以人格和行为变化为特征的bvFTD；以语言功能变化为特征的语义痴呆(semantic dementia,SD)和进行性非流利性失语(progressive nonfluent aphasia,PNFA)；以运动功能障碍为特征的皮质基底节综合征(corticobasal syndrome，CBS)、进行性核上性麻痹综合征和运动神经元疾病(FTD-MND)。
肌萎缩侧索硬化(ALS)是一种神经退行性疾病，其特征在于上和下运动神经元的过早丧失。ALS的进展以致命性麻痹和呼吸衰竭为特征，其病程从诊断到死亡为1至5年。在大多数散发性ALS病例中，该神经病理的特征在于初级运动皮质、脑干运动核、脊髓和相关白质道的神经元和胶质细胞中TDP-43的异常胞质积累。伴有痴呆的ALS涉及运动外新皮质和海马中TDP-43的积累。TDP-43磷酸化在ALS患者中的作用已借助于抗体进行 了探索(Hasegawa et al.，Ann Neurol 2008；64：60-70；Neumannet al.，Acta Neuropathol(2009)117：137-149)。
TDP-43病理状况发生在多至57％的患有阿尔茨海默病的患者的脑中(Josephs KAet al.，Acta Neuropathol.2014；127(6)：811-824；Josephs KA et al.，ActaNeuropathol.2014；127(3)：441-450；McAleese et al.，Brain Pathol.2017Jul；27(4)：472-479)。TDP-43聚集与患者的年龄相关，并且与AD中认知下降、记忆丧失和颞内侧萎缩相关。与TDP-43阴性对象相比，TDP-43阳性患者死亡时认知损伤可能性高10倍。病理性TDP-43遵循常规的进行性沉积模式，TDP-43首先在杏仁核中沉积(I期)，然后在海马、边缘、颞并且最终是额叶纹状体(frontostriatum)(V期)中沉积(Josephs KA et al.，Acta Neuropathol.2014；127(6)：811-824；Josephs KA et al.，Acta Neuropathol.2014；127(3)：441-450)。
最近的证据支持β-淀粉样蛋白、τ、α-突触核蛋白和TDP-43通过朊病毒样机制在神经元组织中进行蛋白质传播的概念(Hasegawa et al.，2017。尽管ALS发作和初始症状在患者间显著不同，但疾病进展的共同特征是病理从最初的病灶区域向大多数神经元扩散。症状的持续恶化可通过TDP-43病理状况的这种进行性扩散来解释。ALS患者脑中的TDP-43病理状况显示以四阶段过程扩散并且认为使用顺行轴突运输通过离皮质轴突投射经突触发生传播(Brettschneider et al.，AnnNeurol.2013 July；74(1)：20-38.)。
一些最近报道涉及TDP-43在多种体外模型中在分子水平上的扩散。来自患者脑的不溶性TDP-43制备物能够在体外诱导胞内聚集体形成(Nonaka et al.，Cell Reports 4(2013)，124-134；Feiler et al.，2015；Porta et al.，Nat.Comm.，2018)。而且，最近已经表明，患者来源的病理性TDP-43在接种到转基因和野生型小鼠中之后可导致内源性TDP-43的广泛沉积(Porta et al.，Nat.Comm.，2018)。此外，已表明胞内TDP-43聚集体在扩散至下一个细胞之前与外泌体联合释放(Nonaka et al.，Cell Reports 4(2013，124-134))。
TDP-43聚集和病理扩散是ALS和FTD目前不可治愈的致命性疾病的主要标志。TDP-43中的突变与ALS和FTD的家族性病例相关，提供了TDP-43错误折叠与疾病进展之间的因果联系。因此，需要旨在寻找促进病理性TDP-43蛋白降解或减少TDP-43聚集体的方法，以治疗病理性TDP-43蛋白相关的疾病。
发明概述
本申请研究发现纤溶酶原能够某种程度上促进神经和肌肉组织中病理性TDP-43蛋白的降解，治疗病理性TDP-43蛋白聚集相关的疾病，例如ALS和额颞叶失智症(又称额颞叶痴呆)。
具体地，本申请涉及以下各项：
1.一种促进病理性TDP-43蛋白降解的方法，包括给药受试者治疗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：促进神经组织中病理性TDP-43蛋白的降解、促进肌肉组织中病理性TDP-43蛋白的降解在一些实施方案中，所述化合物促进肌肉组织细胞内和/或外病理性TDP-43蛋白的降解。在一些实施方案中，所述化合物促进神经组织细胞(例如神经细胞或神经胶质细胞)内和/或外病理性TDP-43蛋白的降解。
5.一种治疗受试者病理性TDP-43蛋白相关疾病的方法，包括给药所述受试者治疗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，所述病理性TDP-43蛋白相关疾病为选自如下的一种或多种：肢体型肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)、延髓型肌萎缩侧索硬化症、Fus基因突变肌萎缩侧索硬化症、阿尔茨海默症(Alzheimer’s disease)、嗜银颗粒性认知症(argyrophilic grain disease)、关岛肌肉萎缩性脊髓侧索硬化-帕金森氏失智症(ALS-parkinsonism dementia complex of Guam)、血管性失智症(vascular dementia)、额颞叶失智症(frontotemporal dementia)、语意失智症(semantic dementia)、雷维体失智(dementia with Lewy bodies)、亨汀顿氏舞蹈症(Huntington’s disease)、小脑萎缩症(Spinocere bellarataxia)、包涵体肌病(inclusion body myopathy)、包涵体肌炎(inclusion body myositis)和帕金森氏症(Parkinson’s disease)。
6.项5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。
7.项5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。
8.项1-7任一项的方法，其中所述化合物为纤溶酶原或纤溶酶。
9.项1-8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。
10.项1-9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。
11.项1-10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。
12.项1-11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。
13.项1-12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。
14.项1-13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。
15.项14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。
