Methods and agents for promoting the degradation of pathological TDP-43 protein

Abstract

The present application relates to a method for promoting the degradation of pathological TDP-43 protein, comprising administering to a subject a therapeutically effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors. The present application also relates to agents that promote the degradation of pathological TDP-43 protein and uses thereof.

Description

[Technical Field]

[0001] The present application relates to a method for promoting the degradation of pathological TDP-43 protein and treating a pathological TDP-43 protein-associated disease, comprising administering to a subject an effective amount of a plasminogen activation pathway-related compound (e.g., plasminogen or plasmin). The present application also relates to a pharmaceutical composition for this use, comprising a plasminogen activation pathway-related compound (e.g., plasminogen or plasmin). [Background technology]

[0002] TDP-43, short for transactive response DNA-binding protein 43, is a protein widely distributed within cells. It binds to DNA and RNA and plays important roles in intracellular RNA transcription, alternative splicing, and mRNA stability regulation. Normal TDP-43 is localized in various subcellular structures, including mitochondria, mitochondrial-associated membranes, RNA granules, and stress granules, and regulates endoplasmic reticulum-mitochondria binding, mitochondrial protein translation, and mRNA transport and translation. Therefore, normal physiological function of TDP-43 is particularly important for cell survival.

[0003] TDP-43 can also bind to itself to form homodimers and polymers. The C-terminus of TDP-43 contains a glutamine-rich region, which is responsible for most of the aggregation. Recent studies have revealed that several factors affect the aggregation process of TDP-43, either by altering the protein structure itself or by altering surrounding proteins, leading to TDP-43 aggregation.

[0004] TDP-43 aggregates have been identified in an increasing number of neurodegenerative diseases (Lagier-Tourenne et al., Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 R46-R64). The diseases include frontotemporal lobe dementia (sporadic or familial, with or without motor-neuron disease (MND), with progranulin (GRN) mutations, with TARDBP mutations, with valosine-containing protein (VCP) mutations, 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 mutations, with angiogenin (ANG) mutations), Alzheimer's disease (AD, sporadic and familial), Down syndrome, Familial British dementia, polyglutamine diseases (Huntington's disease, etc.), and These include, but are not limited to, spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph Disease), hippocampal sclerosis dementia and myopathy (sporadic inclusion body myocilin; 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 the gene encoding desmin (DES).

[0005] Aggregated TDP-43 from patient brains shows a large number of abnormal modifications, including hyperphosphorylation, ubiquitination, acetylation, and proteolytic cleavage of the C-terminal fragment (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). Another hallmark of TDP-43 pathology is the redistribution and accumulation of TDP-43 from the nucleus to the cytoplasm. The characteristic lesions of FTLD-TDP are neuronal cytoplasmic inclusions (NCIs), glial cytoplasmic inclusions (GCIs), and dystrophic neurites (DNs).

[0006] Frontotemporal dementia (FTD) is a clinical term that encompasses a broad group of disorders based on the pathological features of frontal and temporal lobe degeneration—called frontotemporal lobar degeneration (FTLD). FTD is the second leading cause of early degenerative dementia in people under 65 years of age (Le Ber, Revue Neurologique 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 neuron disease (FTD-MND), characterized by motor dysfunction.

[0007] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the early loss of upper and lower motor neurons. ALS progression is characterized by fatal paralysis and respiratory failure, with death occurring 1–5 years from diagnosis. In most cases of sporadic ALS, the neuropathological hallmark is the abnormal cytoplasmic accumulation of TDP-43 in neurons and glial cells in the primary motor cortex, brainstem motor nuclei, spinal cord, and associated white matter tracts. In ALS with dementia, TDP-43 accumulates in the extramotor neocortex and hippocampus. The role of TDP-43 phosphorylation in ALS patients has been investigated using antibodies (Hasegawa et al., Ann Neurol 2008;64:60–70; Neumann et al., Acta Neuropathol (2009) 117:137–149).

[0008] TDP-43 pathology appears in the brains of up to 57% of Alzheimer's disease patients (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 impairment, and medial temporal lobe atrophy in Alzheimer's disease (AD). TDP-43-positive patients are 10 times more likely to have cognitive impairment at death than TDP-43-negative patients. Pathological TDP-43 generally exhibits a progressive deposition pattern, with TDP-43 initially deposited in the amygdala (Stage I), then in the hippocampus, limbic system, temporal lobe, and finally in the 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).

[0009] Recent evidence supports the idea that amyloid-β, tau, α-synuclein, and TDP-43 spread to neuronal tissue via a prion-like mechanism (Hasegawa et al., 2017). Although the onset and early symptoms of ALS vary greatly among patients, a common feature of disease progression is the spread of lesions from the initial lesion site to most neurons. The continued worsening of symptoms can be explained by this progressive spread of TDP-43 lesions. TDP-43 lesions in the brains of ALS patients have been shown to spread in a four-step process, spreading synaptically from the cortex via axonal projections using anterograde axonal transport (Brettschneider et al., Ann Neurol. 2013 July;74(1):20-38).

[0010] Recent reports have demonstrated the molecular distribution of TDP-43 in various in vitro models. Insoluble TDP-43 preparations derived from patient brains can induce intracellular aggregate formation in vitro (Nonaka et al., Cell Reports 4 (2013), pp. 124–134; Feiler et al., 2015; Porta et al., Nat. Comm., 2018). Furthermore, inoculation of patient-derived pathological TDP-43 into transgenic and wild-type mice recently led to widespread deposition of endogenous TDP-43 (Porta et al., Nat. Comm., 2018). Furthermore, intracellular TDP-43 aggregates have been shown to be released with exosomes and disseminate to subsequent cells (Nonaka et al., Cell Reports 4 (2013), pp. 124–134).