16.项1-15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、动脉内或肌肉内给药。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纤溶酶原促进中枢神经病理性蛋白降解机制示意图。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)，小胶质细胞(Microglia)，细胞核(Nucleus)。现有结果显示，纤溶酶原可促进TDP-43、超氧化物歧化酶-1(SOD1)等中枢神经病理性蛋白降解，可改善包括肌萎缩侧索硬化症在内多种神经退行性疾病的临床症状。根据现有数据推测纤溶酶原促进中枢神经病理性蛋白降解机制如下：(1)纤溶酶原穿过血脑屏障，进入中枢神经系统，并在此富集，被活化形成纤溶酶，纤溶酶直接降解细胞外基质异常沉积的中枢神经病理性蛋白比如淀粉样蛋白Aβ，降解形成的蛋白片段进一步被小胶质细胞吞噬，后被溶酶体降解；(2)纤溶酶原经过胞吞作用进入细胞内或细胞核内，经活化形成纤溶酶，促进细胞内的中枢神经病理性蛋白降解比如TDP-43、SOD1、TAU、α-synuclein等；(3)纤溶酶原进入细胞内调节细胞内的蛋白降解系统—泛素蛋白酶体系统(ubiquitin proteasome  system，UPS)的功能，通过UPS系统降解中枢神经病理性蛋白；(4)纤溶酶原进入细胞内调节细胞内的蛋白降解系统—自噬溶酶体系统的功能，通过自噬溶酶体系统系统降解中枢神经病理性蛋白；(5)此外，有研究报道，中枢神经病理性蛋白包括TDP-43和SOD1具有与朊蛋白相似的传染性，纤溶酶原可能具有阻止中枢神经病理性蛋白在细胞间传播的能力。
图2A-B在正常小鼠脑匀浆液中，纤溶酶原对TDP-43蛋白的作用。A为Western blot图片，B为TDP-43蛋白条带光密度定量分析结果。结果显示，重组的TDP-43蛋白单体(monomer)分子量约为43kDa，高分子量的TDP-43蛋白(HMW)分子量为＞55kDa，低分子量TDP-43片段(LMW)分子量为＜40kDa。此外，在正常小鼠脑匀浆液中，纤溶酶原组TDP-43单体、HMW和LMW的量均显著低于溶媒对照组，差异极显著(***代表P＜0.001，*代表P<0.05)。提示纤溶酶原能够促进正常小鼠脑匀浆中TDP-43裂解。
图3A-B在肌萎缩测索硬化症模型小鼠脑匀浆液中，纤溶酶原对TDP-43蛋白的作用。A为Western blot图片，B为TDP-43蛋白条带光密度定量分析结果。结果显示，重组的TDP-43蛋白单体(monomer)分子量约为43kDa，高分子量的TDP-43蛋白(HMW)分子量为＞55kDa，低分子量TDP-43片段(LMW)分子量为＜40kDa。此外，在肌萎缩测索硬化症模型小鼠脑匀浆液中，纤溶酶原组TDP-43单体、HMW和LMW的量均显著低于溶媒对照组，差异极显著(***代表P＜0.001，**代表P<0.01)。提示纤溶酶原能够促进肌萎缩侧索硬化症模型小鼠脑匀浆中TDP-43裂解。
图4A-B纤溶酶原促进肌萎缩测索硬化症模型小鼠脊髓组织中TDP-43蛋白降解。A为Western blot图片，B为TDP-43蛋白条带光密度定量分析结果。结果显示，给药组小鼠脊髓组织中TDP-43单体和低分子量TDP-43的量均明显低于溶媒组，且统计差异显著(*代表P<0.05)。说明纤溶酶原能够促进肌萎缩侧索硬化症模型小鼠脊髓组织中TDP-43降解。
图5A-B纤溶酶原促进病理性TDP-43蛋白脑定位注射的肌萎缩测索硬化症模型小鼠脑组织中TDP-43蛋白降解。A为Western blot图片，B为TDP-43蛋白条带光密度定量分析结果。结果显示，给药组小鼠脑组织中TDP-43单体和低分子量TDP-43的量均明显低于溶媒组，且统计差异显著(*代表P<0.05)。说明纤溶酶原能够促进肌萎缩侧索硬化症模型小鼠脑组织中TDP-43降解。
图6A-I给予纤溶酶原于肌萎缩侧索硬化症模型小鼠后，小鼠脊髓组织纤溶酶原和TDP-43免疫荧光共定位染色代表性图片。A-C：正常对照组，D-F：溶媒组，G-I：给药组。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(如△所示)和细胞核内(如所示)。纤溶酶原与TDP-43(红色荧光)胞质内(如▲所示)和核内共定位(如所示)。且给药组脊髓组织TDP-43水平低于溶媒组，且给药组纤溶酶原与TDP-43共定位多于溶媒组。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TDP-43共定位，并可降解TDP-43。
图7A-I给予纤溶酶原于肌萎缩侧索硬化症模型小鼠后，小鼠肌肉组织纤溶酶原和TDP-43免疫荧光共定位染色代表性图片。A-C：正常对照组，D-F：溶媒组，G-I：给药组。结果显示，给药组纤溶酶原(绿色荧光)在肌肉组织中的阳性着色明显多于溶媒组，说明纤溶酶原可在肌肉组织富集。此外，纤溶酶原存在于胞质(如△所示)和细胞核内(如所示)。纤溶酶原与TDP-43(红色荧光)胞质内(如▲所示)和核内共定位(如所示)。说明在肌萎缩侧索硬化症模型小鼠中纤溶酶原能够在肌肉组织富集，进入细胞内，与TDP-43共定位。
图8A-B给予纤溶酶原于冈田酸诱导的痴呆模型小鼠脑匀浆TDP-43水平WB检测结果。A为Western blot图片，B为TDP-43蛋白条带光密度定量分析结果。结果显示，给药组小鼠脑组织中TDP-43单体和低分子量TDP-43的水平明显低于溶媒组。说明纤溶酶原能够促进冈田酸诱导的痴呆模型小鼠脑组织中TDP-43降解。
图9A-B给予纤溶酶原肌萎缩测索硬化症模型小鼠肾细胞核中TDP-43水平WB检测结果。A为Western blot图片，B为TDP-43蛋白条带光密度定量分析结果。结果显示，给药组肾细胞核中TDP-43的水平显著低于溶媒组(*代表P<0.05)。提示纤溶酶原能够促进肾细胞核中TDP-43降解。
图10A-D给予纤溶酶原冈田酸处理NSC34细胞浆和细胞核中TDP-43水平WB检测结果。A为细胞浆Western blot图片，B为细胞浆TDP-43蛋白条带光密度定量分析结果，C为细胞核Western blot图片，D为细胞核TDP-43蛋白条带光密度定量分析结果。结果显示，给药组细胞浆和细胞核中TDP-43的水平接近显著或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<0.01，***代表P<0.001)。提示纤溶酶原能够细胞浆和细胞核中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11A-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12尾静脉注射纤溶酶原于SOD1-G93A小鼠后，不同时间点血浆纤溶酶原水平ELISA法检测结果。SOD1-G93A小鼠血浆ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，SOD1-G93A小鼠血浆纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全。*代表P<0.05，**代表P<0.01，***代表P<0.001。该结果表明(1)纤溶酶原在血浆中水平具有剂量依赖效应，纤溶酶原施予浓度越高，聚集的越多；(2)纤溶酶原在血浆中水平具有时间依赖效应，先升高，在2至12小时逐渐降低。
图13A-B静脉注射纤溶酶原于SOD1-G93A小鼠后，不同时间点脑组织纤溶酶原水平ELISA法检测结果以及脑组织中纤溶酶原与血浆中纤溶酶原比值。A为脑组织纤溶酶原水平ELISA法检测结果，B为脑组织中纤溶酶原与血浆中纤溶酶原比值。SOD1-G93A小鼠脑ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，SOD1-G93A小鼠脑组织纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全。给药纤溶酶原2、6、12小时后脑组织纤溶酶原水平与血液中纤溶酶原水平比值分别为3.47％、4.94％和6.79％。该结果表明(1)在生理和病理情况下施予纤溶酶原后，可促进纤溶酶原穿过血脑屏障，在脑 组织富集；(2)静脉注射纤溶酶原于小鼠体内，脑组织纤溶酶原水平显著增加；(3)纤溶酶原在脑组织富集具有时间依赖效应，先升高，2至12小时逐渐降低，在12至24小时几乎全部完全代谢；(4)纤溶酶原在脑组织富集具有剂量依赖效应，施予剂量越高，脑组织中纤溶酶原水平越高。**代表P<0.01，***代表P<0.001。
图14静脉注射纤溶酶原于SOD1-G93A小鼠后，不同时间点脊髓组织纤溶酶原水平ELISA法检测结果。SOD1-G93A小鼠脊髓ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，SOD1-G93A小鼠脊髓纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全。*代表P<0.05，**代表P<0.01。该结果表明(1)施予生理剂量水平的纤溶酶原能够穿过SOD1-G93A小鼠血脑屏障，在脊髓组织富集；(2)纤溶酶原在脊髓的富集具有剂量依赖效应，纤溶酶原施予剂量越高，富集的越多；(3)纤溶酶原在脊髓的富集具有时间依赖效应，先升高，在2至12小时逐渐降低，在12至24小鼠基本完全代谢。
图15A-B单次静脉注射纤溶酶原于SOD1-G93A小鼠后，脑组织匀浆中纤溶酶原水平和纤溶酶活性检测结果。A为纤溶酶原水平ELISA法检测结果，B为纤溶酶活性水平酶底物动力学法检测结果。结果显示，给药组小鼠脑组织匀浆中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(*代表P<0.05，***代表P<0.001)。提示静脉施予纤溶酶原能够促进脑组织中纤溶酶原水平和纤溶酶活性增加。
图16连续静脉注射纤溶酶原于SOD1-G93A小鼠7天后，脑组织、脊髓组织和肾组织细胞核纤溶酶原水平检测结果。结果显示，给药纤溶酶原7天后，给药组SOD1-G93A小鼠脑组织、脊髓组织和肾组织细胞核中人纤溶酶原水平显著高于溶媒组，且统计差异极为显著(***代表P<0.001)。提示静脉施予纤溶酶原能够促进脑组织、脊髓组织和肾组织细胞核中人纤溶酶原水平增加。
图17尾静脉注射纤溶酶原于帕金森模型小鼠后，不同时间点血液纤溶酶原水平ELISA法检测结果。结果显示，给药组小鼠血液中纤溶酶原水平 明显高于溶媒组小鼠，纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全。***代表P<0.001。
图18尾静脉注射纤溶酶原于帕金森模型小鼠后，不同时间点血液纤溶酶原水平ELISA法检测结果。结果显示，给药组小鼠脑组织中纤溶酶原水平明显高于溶媒组小鼠，纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全。结果表明尾静脉注射的纤溶酶原能够穿过血脑屏障，促进帕金森模型小鼠脑组织中纤溶酶原水平增加。***代表P<0.001。
图19静脉注射纤溶酶原于帕金森模型小鼠后，不同时间点脊髓组织纤溶酶原水平ELISA法检测结果。结果显示，给药组小鼠脊髓组织中纤溶酶原水平明显高于溶媒组小鼠，纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全。结果表明尾静脉注射的纤溶酶原能够穿过血脑屏障，促进帕金森模型小鼠脊髓组织中纤溶酶原水平增加。**代表P<0.01，***代表P<0.001。
图20尾静脉注射纤溶酶原于帕金森模型小鼠后，不同时间点脊髓或脑组织纤溶酶原水平与血液中纤溶酶原水平比值。结果显示，给药纤溶酶原2、6、12小时后脊髓组织纤溶酶原水平与血液中纤溶酶原水平比值分别为1.24％、1.16％和1.46％，给药纤溶酶原2、6、12小时后脑组织纤溶酶原水平与血液中纤溶酶原水平比值分别为3.47％、4.18％和8.51％。*代表P<0.05，**代表P<0.01。结果表明尾静脉注射的纤溶酶原能够穿过血脑屏障，促进帕金森模型小鼠脑和脊髓组织中纤溶酶原水平增加。
图21尾静脉注射纤溶酶原于帕金森模型小鼠后，脑组织纤溶酶活性水平检测结果。结果显示，给药组小鼠脑组织中纤溶酶活性水平明显高于溶媒组小鼠，且统计差异显著。*代表P<0.05。结果表明尾静脉注射的纤溶酶原能够穿过血脑屏障，促进帕金森模型小鼠脑组织中纤溶酶活性水平增加。
图22尾静脉注射纤溶酶原于FAD小鼠后，不同时间点血浆纤溶酶原水平ELISA法检测结果。FAD小鼠血浆ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，FAD小鼠血浆纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全。该结果表 明(1)纤溶酶原在血浆中水平具有剂量依赖效应，纤溶酶原施予浓度越高，聚集的越多；(2)纤溶酶原在血浆中水平具有时间依赖效应，先升高，在2至12小时逐渐降低。
图23A-B尾静脉注射纤溶酶原于FAD小鼠后，不同时间点脑组织纤溶酶原水平ELISA法检测结果(A),不同时间点脑组织纤溶酶原水平与血液中纤溶酶原水平比值(B)。FAD小鼠脑ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，FAD小鼠脑组织纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全。6mg/kg纤溶酶原组小鼠在接受纤溶酶原注射2和6小时后脑组织中纤溶酶原与血液中纤溶酶原比值分别为3.59％和4.23％；50mg/kg纤溶酶原组小鼠在接受纤溶酶原注射2、6和12小时后脑组织中纤溶酶原与血液中纤溶酶原比值分别为2.49％、2.31％和3.32％。该结果表明(1)在生理和病理情况下施予纤溶酶原后，可促进纤溶酶原穿过血脑屏障，在脑组织富集；(2)静脉注射纤溶酶原于小鼠体内，脑组织纤溶酶原水平显著增加；(3)纤溶酶原在脑组织富集具有时间依赖效应，先升高，2至12小时逐渐降低，在12至24小时几乎全部完全代谢；(4)纤溶酶原在脑组织富集具有剂量依赖效应，施予剂量越高，脑组织中纤溶酶原水平越高。
图24尾静脉注射纤溶酶原于FAD小鼠2小时后，酶底物动力学法检测脑匀浆中纤溶酶活性结果。结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，FAD小鼠脑组织纤溶酶活性水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。该结果表明，注射纤溶酶原于小鼠体内，脑组织纤溶酶水平显著增加，此外，纤溶酶在脑组织中活性具有剂量依赖效应，施予剂量越高，脑组织中纤溶酶原水平越高。
图25A-B尾静脉注射纤溶酶原于FAD小鼠后，不同时间点脊髓组织纤溶酶原水平ELISA法检测结果(A),不同时间点脊髓组织纤溶酶原水平与血液中纤溶酶原水平比值(B)。FAD小鼠脊髓ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，FAD小鼠脊髓组织纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完 全。6mg/kg纤溶酶原组小鼠在接受纤溶酶原注射2和6小时后脊髓组织中纤溶酶原与血液中纤溶酶原比值分别为0.93％和1.62％；50mg/kg纤溶酶原组小鼠在接受纤溶酶原注射2、6和12小时后脊髓组织中纤溶酶原与血液中纤溶酶原比值分别为0.33％、0.40％和1.56％。该结果表明(1)在生理和病理情况下施予纤溶酶原后，可促进纤溶酶原穿过血脑屏障，在脊髓组织富集；(2)静脉注射纤溶酶原于小鼠体内，脊髓组织纤溶酶原水平显著增加；(3)纤溶酶原在脊髓组织富集具有时间依赖效应，先升高，2至12小时逐渐降低，在12至24小时几乎全部完全代谢；(4)纤溶酶原在脊髓组织富集具有剂量依赖效应，施予剂量越高，脊髓组织中纤溶酶原水平越高。
图26A-C用药纤溶酶原前后ALS患者临床表型变化。A:用药纤溶酶原前后9名ALS患者ALSFRS-R评分，B:患者5第二疗程用药过程中最大行走步数，C:9名ALS患者用药纤溶酶原0.5个月中ALSFRS-R评分(实线表示)以及ALS患者用药Riluzole或Edaravone 6个月ALSFRS-R评分(虚线表示)。
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(February 2008)."Tissue-type plasminogen activator as a therapeutic target in stroke".Expert Opinion on Therapeutic Targets.12(2):159–70；Geiger M,Huber K,Wojta J,Stingl L,Espana F,Griffin JH,Binder BR(Aug 1989)."Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo".Blood.74(2):722–8.