[0011] TDP-43 aggregation and pathological proliferation are key features of ALS and FTD, which are currently incurable and fatal diseases. TDP-43 mutations are associated with familial cases of ALS and FTD, suggesting a causal relationship between TDP-43 misfolding and disease progression. Therefore, to treat diseases associated with pathological TDP-43 protein, it is necessary to find methods to promote the degradation of pathological TDP-43 protein or reduce TDP-43 aggregation. Summary of the Invention

[0012] The present application states that research has shown that plasminogen can promote the degradation of pathological TDP-43 protein in nerve and muscle tissue to some extent, and can treat diseases associated with the aggregation of pathological TDP-43 protein, such as ALS and frontotemporal dementia (also known as FTD).

[0013] Specifically, the present application relates to the following sections:

[0014] 1. A method for promoting the degradation of pathological TDP-43 protein, comprising administering to a subject a therapeutically effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

[0015] 2. The method of claim 1, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA, and uPA.

[0016] 3. The method according to item 1, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, such as an antibody.

[0017] 4. The method according to any one of items 1 to 3, wherein the compound has one or more of the following activities: promoting the degradation of pathological TDP-43 protein in nervous tissue; promoting the degradation of pathological TDP-43 protein in muscle tissue. In some embodiments, the compound promotes the degradation of pathological TDP-43 protein within and / or outside muscle tissue cells. In some embodiments, the compound promotes the degradation of pathological TDP-43 protein within and / or outside nervous tissue cells (e.g., neurons or glial cells).

[0018] 5. A method for treating a pathological TDP-43 protein-associated disease in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds capable of upregulating the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors, wherein the pathological TDP-43 protein-associated disease is selected from amyotrophic lateral sclerosis (ALS), bulbar amyotrophic lateral sclerosis, Fus mutant amyotrophic lateral sclerosis, Alzheimer's disease, argyrophilic grain syndrome, and the like. disease, ALS-parkinsonism dementia complex of Guam, vascular dementia, frontotemporal dementia (FTD), semantic dementia, dementia with Lewy bodies, Huntington's disease, spinocerebellar degeneration, inclusion body myopathy, inclusion body myositis, and Parkinson's disease.

[0019] 6. The method of claim 5, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA, and uPA.

[0020] 7. The method according to item 5, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, such as an antibody.

[0021] 8. The method according to any one of items 1 to 7, wherein the compound is plasminogen or plasmin.

[0022] 9. The method according to any one of items 1 to 8, wherein the plasminogen is Glu-plasminogen, Lys-plasminogen, or a conservatively substituted variant thereof.

[0023] 10. The method according to any one of items 1 to 9, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2 and has lysine-binding activity and / or proteolytic activity of plasminogen.

[0024] 11. The method according to any one of items 1 to 10, wherein the plasminogen comprises one or more selected from the group consisting of: 1) containing the serine protease domain set forth in SEQ ID NO: 14; 2) a serine protease domain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 and retaining proteolytic activity; 3) one or more Kringle domains selected from the group consisting of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5; and 4) A Kringle domain that has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to one or more selected from the group consisting of Kringle 1, Kringle 2, Kringle 3, Kringle 4 and Kringle 5 and retains lysine-binding activity.

[0025] 12. The method according to any one of items 1 to 11, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or a mutant thereof that retains the proteolytic activity of plasminogen.

[0026] 13. The method according to any one of items 1 to 12, wherein the plasminogen comprises the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12, or comprises a conservatively substituted variant of the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12.

[0027] 14. The method according to any one of items 1 to 13, wherein the plasminogen is used in combination with one or more other therapeutic methods or drugs.

[0028] 15. The method according to item 14, wherein the other treatment methods include cell therapy (including stem cell therapy), supportive therapy, and physical therapy.

[0029] 16. The method of any one of paragraphs 1 to 15, wherein the plasminogen is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial, or intramuscular administration. In certain embodiments, the plasminogen pathway activator is administered in combination with one or more other drugs and / or therapeutic methods, preferably including cell therapy (e.g., stem cell therapy) and gene therapy (e.g., antisense RNA, small molecule splicing modifiers).

[0030] In certain embodiments, the plasminogen pathway activator is a component of the plasminogen activation pathway, such as plasminogen, which comprises or has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12, and has plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen is a protein having 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid added, deleted, and / or substituted based on SEQ ID NO: 2, 6, 8, 10, or 12, and having plasminogen activity and / or lysine-binding activity. In some specific embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In some specific embodiments, the plasminogen is a protein comprising a plasminogen activity fragment and having plasminogen activity and / or lysine-binding activity. In some specific embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In certain embodiments, the plasminogen active fragment comprises or has a plasminogen serine protease domain or a plasminogen protease domain. In certain embodiments, the amino acid sequence of the plasminogen active fragment is set forth in SEQ ID NO: 14. In certain embodiments, the plasminogen is selected from Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (human full-length plasminogen after cleavage between amino acids 76 and 77), miniplasminogen (comprising Kringle 5 (K5) and a serine protease domain), microplasminogen (comprising a serine protease domain), delta-plasminogen (comprising Kringle 1 and a serine protease domain), or a variant thereof that retains plasminogen activity.In certain embodiments, the plasminogen is human full-length plasminogen, or a variant or fragment that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen is an ortholog of human plasminogen derived from a primate or rodent, or a variant or fragment that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen comprises the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human native plasminogen.