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。
“病理性TDP-43蛋白”是和“生理功能性TDP-43蛋白”相对的术语。生理功能性TDP-43蛋白是指在体内细胞环境中处于能够表现出其所期望功能的状态中的TDP-43蛋白。相比之下，“病理性TDP-43蛋白”是指在体内细胞环境中不能够表现出其所期望功能的TDP-43蛋白。病理性TDP-43蛋白的实例包括但不限于：发生突变而失去其生理功能(例如丧失超过50％、60％、70％、80％、90％的其相关生理功能)的TDP-43蛋白、形成蛋白聚集体的TDP-43蛋白、错误折叠的TDP-43蛋白、发生异常修饰的TDP-43蛋白(包括过度磷酸化、泛素化、乙酰化和通过蛋白水解切割的C-末端片段)、以及发生蛋白变性的TDP-43蛋白。TDP-43病理状况的另一个特征性特征是TDP-43从细胞核到胞质的重分布和积累，这样的蛋白也涵盖在本申请病理性TDP-43蛋白的范围内。在本申请中，在纤溶酶原“促进TDP-43(蛋白)降解”的语境下，意指纤溶酶原促进病理性TDP-43(蛋白)的降解。
“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.2006 Mini Rev.Med Chem.6:3-10和Camarero JA等2005 Protein 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。
本申请的“肌肉萎缩”为各种原因导致的肌肉组织的量的减少、结构异常或降低或丧失和/或功能异常或减弱或丧失。肌肉萎缩的病因主要有各种肌肉疾病或外伤，包括脊髓空洞症、脊髓炎、神经根型脊椎关节病、脑底蛛网膜炎、脑干病变以及脑、脊神经病变等。
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.1976 Jun 25；251(12):3693-9；HAGAN JJ,ABLONDI FB,DE RENZO EC.Purification and biochemical properties of human plasminogen.J Biol Chem.1960 Apr；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(Plasminogen)促进病理性TDP-43蛋白在正常小鼠脑匀浆中裂解
取11～12周龄、18-25g的C57BL/6J雄性小鼠4只，处死后取整个脑部并称重，按150mg组织/mLPBS分别加入1×PBS(Thermo Fisher，pH7.4； 10010-031，4℃下匀浆(1min，3-4次)，匀浆后于4℃离心(12000rpm，20min)，取上清液即脑匀浆液置于新的EP管中。
取Eppendorf(EP)管分别设为①空白组、②空白对照组、③溶媒对照组、④纤溶酶原组，各组设置5个平行。空白组加入21.5μL生理盐水、4.6μL溶媒溶液(10mM柠檬酸钠，2％盐酸精氨酸，3％甘露醇，pH 7.4)、23.9μL小鼠脑匀浆；空白对照组加入21.5μL生理盐水、2.3μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆；溶媒对照组加入20.5μL TDP-43(南京金斯瑞生物科技有限公司，定制表达人TDP-43，C134WHE160-2/P5HF001，1.05mg/mL)、4.6μL溶媒溶液、23.9μL小鼠脑匀浆；纤溶酶原组加入20.5μL TDP-43(1.05mg/mL)、2.3μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆。各组样品加好后，37℃温育3h后分别加入50μL 0.1％三氟乙酸溶液终止反应。
根据SDS-PAGE配胶说明制备12％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，加入兔抗人TDP-43抗体(Proteintech(China),12892-1-AP)和亲环素抗体室温孵育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，重组的TDP-43蛋白单体(monomer)分子量约为43kDa，高分子量的TDP-43蛋白(HMW)分子量为＞55kDa，低分子量TDP-43片段(LMW)分子量为＜40kDa。此外，在正常小鼠脑匀浆液中，纤溶酶原组TDP-43单体、HMW和LMW的量均显著低于溶媒对照组，差异极显著(***代表P＜0.001，*代表P<0.05)(图2)。提示纤溶酶原能够促进正常小鼠脑匀浆中TDP-43裂解。
实施例2纤溶酶原(Plasminogen)促进病理性TDP-43蛋白在肌萎缩测索硬化症模型小鼠脑匀浆中裂解
取B6.Cg-Tg(SOD1-G93A)1Gur/J转基因雄性小鼠(简称SOD1-G93A转基因小鼠)4只，处死后取整个脑部并称重，按150mg组织/mLPBS分别加入1×PBS(Thermo Fisher，pH7.4；10010-031，4℃下匀浆(1min，3-4次)，匀浆后于4℃离心(12000rpm，20min)，取上清液即脑匀浆液置于新的EP管中。
取Eppendorf(EP)管分别设为①空白组、②空白对照组、③溶媒对照组、④纤溶酶原组，各组设置5个平行。空白组加入21.5μL生理盐水、4.6μL溶媒溶液(10mM柠檬酸钠，2％盐酸精氨酸，3％甘露醇，pH 7.4)、23.9μL小鼠脑匀浆；空白对照组加入21.5μL生理盐水、2.3μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆；溶媒对照组加入20.5μL TDP-43(南京金斯瑞生物科技有限公司，定制表达人TDP-43，C134WHE160-2/P5HF001，1.05mg/mL)、4.6μL溶媒溶液、23.9μL小鼠脑匀浆；纤溶酶原组加入20.5μL TDP-43(1.05mg/mL)、2.3μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆。各组样品加好后，37℃温育3h后分别加入50μL 0.1％三氟乙酸溶液终止反应。
根据SDS-PAGE配胶说明制备12％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，加入兔抗人TDP-43抗体(Proteintech,12892-1-AP)和亲环素抗体室温孵育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，重组的TDP-43蛋白单体(monomer)分子量约为43kDa，高分子量的TDP-43蛋白(HMW)分子量为＞55kDa，低分子量TDP-43片段(LMW)分子量为＜40kDa。此外，在肌萎缩测索硬化症模型小鼠脑匀浆液中，纤溶酶原组TDP-43单体、HMW和LMW的量均显著低于溶媒对照 组，差异极显著(***代表P＜0.001，**代表P<0.01)(图3)。提示纤溶酶原能够促进肌萎缩侧索硬化症模型小鼠脑匀浆中TDP-43裂解。
实施例3纤溶酶原促进肌萎缩侧索硬化症模型小鼠脊髓组织中TDP-43降解
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TDP-43 western blot检测。
结果显示，给药组小鼠脊髓组织中TDP-43单体和低分子量TDP-43的量均明显低于溶媒组，且统计差异显著(*代表P<0.05)(图4)。说明纤溶酶原能够促进肌萎缩侧索硬化症模型小鼠脊髓组织中TDP-43降解。
实施例4纤溶酶原促进肌萎缩侧索硬化症模型小鼠脑组织中TDP-43降解
取6-7周龄的C57BL/6J雌性小鼠9只，造模前进行称重，称重后所有小鼠随机分为2组，空白对照组,3只，模型组6只。分组完成后，假手术组和模型组小鼠使用腹腔注射三溴乙醇麻醉，麻醉剂量20mL/kg；模型小鼠根据小鼠立体定位图谱，定位于海马(根据前卤点的坐标定位：AP-2.54mm，ML±2mm，DV-2.4mm)，每只小鼠双侧分别缓慢微量注射，假手术组小鼠仅在坐标定位点钻孔，不进行注射[4]。模型组小鼠注射TDP-43溶液，注射速率为0.5μL/min，注射体积3μL/侧，注射完毕后，注射器停留5min后缓慢退出。脑定位注射3天后，所有小鼠称重，模型组小鼠根据体重腹腔注射5mg/kg LPS溶液，随后将模型组小鼠随机分为两组，给药组3只，溶媒组3只，LPS注射24小时后，假手术组小鼠、溶媒组小鼠尾静脉注射溶媒5mL/kg，给药组小鼠尾静脉注射纤溶酶原50mg/kg，连续给药3天。第三次给药2小时后处死小鼠取材脑组织。匀浆后行TDP-43 western blot检测。