[0031] In some specific embodiments, the plasminogen pathway activator is administered systemically or locally, for example, intravenously, intramuscularly, as a nasal inhalation spray, or in the form of nasal drops. In some embodiments, the subject is human. In some embodiments, the subject has a plasminogen deficiency or deficiency. In some embodiments, the deficiency or deficiency is congenital, inherited, and / or localized. In some embodiments, the plasminogen is administered at a daily dose of 0.0001-2000 mg / kg, 0.001-800 mg / kg, 0.01-600 mg / kg, 0.1-400 mg / kg, 1-200 mg / kg, 1-100 mg / kg, 10-100 mg / kg (calculated per kilogram of body weight), or 0.0001-2000 mg / cm. 2 , 0.001~800mg / cm 2 , 0.01 to 600 mg / cm 2 , 0.1 to 400 mg / cm 2 , 1-200mg / cm 2 , 1-100mg / cm 2 , 10-100mg / cm 2 (calculated per square centimeter of body surface area) administered daily, every two days, or every three days continuously.

[0032] In one aspect, the present application also relates to pharmaceutical compositions, medicaments, formulations, kits, and articles of manufacture for use in the above methods, comprising the above plasminogen pathway activators, e.g., the above plasminogens.

[0033] In some embodiments, the pharmaceutical compositions, medicaments, and formulations comprise a pharmaceutically acceptable carrier and a component of the plasminogen activation pathway, e.g., a plasminogen pathway activator such as plasminogen. In some embodiments, the kits and articles of manufacture comprise one or more containers containing the pharmaceutical composition, medicament, or formulation. In some embodiments, the kit or article of manufacture also further comprises a label or protocol instructing use of the plasminogen pathway activator, e.g., a component of the plasminogen activation pathway, such as plasminogen, in the above-described methods. In some embodiments, the kit or article of manufacture also further comprises one or more additional containers containing one or more other agents.

[0034] In one aspect, the present application also relates to a plasminogen pathway activator, such as the plasminogen described above, for the uses described above.

[0035] In one aspect, the present application also relates to the use of a therapeutically effective amount of the above-described plasminogen pathway activator in the manufacture of a pharmaceutical composition, medicament, formulation, kit, or article of manufacture for use in the above-described method.

[0036] In some embodiments, the plasminogen pathway activator is one or more selected from a component of the plasminogen activation pathway, a compound that can activate plasminogen directly or indirectly by activating an upstream component of the plasminogen activation pathway, a compound that mimics the activity of plasminogen or plasmin, a compound that can upregulate the expression of plasminogen or plasminogen activators, a plasminogen analog, a plasmin analog, a tPA or uPA analog, and an antagonist of a fibrinolysis inhibitor.

[0037] In certain embodiments, the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasmin, plasminogen activators, tPA, and uPA. In certain embodiments, the antagonist of the fibrinolysis inhibitor is an antagonist of PAI-1, complement C1 inhibitor, α2-antiplasmin, or α2-macroglobulin, e.g., an antibody against PAI-1, complement C1 inhibitor, α2-antiplasmin, or α2-macroglobulin.

[0038] In certain embodiments, the plasminogen pathway activator is administered in combination with one or more other agents and / or therapeutic methods, preferably including cell therapy (e.g., stem cell therapy) and gene therapy (e.g., antisense RNA, small molecule splicing modifiers).

[0039] In certain embodiments, the plasminogen pathway activator is a component of the plasminogen activation pathway, such as plasminogen, which comprises or has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12, and has plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen is a protein having 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid added, deleted, and / or substituted based on SEQ ID NO: 2, 6, 8, 10, or 12, and having plasminogen activity and / or lysine-binding activity. In some specific embodiments, the plasminogen activity is the proteolytic activity of the plasminogen. In some specific embodiments, the plasminogen is a protein comprising a plasminogen activity fragment and having plasminogen activity and / or lysine-binding activity. In some specific embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In certain embodiments, the plasminogen active fragment comprises or has a plasminogen serine protease domain or a plasminogen protease domain. In certain embodiments, the amino acid sequence of the plasminogen active fragment is set forth in SEQ ID NO: 14. In certain embodiments, the plasminogen is Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (human full-length plasminogen after cleavage between amino acids 76 and 77), miniplasminogen (comprising Kringle 5 (K5) and a serine protease domain), microplasminogen (comprising a serine protease domain), delta-plasminogen (comprising Kringle 1 and a serine protease domain), or a variant thereof that retains plasminogen activity.In certain embodiments, the plasminogen is human full-length plasminogen, or a variant or fragment that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen is an ortholog of human plasminogen derived from a primate or rodent, or a variant or fragment that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen comprises the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human native plasminogen.

[0040] In some specific embodiments, the plasminogen pathway activator, e.g., a component of the plasminogen activation pathway, e.g., plasminogen, is administered in combination with one or more other drugs and / or therapeutic methods. In some embodiments, the plasminogen pathway activator, e.g., a component of the plasminogen activation pathway, e.g., plasminogen, is administered intravenously, intramuscularly, intrathecally, intranasally, aerosolized, as nose drops, or as eye drops.

[0041] In some embodiments, the pharmaceutical compositions, medicaments, and formulations comprise a pharmaceutically acceptable carrier and a component of the plasminogen activation pathway, e.g., a plasminogen pathway activator such as plasminogen. In some embodiments, the kits and articles of manufacture comprise one or more containers containing the pharmaceutical composition, medicament, or formulation. In some embodiments, the kit or article of manufacture also further comprises a label or protocol instructing use of the plasminogen pathway activator, e.g., a component of the plasminogen activation pathway, such as plasminogen, in the above-described methods.

[0042] In some embodiments, the kit or article of manufacture also further comprises one or more separate containers containing one or more other agents.

[0043] The present invention explicitly covers all combinations of the technical features belonging to the embodiments of the present invention, and the technical configurations after these combinations are expressly disclosed in this application as if the above technical configurations were expressly disclosed separately. Furthermore, the present invention also explicitly covers the combinations between each embodiment and their elements, and the technical configurations after the combinations are expressly disclosed in this specification. [Brief explanation of the drawings]

[0044] Figure 1 shows a schematic diagram of the mechanism by which plasminogen promotes the degradation of pathological proteins 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 (PGPF), plasmin degradation products (PDP), lysosomes, ubiquitin (UBI), ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin ligase (E3), proteasome, microglia, and nucleus. Existing evidence suggests that plasminogen can promote the degradation of pathological CNS proteins, such as TDP-43 and superoxide dismutase-1 (SOD1), and improve the clinical symptoms of various neurodegenerative diseases, including amyotrophic lateral sclerosis (AMSC). Based on existing data, the mechanism by which plasminogen promotes the degradation of pathological CNS proteins is hypothesized as follows: (1) Plasminogen crosses the blood-brain barrier to enter the CNS, where it is concentrated and activated to form plasmin, which then directly degrades pathological CNS proteins abnormally deposited in the extracellular matrix, such as the amyloid protein Aβ. The resulting protein fragments are then phagocytosed by microglia and subsequently degraded by lysosomes. (2) Plasminogen enters cells and nuclei via endocytosis, where it is activated to form plasmin, promoting the degradation of pathological CNS proteins, such as TDP-43, SOD1, TAU, and α-synuclein.(3) Plasminogen enters cells and regulates the function of the ubiquitin proteasome system (UPS), an intracellular protein degradation system, which degrades CNS pathological proteins via the UPS. (4) Plasminogen enters cells and regulates the function of the autophagy-lysosomal system, an intracellular protein degradation system, which degrades CNS pathological proteins via the autophagy-lysosomal system. (5) In addition, some studies have reported that CNS pathological proteins, such as TDP-43 and SOD1, have the same infectious properties as prions, and plasminogen may have the ability to prevent CNS pathological proteins from spreading between cells. Figure 2 (A-B) shows the effect of plasminogen on TDP-43 protein in normal mouse brain homogenates. A is a Western blot image, and 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 approximately 43 kDa, the molecular weight of the high-molecular-weight TDP-43 protein (HMW) was greater than 55 kDa, and the molecular weight of the low-molecular-weight TDP-43 fragment (LMW) was less than 40 kDa. Furthermore, in normal mouse brain homogenates, the amounts of TDP-43 monomer, HMW, and LMW in the plasminogen group were significantly lower than those in the solvent control group, and the difference was highly significant (*** indicates P<0.001, * indicates P<0.05). This suggests that plasminogen can promote the cleavage of TDP-43 in normal mouse brain homogenates. Figure 3 (A-B) shows the effect of plasminogen on TDP-43 protein in brain homogenates from amyotrophic lateral sclerosis (ALS) model mice. A is a Western blot image, and 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 approximately 43 kDa, the molecular weight of the high-molecular-weight TDP-43 protein (HMW) was greater than 55 kDa, and the molecular weight of the low-molecular-weight TDP-43 fragment (LMW) was less than 40 kDa. Furthermore, in brain homogenates from ALS model mice, the amounts of TDP-43 monomer, HMW, and LMW were significantly lower in the plasminogen group than in the solvent control group, and the differences were highly significant (*** indicates P<0.001, ** indicates P<0.01). This suggests that plasminogen can promote the cleavage of TDP-43 in brain homogenates from amyotrophic lateral sclerosis model mice. Figure 4 (A-B) shows that plasminogen promotes the degradation of TDP-43 protein in the spinal cord tissue of amyotrophic lateral sclerosis model mice. A is a Western blot image, and B is the result of quantitative analysis of the optical density of the TDP-43 protein band. The results show 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 difference was statistically significant (* indicates P<0.05). This indicates that plasminogen can promote the degradation of TDP-43 protein in the spinal cord tissue of amyotrophic lateral sclerosis model mice. Figure 5 (A-B) shows that plasminogen promotes the degradation of TDP-43 protein in the brain tissue of amyotrophic lateral sclerosis (AMLS) model mice, which were locally injected with pathological TDP-43 protein. A is a Western blot image, and B is the result of quantitative analysis of the optical density of the TDP-43 protein band. The results show that the amounts of TDP-43 monomer and low-molecular-weight TDP-43 in the brain tissue of mice treated with the drug were significantly lower than those in the vehicle group, and the difference was statistically significant (* indicates P<0.05). This indicates that plasminogen can promote the degradation of TDP-43 in the brain tissue of AMLS model mice. (Figure 6) JPEG2025539229000001.jpg64127 (Figure 7) Figure 8 (A-B) shows the results of WB detection of TDP-43 levels in brain homogenates from plasminogen-treated okadaic acid-induced dementia model mice. A is a Western blot image, and B is the result of quantitative analysis of the optical density of the TDP-43 protein band. The results show that the levels of TDP-43 monomer and low-molecular-weight TDP-43 in the brain tissue of mice treated with the drug 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 okadaic acid-induced dementia model mice. Figure 9. A-B shows the results of WB detection of TDP-43 levels in the kidney cell nuclei of plasminogen-administered amyotrophic lateral sclerosis model mice. A is a Western blot image, and B is the result of quantitative analysis of the optical density of the TDP-43 protein band. The results show that the TDP-43 levels in the kidney cell nuclei of the drug-administered group were significantly lower than those of the vehicle group (* indicates P<0.05). This suggests that plasminogen can promote the degradation of TDP-43 in the kidney cell nuclei. Figure 10 (A–D) shows the results of WB analysis of TDP-43 levels in the cytoplasm and nucleus of okadaic acid-treated NSC34 cells after plasminogen administration. A is a Western blot image of the cytoplasm, B is a quantitative analysis of the optical density of the cytoplasmic TDP-43 protein band, C is a Western blot image of the nucleus, and D is a quantitative analysis of the optical density of the nuclear TDP-43 protein band. The results show that the TDP-43 levels in the cytoplasm and nucleus of the drug-treated group were significantly lower than or close to the nuclear levels in the vehicle group, indicating that the addition of EACA completely inhibited the effect of plasminogen on TDP-43 (** indicates P<0.01, *** indicates P<0.001). These results suggest that plasminogen can promote the degradation of TDP-43 in the cytoplasm and nucleus, and that this effect of plasminogen is closely related to the lysine-binding site in its structure. Figure 11 (A-D) shows the results of measuring plasminogen and plasmin activity levels in the cytoplasm and nucleus of okadaic acid-treated NSC34 cells after plasminogen administration. A shows the cytoplasmic plasminogen level measured by ELISA, B shows the nuclear plasminogen level measured by ELISA, C shows the enzyme substrate kinetics of cytoplasmic plasmin activity levels, and D shows the enzyme substrate kinetics of nuclear plasmin activity levels. The results show that the human plasminogen and plasmin activity levels in the cytoplasm and nucleus of the drug-treated group were significantly higher than those of the vehicle group, and the difference was statistically significant. The addition of EACA completely inhibited these effects of plasminogen (** indicates P<0.01, *** indicates P<0.001). These results suggest that plasminogen can enter cells and even the nucleus to enhance plasmin activity, and that plasminogen entry into cells and the nucleus is closely related to its lysine-binding activity. Figure 12 shows the results of ELISA detection of plasma plasminogen levels at different time points after tail vein injection of plasminogen into SOD1-G93A mice. ELISA detection of plasma plasminogen levels in SOD1-G93A mice showed a significant increase after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen level in the 50 mg / kg group being significantly higher than that in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after administration and were nearly completely metabolized between 12 and 24 hours. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. These results indicate that (1) plasma plasminogen levels exhibit a dose-dependent effect, with higher administered plasminogen concentrations resulting in more aggregation, and (2) plasma plasminogen levels exhibit a time-dependent effect, with an initial increase followed by a gradual decrease over the course of 2 to 12 hours. Figure 13A-B shows the ELISA results for plasminogen levels in brain tissue and the ratio of plasminogen in brain tissue to plasma at different time points after intravenous injection of plasminogen into SOD1-G93A mice. A shows the ELISA results for plasminogen levels in brain tissue, and B shows the ratio of plasminogen in brain tissue to plasma. According to the ELISA results for the brain of SOD1-G93A mice, the plasminogen levels in brain tissue of SOD1-G93A mice significantly increased after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen level in the 50 mg / kg group being significantly higher than that in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after administration and were almost completely metabolized within 12 to 24 hours. The ratios of brain tissue plasminogen levels to blood plasminogen levels were 3.47%, 4.94%, and 6.79% at 2, 6, and 12 hours after plasminogen administration, respectively. These results indicate that (1) plasminogen administration under physiological and pathological conditions can promote plasminogen crossing the blood-brain barrier and its concentration in brain tissue; (2) intravenous injection of plasminogen into mice significantly increased plasminogen levels in brain tissue; (3) plasminogen concentration in brain tissue showed a time-dependent effect, initially increasing, gradually decreasing between 2 and 12 hours, and being almost completely metabolized between 12 and 24 hours; and (4) plasminogen concentration in brain tissue showed a dose-dependent effect, with higher doses resulting in higher plasminogen levels in brain tissue. ** indicates P<0.01, and *** indicates P<0.001. Figure 14 shows the results of ELISA detection of spinal cord tissue plasminogen levels at different time points after intravenous injection