结果显示，给药组小鼠脑组织中TDP-43单体和低分子量TDP-43的量均明显低于溶媒组，且统计差异显著(*代表P<0.05)(图5)。说明纤溶酶原能够促进肌萎缩侧索硬化症模型小鼠脑组织中TDP-43降解。
实施例5纤溶酶原在肌萎缩侧索硬化症模型小鼠脊髓组织中富集且与TDP-43在细胞内共定位
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XTSA520 IHC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TDP-43抗体(Proteintech,12892-1-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TDP-43二抗的红色荧光显色。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。
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(如△所示)和细胞核内(如所示)。纤溶酶原与TDP-43(红色荧光)胞质内(如▲所示)和核内共定位(如所示)。且给药组脊髓组织TDP-43水平低于溶媒组，且给药组纤溶酶原与TDP-43共定位多于溶媒组(图6)。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TDP-43共定位，并可降解TDP-43。
实施例6纤溶酶原在肌萎缩侧索硬化症模型小鼠肌肉组织中富集且与TDP-43在细胞内共定位
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。取材臀肌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XTSA520 IHC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TDP-43抗体(Proteintech,12892-1-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TDP-43二抗的红色荧光显色。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。
结果显示，给药组纤溶酶原(绿色荧光)在肌肉组织中的阳性着色明显多于溶媒组，说明纤溶酶原可在肌肉组织富集。此外，纤溶酶原存在于胞质(如△所示)和细胞核内(如所示)。纤溶酶原与TDP-43(红色荧 光)胞质内(如▲所示)和核内共定位(如所示)(图7)。说明在肌萎缩侧索硬化症模型小鼠中纤溶酶原能够在肌肉组织富集，进入细胞内，与TDP-43共定位。
实施例7纤溶酶原促进冈田酸诱导的痴呆模型小鼠脑组织中TDP-43降解
取30-32周龄的B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax(以下简称FAD)雌性小鼠(购自Jackson lab，品系号：Stock Number：034840)10只，造模前进行称重，做为模型组，选取6-7周龄的C57雌性小鼠5只做为空白组。分组完成后，空白组组和模型组小鼠使用腹腔注射三溴乙醇麻醉，麻醉剂量20mL/kg；模型小鼠根据小鼠立体定位图谱，定位于基底外侧杏仁核(根据前卤点的坐标定位：AP-1.94mm，ML±3.15mm，DV-4.5mm)，每只小鼠双侧分别缓慢微量注射，空白组小鼠仅在坐标定位点钻孔，不进行注射[5]。模型组小鼠注射50ng/μL冈田酸(厂商：上海源叶生物科技有限公司，货号S30686-25ug：)溶液，注射速率为0.5μL/min，注射体积2μL，注射完毕后，注射器停留6min后缓慢退出。脑定位注射3天后，所有小鼠称重，模型组小鼠根据体重将模型组随机分为两组，给药组5只，溶媒组5只，空白组小鼠、溶媒组小鼠尾静脉注射溶媒5mL/kg，给药组小鼠尾静脉注射纤溶酶原50mg/k。单次给药6小时后处死小鼠取材脑组织。匀浆后行TDP-43western blot检测。
结果显示，给药组小鼠脑组织中TDP-43单体和低分子量TDP-43的水平明显低于溶媒组(图8)。说明纤溶酶原能够促进冈田酸诱导的痴呆模型小鼠脑组织中TDP-43降解。
实施例8纤溶酶原促进肌萎缩侧索硬化症模型小鼠肾细胞核中TDP-43降解
取9-10周龄的SOD1-G93A小鼠(Jackson Laboratory,Stock Number:004435)和C57BL/6J小鼠。SOD1-G93A小鼠被随机分为两组，溶媒组和给药，C57BL/6J小鼠作为正常对照组，每组各3只。溶媒组小鼠每天尾静脉注射5mL/kg溶媒(10mM citric acid sodium citrate solution,pH 7.4)，给药组小鼠每天尾静脉注射50mg/kg的纤溶酶原。正常对照小鼠不做给药处理。7天后处死小鼠，取材肾组织。取材的肾组织被放置在冰上预冷RPMI-1640(Sigma-Aldrich)培养基中。PBS冲洗后，肾组织被切成小块，然后孵育 0.25％的胰酶进行消化，37℃，10分钟，每2分钟晃匀一次。加入含有10％胎新生牛血清的DMEM培养基终止消化，离心后(1500rpm,5分钟)，去掉上清，获取单个细胞沉淀。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。
结果显示，给药组肾细胞核中TDP-43的水平显著低于溶媒组(图9A-B)。提示纤溶酶原能够促进肾细胞核中TDP-43降解。
实施例9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[6]。
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10A-D)。提示纤溶酶原能够细胞浆和细胞核中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10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11A-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11给予纤溶酶原促进SOD1-G93A小鼠血液中纤溶酶原水平增加
取10-15周龄B6.Cg-Tg(SOD1-G93A)1Gur/J(SOD1-G93A)小鼠(谱系号：004435)(简称SOD1-G93A小鼠)(种鼠购自美国Jackson Laboratory实验室)27只，随机分为3组，溶媒对照组3只，6mg/kg纤溶酶原组12只，50mg/kg纤溶酶原组12只。溶媒对照组小鼠按照5ml/kg尾 静脉注射施予溶媒，6mg/kg纤溶酶原组小鼠按照6mg/kg体重尾静脉注射施予纤溶酶原(1.2mg/ml)，50mg/kg纤溶酶原组小鼠按照6mg/kg体重尾静脉注射施予纤溶酶原(10mg/ml)。溶媒对照组小鼠在给药2小时后处死，取材血液。6mg/kg纤溶酶原组和50mg/kg纤溶酶原组小鼠在给药2、6、12和24小时各处死3只，取材血液。血液离心后(3500rpm,10min,4℃)取上清，按照Human Plasminogen ELISA Kit(厂家：AssayMax，货号：EP1200-1)说明书操作进行检测。以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。
SOD1-G93A小鼠血浆ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，SOD1-G93A小鼠血浆纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全(图12)。
该结果表明(1)纤溶酶原在血浆中水平具有剂量依赖效应，纤溶酶原施予浓度越高，血浆纤溶酶原水平越高；(2)纤溶酶原在血浆中水平具有时间依赖效应，先升高，在2至12小时逐渐降低。
实施例12给予纤溶酶原促进SOD1-G93A小鼠脑组织中纤溶酶原水平增加
从实施例11的处死小鼠取材脑组织，匀浆后按照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。