of plasminogen into SOD1-G93A mice. ELISA detection of spinal cord levels in SOD1-G93A mice showed that spinal cord plasminogen levels significantly increased after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen level in the 50 mg / kg group being significantly higher than that in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after administration and were almost completely metabolized within 12 to 24 hours. * indicates P<0.05, ** indicates P<0.01. These results indicate that (1) plasminogen administered at physiological doses can cross the blood-brain barrier and concentrate in the spinal cord tissue of SOD1-G93A mice, (2) plasminogen concentration in the spinal cord exhibits a dose-dependent effect, with the higher the administered dose of plasminogen, the greater the concentration, and (3) plasminogen concentration in the spinal cord exhibits a time-dependent effect, initially increasing, gradually decreasing over 2 to 12 hours, and being almost completely metabolized over 12 to 24 hours. Figure 15A-B shows the results of detecting plasminogen levels and plasmin activity in brain tissue homogenates after a single intravenous administration of plasminogen to SOD1-G93A mice. A shows the plasminogen level detected by ELISA, and B shows the enzyme substrate kinetics detection of plasmin activity levels. The results show that the plasminogen and plasmin activity levels in brain tissue homogenates of mice in the drug-treated group were significantly higher than those in the vehicle group, and the difference was statistically significant (* indicates P<0.05, *** indicates P<0.001). These results suggest that intravenous administration of plasminogen can promote an increase in plasminogen levels and plasmin activity in brain tissue. (Figure 16) This figure shows the results of detecting plasminogen levels in the cell nuclei of brain, spinal cord, and kidney tissues of SOD1-G93A mice after continuous 7-day intravenous administration of plasminogen. The results show that 7 days after plasminogen administration, the human plasminogen levels in the cell nuclei of brain, spinal cord, and kidney tissues of SOD1-G93A mice in the drug-treated group were significantly higher than those in the vehicle group, and the difference was statistically highly significant (*** indicates P<0.001). This suggests that intravenous administration of plasminogen can promote an increase in human plasminogen levels in the cell nuclei of brain, spinal cord, and kidney tissues. Figure 17 shows the results of ELISA assay of blood plasminogen levels at different time points after tail vein injection of plasminogen into Parkinson's disease model mice. The results show that the blood plasminogen levels of mice in the drug-treated group were significantly higher than those of mice in the vehicle group, and that plasminogen levels gradually decreased within 2 hours after administration and were essentially completely metabolized between 12 and 24 hours. *** indicates P<0.001. Figure 18 shows the results of ELISA assay of blood plasminogen levels at different time points after tail vein injection of plasminogen into Parkinson's disease model mice. The results show that plasminogen levels in the brain tissue of mice in the drug-treated group were significantly higher than those in the vehicle-treated group. Plasminogen levels gradually decreased within 2 hours of administration and were essentially completely metabolized within 12 to 24 hours. These results indicate that plasminogen injected via the tail vein can cross the blood-brain barrier and promote an increase in plasminogen levels in the brain tissue of Parkinson's disease model mice. *** indicates P<0.001. Figure 19 shows the results of ELISA assay of plasminogen levels in spinal cord tissue at different time points after intravenous injection of plasminogen into Parkinson's disease model mice. The results show that plasminogen levels in spinal cord tissue of mice in the drug-treated group were significantly higher than those in the vehicle-treated group. Plasminogen levels gradually decreased within 2 hours after administration and were essentially completely metabolized within 12 to 24 hours. These results indicate that plasminogen injected via the tail vein can cross the blood-brain barrier and promote the increase of plasminogen levels in spinal cord tissue of Parkinson's disease model mice. ** indicates P<0.01, *** indicates P<0.001. Figure 20 shows the ratios of plasminogen levels in spinal cord or brain tissue to blood plasminogen levels at different time points after tail vein injection of plasminogen into Parkinson's disease model mice. The ratios of plasminogen levels in spinal cord tissue to blood plasminogen levels were 1.24%, 1.16%, and 1.46%, respectively, 2, 6, and 12 hours after plasminogen administration. The ratios of plasminogen levels in brain tissue to blood plasminogen levels were 3.47%, 4.18%, and 8.51%, respectively, 2, 6, and 12 hours after plasminogen administration. * indicates P<0.05, ** indicates P<0.01. These results indicate that plasminogen injected via 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 disease model mice. (Figure 21) This figure shows the results of detecting plasmin activity levels in brain tissue after tail vein injection of plasminogen into Parkinson's disease model mice. The results showed that the plasminogen activity levels in brain tissue of mice in the drug-treated group were significantly higher than those of mice in the vehicle group, and the difference was statistically significant. * indicates P<0.05. This result indicates that plasminogen injected via the tail vein can cross the blood-brain barrier and promote an increase in plasminogen activity levels in brain tissue of Parkinson's disease model mice. Figure 22 shows the results of ELISA assay of plasma plasminogen levels at different time points after tail vein injection of plasminogen into FAD mice. ELISA results showed that plasma plasminogen levels in FAD mice significantly increased after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen level in the 50 mg / kg group being significantly higher than that in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after administration and were almost completely metabolized between 12 and 24 hours. These results indicate that (1) plasma plasminogen levels exhibited a dose-dependent effect, with higher concentrations of administered plasminogen resulting in greater aggregation; and (3) plasma plasminogen levels exhibited a time-dependent effect, initially increasing and then gradually decreasing between 2 and 12 hours. Figure 23A-B shows the ELISA results for plasminogen levels in brain tissue at different time points after tail vein injection of plasminogen into FAD mice (A), and the ratio of plasminogen levels in brain tissue to blood plasminogen levels at different time points (B). According to the brain ELISA results for FAD mice, plasminogen levels in brain tissue significantly increased after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen level in the 50 mg / kg group being significantly higher than that in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after injection and were almost completely metabolized within 12 to 24 hours. The ratios of plasminogen in brain tissue to blood plasminogen in the 6 mg / kg plasminogen-treated mice were 3.59% and 4.23%, respectively, 2 and 6 hours after plasminogen administration, and the ratios of plasminogen in brain tissue to blood plasminogen in the 50 mg / kg plasminogen-treated mice were 2.49%, 2.31%, and 3.32%, respectively, 2, 6, and 12 hours after plasminogen administration. These results indicate that (1) plasminogen administration under physiological and pathological conditions can promote plasminogen crossing the blood-brain barrier and its concentration in brain tissue, (2) intravenous injection of plasminogen into mice significantly increases plasminogen levels in brain tissue, (3) plasminogen concentration in brain tissue exhibits a time-dependent effect, initially increasing, gradually decreasing over 2 to 12 hours, and being almost completely metabolized over 12 to 24 hours, and (4) plasminogen concentration in brain tissue exhibits a dose-dependent effect, with the higher the dose, the higher the plasminogen level in brain tissue. Figure 24 shows the results of measuring plasmin activity in brain homogenates using enzyme substrate kinetics 2 hours after tail vein injection of plasminogen into FAD mice. The results showed that plasmin activity in brain tissue of FAD mice significantly increased 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. These results indicate that plasminogen injection into mice significantly increased plasmin levels in brain tissue and that plasmin activity in brain tissue exhibited a dose-dependent effect, with higher doses resulting in higher plasminogen levels in brain tissue. Figure 25A-B shows the results of ELISA detection of plasminogen levels in spinal cord tissue at different time points after tail vein injection of plasminogen into FAD mice (A), and the ratio of plasminogen levels in spinal cord tissue to blood plasminogen levels at different time points (B). ELISA detection of spinal cord levels in FAD mice showed that plasminogen levels in spinal cord tissue significantly increased after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen level in the 50 mg / kg group being significantly higher than that in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after injection and were almost completely metabolized within 12 to 24 hours. The ratios of plasminogen in the spinal cord tissue to blood plasminogen in the 6 mg / kg plasminogen-treated mice were 0.93% and 1.62%, respectively, 2 and 6 hours after plasminogen administration, and the ratios of plasminogen in the spinal cord tissue to blood plasminogen in the 50 mg / kg plasminogen-treated mice were 0.33%, 0.40%, and 1.56%, respectively, 2, 6, and 12 hours after plasminogen administration. These results indicate that (1) plasminogen administration under physiological and pathological conditions can promote plasminogen crossing the blood-brain barrier and concentration in spinal cord tissue, (2) intravenous injection of plasminogen into mice significantly increased plasminogen levels in spinal cord tissue, (3) plasminogen concentration in spinal cord tissue exhibited a time-dependent effect, initially increasing, gradually decreasing over 2 to 12 hours, and being almost completely metabolized over 12 to 24 hours, and (4) plasminogen concentration in spinal cord tissue had a dose-dependent effect, with the higher the dose, the higher the plasminogen level in spinal cord tissue. (Figure 26) A to C show changes in the clinical phenotype of ALS patients before and after plasminogen administration. A: ALSFRS-R scores of nine ALS patients before and after plasminogen administration, B: maximum number of steps taken by patient 5 during the second treatment course, C: ALSFRS-R scores of nine ALS patients who took plasminogen for 0.5 months (shown by solid line) and ALSFRS-R scores of ALS patients who took riluzole or edaravone for 6 months (shown by dotted line). Detailed Description of the Invention

[0045] The fibrinolytic system, also known as the fibrinolytic system, is a system of chemicals involved in the process of fibrinolysis (fibrinolysis), primarily including plasminogen (PLG), plasmin, plasminogen activators, and fibrinolysis inhibitors. Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA is a serine protease synthesized by vascular endothelial cells. t-PA activates plasminogen, a process that primarily occurs with fibrin. Urokinase-type plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells and can directly activate plasminogen without requiring fibrin as a cofactor. Plasminogen (PLG) is synthesized in the liver. During blood clot formation, PLG is absorbed in large quantities into the fibrin network and activated to plasmin by t-PA or u-PA, promoting fibrinolysis. Plasminase (PL) is a serine protease that degrades fibrin and fibrinogen, hydrolyzes various coagulation factors (e.g., V, VIII, X, VII, XI, and II), converts plasminogen to plasmin, and hydrolyzes complement. Fibrinolysis inhibitors include plasminogen activator inhibitor (PAI) and α2-antiplasmin (α2-AP). PAIs exist in two forms, PAI-1 and PAI-2, which specifically bind to t-PA in a 1:1 ratio, thereby inactivating it and simultaneously activating PLG. α2-AP is synthesized in the liver and binds to PL in a 1:1 ratio to form a complex, thereby inhibiting PL activity. FXIII covalently binds α2-AP to fibrin, thereby reducing its susceptibility to PL. Substances that inhibit the activity of the fibrinolytic system in vivo include PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin.