SOD1-G93A小鼠脑ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，SOD1-G93A小鼠脑组织纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全(图13A)。给药纤溶酶原2、6、12小时后脑组织纤溶酶原水平与血液中纤溶酶原水平比值分别为3.47％、4.94％和6.79％(图13B)。该结果表明(1)在生理和病理情况下施予纤溶酶原后，可促进纤溶酶原穿过血脑屏障，在 脑组织富集；(2)静脉注射纤溶酶原于小鼠体内，脑组织纤溶酶原水平显著增加；(3)纤溶酶原在脑组织富集具有时间依赖效应，先升高，2至12小时逐渐降低，在12至24小时几乎全部完全代谢；(4)纤溶酶原在脑组织富集具有剂量依赖效应，施予剂量越高，脑组织中纤溶酶原水平越高。
实施例13给予纤溶酶原促进SOD1-G93A小鼠脊髓组织中纤溶酶原水平增加
从实施例11的处死小鼠取材脊髓组织，匀浆后按照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。
SOD1-G93A小鼠脊髓ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，SOD1-G93A小鼠脊髓纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全(图14)。
该结果表明(1)施予生理剂量水平的纤溶酶原能够穿过SOD1-G93A小鼠血脑屏障，在脊髓组织富集；(2)纤溶酶原在脊髓的富集具有剂量依赖效应，纤溶酶原施予剂量越高，富集得越多；(3)纤溶酶原在脊髓的富集具有时间依赖效应，先升高，在2至12小时逐渐降低，在12至24小鼠基本完全代谢。
实施例14给予纤溶酶原促进SOD1-G93A小鼠脑组织中纤溶酶原水平和纤溶酶活性水平增加
取22周龄的SOD1-G93A小鼠8只，随机分为两组，溶媒组和给药组，每组4只。将2.5mg/mL细菌脂多糖(LPS)(北京索莱宝科技有限公司，L8880)经气管滴注施予于溶媒组和给药组小鼠，造模剂量5mg/kg。另取4只同周龄的C57作为正常对照小鼠。LPS处理3天后开始给药。空白组小鼠、溶媒组小鼠尾静脉注射溶媒5mL/kg，给药组小鼠尾静脉注射纤溶酶原50mg/k。单次给药6小时后处死小鼠取材脑组织。匀浆后行纤溶酶原水平ELISA检测和纤溶酶活性酶底物法检测。
结果显示，给药组小鼠脑组织匀浆中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(图15A-D)。提示静脉施予纤溶酶原能够促进脑组织中纤溶酶原水平和纤溶酶活性增加。
实施例15给予纤溶酶原促进SOD1-G93A小鼠脑组织、脊髓组织和肾组织细胞核中纤溶酶原水平增加
取9周龄的SOD1-G93A小鼠10只，随机分为两组，溶媒组和给药组，每组5只。另取5只同周龄的C57作为正常对照小鼠。正常组小鼠、溶媒组小鼠尾静脉注射溶媒5mL/kg，给药组小鼠每天尾静脉注射纤溶酶原50mg/kg，连续给药7天。7天后处死小鼠取材脑组织、脊髓组织和肾组织。将组织切成小块，用0.25％的胰酶(Beyotime Biotechnology，C0201-500mL)消化后，用200目的细胞筛过滤，获得单个细胞悬液。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。提取的核蛋白行人纤溶酶原水平ELISA检测。
结果显示，给药纤溶酶原7天后，给药组SOD1-G93A小鼠脑组织、脊髓组织和肾组织细胞核中人纤溶酶原水平显著高于溶媒组，且统计差异极为显著(***代表P<0.001)(图16)。提示静脉施予纤溶酶原能够促进脑组织、脊髓组织和肾组织细胞核中人纤溶酶原水平增加。
实施例16给予纤溶酶原促进帕金森模型小鼠血液中纤溶酶原水平增加
取6周龄雄性C57BL/6J小鼠18只，小鼠按照35mg/kg/只腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine，MPTP)溶液，连续注射5天，建立帕金森模型[7]。MPTP溶液配制：取45mg MPTP(Sigma，M0896)溶解在9ml生理盐水溶液中，配制最终浓度为5mg/ml。小鼠按体重随机分为2组，溶媒组6只小鼠和给药组12只小鼠，并开始给药，记为第1天，给药组小鼠尾静脉注射 50mg/kg体重尾静脉注射给予纤溶酶原溶液，溶媒组注射5mL/kg体重尾静脉注射溶媒溶液(10mM柠檬酸-柠檬酸钠溶液，pH7.4)。溶媒组小鼠于给药后2和24小时各处死3只小鼠，取材血液，给药组小鼠于给药后2、6、12、24小时后处死，取材血液。血液离心后(3500rpm,10min,4℃)取上清，按照Human Plasminogen ELISA Kit(厂家：AssayMax，货号：EP1200-1)说明书操作进行检测。以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。
结果显示，给药组小鼠血液中纤溶酶原水平明显高于溶媒组小鼠，纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全(图17)(***代表P<0.001)。
实施例17给予纤溶酶原促进帕金森模型小鼠脑组织中纤溶酶原水平增加
从实施例16的处死小鼠取材脑组织，匀浆后按照Human Plasminogen ELISA Kit(厂家：AssayMax，货号：EP1200-1)说明书操作进行检测。以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。
结果显示，给药组小鼠脑组织中纤溶酶原水平明显高于溶媒组小鼠，纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全(图18)(***代表P<0.001)。结果表明尾静脉注射的纤溶酶原能够穿过血脑屏障，促进帕金森模型小鼠脑组织中纤溶酶原水平增加。
实施例18给予纤溶酶原促进帕金森模型小鼠脊髓组织中纤溶酶原水平增加
从实施例16的处死小鼠取材脊髓组织，匀浆后按照Human Plasminogen ELISA Kit(厂家：AssayMax，货号：EP1200-1)说明书操作进行检测。以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。
结果显示，给药组小鼠脊髓组织中纤溶酶原水平明显高于溶媒组小鼠，纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全(图19)(**代表P<0.01，***代表P<0.001)。结果表明尾静脉注射 的纤溶酶原能够穿过血脑屏障，促进帕金森模型小鼠脊髓组织中纤溶酶原水平增加。
实施例19给予纤溶酶原促进帕金森模型小鼠脑和脊髓组织中纤溶酶原水平增加
从实施例16的处死小鼠取材脑和脊髓组织，匀浆后按照Human Plasminogen ELISA Kit(厂家：AssayMax，货号：EP1200-1)说明书操作进行检测。以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。
结果显示，给药纤溶酶原2、6、12小时后脊髓组织纤溶酶原水平与血液中纤溶酶原水平比值分别为1.24％、1.16％和1.46％，给药纤溶酶原2、6、12小时后脑组织纤溶酶原水平与血液中纤溶酶原水平比值分别为3.47％、4.18％和8.51％(图20)(*代表P<0.05，**代表P<0.01)。结果表明尾静脉注射的纤溶酶原能够穿过血脑屏障，促进帕金森模型小鼠脑和脊髓组织中纤溶酶原水平增加。
实施例20纤溶酶原促进帕金森模型小鼠脑组织中纤溶酶活性增加
取雌性小鼠15只，造模前进行称重，根据体重随机分为两组，空白对照组5只，模型组10只，模型组所有小鼠按照体重35mg/kg/只腹腔注射5mg/mL MPTP溶液，空白对照组不鼠腹腔注射7mL/kg生理盐水，造模时间定在每日早上9点，连续注射5天。MPTP最后一次注射24小时后，所有小鼠称重，并腹腔注射5mg/kg LPS溶液。腹腔注射LPS 24小时后，模型组小鼠根据体重随机分为两组，给药组小鼠5只，溶媒组小鼠5只，溶媒组小鼠尾静脉注射溶媒，给药组小鼠尾静脉注射纤溶酶原50mg/kg，单次给药，给药2小时后进行解剖取材。脑组织匀浆行纤溶酶活性酶底物动力学法检测。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15μL20mM 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，给药组小鼠脑组织中纤溶酶活性水平明显高于溶媒组小鼠，且统计差异显著(图21)(*代表P<0.05)。结果表明尾静脉注射的纤溶酶原能够穿过血脑屏障，促进帕金森模型小鼠脑组织中纤溶酶活性水平增加。