[0046] The term "fibrin plasminogen pathway activator" or "plasminogen pathway activator" of the present invention covers components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolysis inhibitors.

[0047] As used herein, the term "component of the fibrin plasminogen activation pathway" or "component of the plasminogen activation pathway" refers to a 1. Plasminogen, Lys-plasminogen, Glu-plasminogen, microplasminogen, delta-plasminogen, their variants or analogs; 2. Plasmin and its variants or analogs; and 3. Plasminogen activators, such as tPA and uPA, as well as tPA or uPA variants and analogs that contain one or more domains of tPA or uPA, such as one or more kringle domains and proteolytic domains.

[0048] The term "antagonist of a fibrinolysis inhibitor" covers antagonists of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, for example antibodies of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0049] The above-mentioned "variants" of plasminogen, plasmin, tPA and uPA include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as proteins having, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid added, deleted and / or substituted and still having plasminogen activity, plasmin activity, tPA or uPA activity. For example, a "variant" of plasminogen activity, plasmin activity, tPA, or uPA includes, for example, mutants of these proteins resulting from substitution of 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 conservative amino acid.

[0050] The "plasminogen variant" of the present invention includes or covers proteins having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12, and having plasminogen activity and / or lysine-binding activity. For example, the "plasminogen variant" of the present invention may be a protein in which 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is added, deleted, and / or substituted based on SEQ ID NO: 2, 6, 8, 10, or 12, and still has plasminogen activity and / or lysine-binding activity. In particular, the plasminogen variants of the present invention include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as mutants of these proteins obtained by conservative substitution of, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acids.

[0051] The plasminogen of the present invention may be an orthologue of human plasminogen derived from a primate or rodent, or a variant that still retains plasminogen activity and / or lysine-binding activity, such as the plasminogen shown in SEQ ID NO: 2, 6, 8, 10 or 12, e.g., the human native plasminogen shown in SEQ ID NO: 2.

[0052] The above "analogs" of plasminogen, plasmin, tPA and uPA include compounds that provide substantially the same effects as plasminogen, plasmin, tPA or uPA, respectively.