实施例21给予纤溶酶原促进阿尔兹海默模型小鼠血液中纤溶酶原水平增加
取16周龄B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas Mmjax(Stock Number：034840)(简称FAD小鼠)(种鼠购自美国Jackson Laboratory实验室)27只，随机分为3组，溶媒对照组3只，6mg/kg纤溶酶原组12只，50mg/kg纤溶酶原组12只。溶媒对照组小鼠按照5ml/kg尾静脉注射施予溶媒，6mg/kg纤溶酶原组小鼠按照6mg/kg体重尾静脉注射施予纤溶酶原(1.2mg/ml),50mg/kg纤溶酶原组小鼠按照6mg/kg体重尾静脉注射施予纤溶酶原(10mg/ml)。溶媒对照组小鼠在给药2小时后处死，取材血液。6mg/kg纤溶酶原组和50mg/kg纤溶酶原组小鼠在给药2、6、12和24小时各处死3只，取材血液。血液离心后(3500rpm,10min,4℃)取上清，按照Human Plasminogen ELISA Kit(厂家：AssayMax，货号：EP1200-1)说明书操作进行检测。以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。
FAD小鼠血浆ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，FAD小鼠血浆纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全(图22)。
该结果表明(1)纤溶酶原在血浆中水平具有剂量依赖效应，纤溶酶原施予浓度越高，聚集的越多；(2)纤溶酶原在血浆中水平具有时间依赖效应，先升高，在2至12小时逐渐降低。
实施例22给予纤溶酶原促进阿尔兹海默模型小鼠脑组织中纤溶酶原水平增加
从实施例21的处死小鼠取材脑组织，匀浆后按照Human Plasminogen ELISA Kit(厂家：AssayMax，货号：EP1200-1)说明书操作进行检测。以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。
FAD小鼠脑ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，FAD小鼠脑组织纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全(图23A)。6mg/kg纤溶酶原组小鼠在接受纤溶酶原注射2和6小时后脑组织中纤溶酶原与血液中纤溶酶原比值分别为3.59％和4.23％；50mg/kg纤溶酶原组小鼠在接受纤溶酶原注射2、6和12小时后脑组织中纤溶酶原与血液中纤溶酶原比值分别为2.49％、2.31％和3.32％(图23B)。
该结果表明(1)在生理和病理情况下施予纤溶酶原后，可促进纤溶酶原穿过血脑屏障，在脑组织富集；(2)静脉注射纤溶酶原于FAD小鼠体内纤溶酶原水平显著增加；(3)纤溶酶原在脑组织富集具有时间依赖效应，先升高，2至12小时逐渐降低，在12至24小时几乎全部完全代谢；(4)纤溶酶原在脑组织富集具有剂量依赖效应，施予剂量越高，脑组织中纤溶酶原水平越高。
实施例23给予纤溶酶原促进阿尔兹海默模型小鼠脑组织中纤溶酶原水平增加
从实施例21的处死小鼠取材脑组织，匀浆后进行纤溶酶的酶底物动力学法检测。依次向酶标板(厂家：NUNC，货号：446469)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(厂家：Chromogenix，货号：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。计算出每个样本单位总蛋白量中纤溶酶的活性。
结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，FAD小鼠脑组织纤溶酶活性水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组(图24)。
该结果表明，注射纤溶酶原于小鼠体内，脑组织纤溶酶水平显著增加，此外，纤溶酶在脑组织中活性具有剂量依赖效应，施予剂量越高，脑组织中纤溶酶原水平越高。
实施例24给予纤溶酶原促进阿尔兹海默模型小鼠脊髓组织中纤溶酶原水平增加
从实施例21的处死小鼠取材脊髓组织，匀浆后按照Human Plasminogen ELISA Kit(厂家：AssayMax，货号：EP1200-1)说明书操作进行检测。以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。
FAD小鼠脊髓ELISA水平检测结果显示，尾静脉注射给予50mg/kg和6mg/kg纤溶酶原，FAD小鼠脊髓组织纤溶酶原水平明显增加，且50mg/kg给药组纤溶酶原水平明显高于6mg/kg给药组。纤溶酶原水平在给药后2小时后逐渐降低，在12至24小时基本代谢完全(图25A)。6mg/kg纤溶酶原组小鼠在接受纤溶酶原注射2和6小时后脊髓组织中纤溶酶原与血液中纤溶酶原比值分别为0.93％和1.62％；50mg/kg纤溶酶原组小鼠在接受纤溶酶原注射2、6和12小时后脊髓
组织中纤溶酶原与血液中纤溶酶原比值分别为0.33％、0.40％和1.56％(图25B)。
该结果表明(1)在生理和病理情况下施予纤溶酶原后，可促进纤溶酶原穿过血脑屏障，在脊髓组织富集；(2)静脉注射纤溶酶原于小鼠体内，脊髓组织纤溶酶原水平显著增加；(3)纤溶酶原在脊髓
组织富集具有时间依赖效应，先升高，2至12小时逐渐降低，在12至24小时几乎全部完全代谢；(4)纤溶酶原在脊髓组织富集具有剂量依赖效应，施予剂量越高，脊髓组织中纤溶酶原水平越高。
实施例25纤溶酶原改善肌萎缩侧索硬化症患者状况
本试验募集了39-60岁的肌萎缩侧索硬化症(ALS)患者9名，这9名患者包括肢体型患者8名，延髓型患者1名，其中1名患者携带有FUS基因突变。该治疗获得医院伦理委员会批准。所有患者均签署知情同意书。
人纤溶酶原冻干粉溶解于无菌水中，浓度为5mg/ml，经静脉注射或雾化器雾化后施予患者。患者的基本情况和纤溶酶原使用情况见表1。
表1 ALS患者的基本情况和纤溶酶原使用情况
结果：纤溶酶原改善ALS患者的运动功能
修订的ALS功能评定量表(ALS Functional Rating Scale–Revised(ALSFRS-R,ALS FRS-R)是一种广泛使用和验证的评估工具，用于监测ALS患者的残疾进展[8]。
结果显示，给药前9个病人的ALSFRS-R评分为20.22±10.01，给药纤溶酶原0.5-4个月后增加至23.13±10.82，增加了4.11±5.30分。此外， 患者8为延髓型ALS患者，仅使用纤溶酶原治疗2周，该患者的ALSFRS-R评分迅速从20分增加至36分。患者6携带有FUS基因突变，使用纤溶酶原治疗12天后，该患者的ALSFRS-R评分从27分增加至29分。患者5，在第1个疗程结束8周后该患者的ALSFRS-R评分从29分降至25分，但是在第二个疗程中仅接受纤溶酶原雾化吸入治疗4周后，评分升至29分。此外，在第二个疗程中患者5的最大步行步数从40多步增加至200多步(图26A-B)。
此外，用药纤溶酶原后，患者的呼吸功能、书写能力、说话、吞咽、焦虑和抑郁以及睡眠均有显著改善(表2)。
比较了纤溶酶原与现有的获得FDA批准的2个用于治疗ALS药物Riluzole和Edaravone在ALS中的治疗效果。根据文献报道，用Riluzole治疗6个月后，ALS患者的ALSFRS-R评分减少了-7.0±7.1分；用Edaravone治疗6个月后，ALS患者的ALSFRS-R评分减少了-5.01±0.64分[9,10]。而用纤溶酶原治疗0.5个月后，9名ALS患者的ALSFRS-R评分增加了4.11±5.30分，并且纤溶酶原用药期间未观察到副作用事件(图26C和表3)。
以上结果表明纤溶酶原能够安全有效的治疗ALS。
表2给药纤溶酶原前后ALS患者临床表型


/：表示未观察或未记录
表3纤溶酶原、Riluzole或Edaravone治疗前后ALSFRS-R评分

参考文献
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[2]Summaria L,Spitz F,Arzadon L et al.Isolation and characterization of the affinity chromatography forms of human Glu-and Lys-plasminogens and plasmins.J Biol Chem.1976Jun 25；251(12):3693-9.
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[5]Kamat P K,Rai S,Nath C.Okadaic acid induced neurotoxicity:An emerging tool to study Alzheimer's disease pathology[J].Neurotoxicology,2013,37:163-172.
[6]Sun Z,Chen YH,Wang P,Zhang J,Gurewich V,Zhang P,Liu JN.The blockage of the high-affinity lysine binding sites of plasminogen by EACA significantly inhibits prourokinase-induced plasminogen activation.Biochim Biophys Acta.2002Apr 29；1596(2):182-92.
[7]Ding H,Underwood R,Lavalley N,et al.14-3-3inhibition promotes dopaminergic neuron loss and 14-3-3θoverexpression promotes recovery in the MPTP mouse model of Parkinson's disease[J].Neuroscience,2015,307:73-82.
[8]Cedarbaum JM,Stambler N,Malta E,Fuller C,Hilt D,Thurmond B,Nakanishi A.The ALSFRS-R:a revised ALS functional rating scale that incorporates assessments of respiratory function.BDNF ALS Study Group(Phase III).J Neurol Sci.1999Oct 31；169(1-2):13-21.
[9]Shibuya K,Misawa S,Kimura H et al.A single blind randomized controlled clinical trial of mexiletine in amyotrophic lateral sclerosis:Efficacy and safety of sodium channel blocker phase II trial.Amyotroph Lateral Scler Frontotemporal Degener.2015；16(5-6):353-8.
[10]Writing Group；Edaravone(MCI-186)ALS 19Study Group.Safety and efficacy of edaravone in well defined patients with amyotrophic lateral sclerosis:a randomised,double-blind,placebo-controlled trial.Lancet Neurol.2017Jul；16(7):505-512.
序列表

Claims (16)

  1. 一种促进病理性TDP-43蛋白降解的方法，包括给药受试者治疗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. 权利要求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. 权利要求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. 权利要求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：促进神经组织中病理性TDP-43蛋白的降解、促进肌肉组织中病理性TDP-43蛋白的降解，促进细胞中及细胞核中TDP-43降解。
  5. 一种治疗受试者病理性TDP-43蛋白相关疾病的方法，包括给药所述受试者治疗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，所述病理性TDP-43蛋白相关疾病为选自如下的一种或多种：肢体型肌萎缩侧索硬化症(amyotrophic  lateral sclerosis，ALS)、延髓型肌萎缩侧索硬化症、Fus基因突变肌萎缩侧索硬化症、阿尔茨海默症(Alzheimer’s disease)、嗜银颗粒性认知症(argyrophilic grain disease)、关岛肌肉萎缩性脊髓侧索硬化-帕金森氏失智症(ALS-parkinsonism dementia complex of Guam)、血管性失智症(vascular dementia)、额颞叶失智症(也称额颞痴呆)(frontotemporal dementia，FTD)、语意失智症(semantic dementia)、雷维体失智(dementia with Lewy bodies)、亨汀顿氏舞蹈症(Huntington’s disease)、小脑萎缩症(Spinocere bellarataxia)、包涵体肌病(inclusion body myopathy)、包涵体肌炎(inclusion body myositis)和帕金森氏症(Parkinson’s disease)。
  6. 权利要求5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。
  7. 权利要求5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。
  8. 权利要求1-7任一项的方法，其中所述化合物为纤溶酶原或纤溶酶。
  9. 权利要求1-8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。
  10. 权利要求1-9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。
  11. 权利要求1-10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。
  12. 权利要求1-11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。
  13. 权利要求1-12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。
  14. 权利要求1-13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。
  15. 权利要求14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。
  16. 权利要求1-15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、动脉内或肌肉内给药。
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WO2001024784A2 (en) * 1999-10-04 2001-04-12 Fujisawa Pharmaceutical Co., Ltd. New use
WO2011062762A2 (en) * 2009-11-19 2011-05-26 President And Fellows Of Harvard College Angiogenin and variants thereof for treatment of neurodegenerative diseases
WO2012145428A2 (en) * 2011-04-21 2012-10-26 Washington University Altering protein concentrations in cerebrospinal fluid and/or brain interstitial fluid
CN111905103A (zh) * 2019-05-10 2020-11-10 深圳瑞健生命科学研究院有限公司 一种治疗肌萎缩侧索硬化的方法和药物
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