[0053] The above-mentioned "variants" and "analogs" of plasminogen, plasmin, tPA, and uPA include "variants" and "analogs" of plasminogen, plasmin, tPA, and uPA that contain one or more domains (e.g., one or more kringle domains and proteolytic domains). For example, "variants" and "analogs" of plasminogen include plasminogen variants and analogs, such as mini-plasminogen, that contain one or more plasmin domains (e.g., one or more kringle (k) domains and proteolytic domains (also called serine protease domains or plasminogen protease domains)). "Variants" and "analogs" of plasmin include plasmin "variants" and "analogs," such as mini-plasmin and delta-plasmin, that contain one or more plasmin domains (e.g., one or more kringle domains and proteolytic domains).

[0054] Whether the above-mentioned "variants" or "analogs" of plasminogen, plasmin, tPA, or uPA have the activity of plasminogen, plasmin, tPA, or uPA, respectively, or whether they provide substantially the same effect as plasminogen, plasmin, tPA, or uPA, respectively, can be measured by measuring the level of activated plasmin activity using methods known in the art, such as enzymography, ELISA (enzyme-linked immunosorbent assay), and FACS (fluorescence-activated cell sorting). For example, this can be measured by referring to the methods described in the following documents: Ny, A., Leonardsson, G., Hagglund, AC, Hagglof, P., Ploplis, VA, Carmeliet, P. and Ny, T. (1999). 1984).“Complex formation of platelet thrombospondin with plasminogen.Modulation of activation by tissue activator”.J.Clin.Invest.74(5):1625-33;Gravanis I,Tsirka SE (February 2008).“Tissue-type plasminogen activator as a therapeutic target in stroke”.Expert Opinion on Therapeutic Targets.12(2):159-70;Geiger M,Huber K, Wojta J, Stingl L, Espana F, Griffin JH, Binder BR (Aug 1989). “Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo”. Blood. 74(2):722-8.

[0055] In some embodiments of the present invention, the "component of the plasminogen activation pathway" of the present invention is plasminogen, and is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or variants thereof that retain plasminogen activity. In some embodiments, the plasminogen is natural or synthetic human plasminogen, or a conservative mutant or fragment thereof that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen is an ortholog of human plasminogen derived from a primate or rodent, or a conservative mutant or fragment thereof that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the amino acid sequence of the plasminogen comprises or has the amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human full-length plasminogen. In some embodiments, the plasminogen is human full-length plasminogen as set forth in SEQ ID NO:2.

[0056] "A compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway" refers to any compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway, including, for example, tPA, uPA, streptokinase, saruplase, alteplase, reteplase, tenecteplase, anistreplase, monteplase, lanoteplase, pamiteplase, and staphylokinase.

[0057] The "antagonist of fibrinolysis inhibitors" of the present invention is a compound that antagonizes, weakens, blocks, or prevents the action of fibrinolysis inhibitors, such as PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin. The antagonists are antibodies to PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or antisense RNA or mini-RNA that, for example, blocks or downregulates the expression of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that occupy the binding site of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin but do not have the function of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that block the binding and / or activation domains of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0058] Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease capable of hydrolyzing several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans. Plasmin can also activate some pro-matrix metalloproteinases (pro-MMPs) to form active matrix metalloproteinases (MMPs). Thus, plasmin is an important upstream regulator of extracellular proteolysis. Plasmin is formed by the proteolysis of plasminogen from two physiological PAs: tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). Because plasminogen is present at relatively high levels in plasma and other body fluids, it has traditionally been thought that regulation of the PA system is primarily achieved by PA synthesis and activity levels. The synthesis of PA system components is tightly regulated by various factors, such as hormones, growth factors, and cytokines. In addition, there are specific physiological inhibitors of plasmin and PAs. The main inhibitor of plasmin is α2-antiplasmin. The activity of PAs is simultaneously inhibited by plasminogen activator inhibitor-1 (PAI-1), which inhibits uPA and tPA, and is regulated by plasminogen activator inhibitor-2 (PAI-2), which primarily inhibits uPA. Some cells have a uPA-specific cell surface receptor (uPAR) that is active in direct hydrolysis.

[0059] Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. It is synthesized primarily in the liver and present in large amounts in extracellular fluids. Plasminogen contains approximately 2 μM of plasminogen in plasma. Therefore, plasminogen is a significant potential source of proteolytic activity in tissues and body fluids. Two molecular forms of plasminogen exist: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). The naturally secreted and undegraded form of plasminogen contains a single amino-terminal (N-terminal) glutamic acid and is therefore called glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys77. Compared with glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PA at a higher rate. The Arg560-Val561 peptide bond in these two forms of plasminogen is cleaved by uPA or tPA, resulting in the formation of the disulfide-linked double-chain protease plasmin. The amino-terminal portion of plasminogen contains five homologous tricyclic rings, or kringles, and the carboxyl-terminal portion contains the protease domain. Some kringles contain lysine-binding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor, α2-AP. The most recently discovered 38 kDa fibrin-plasminogen fragment, kringlel-4, is a potent inhibitor of angiogenesis. This fragment, named angiostatin, is produced by hydrolysis of plasminogen with several proteases.

[0060] The main substrate of plasmin is fibrin, and fibrin lysis is a key factor in preventing the formation of pathological thrombi. [Plasmin also has substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycans, and gelatin, indicating that plasmin plays an important role in ECM remodeling. Plasmin can also indirectly degrade other components of the ECM by converting several protease precursors, including MMP-1, MMP-2, MMP-3, and MMP-9, into active proteases. Therefore, plasmin has been proposed to be an important upstream regulator of extracellular protein hydrolysis. Plasmin also has the ability to activate several latent forms of growth factors. In vitro, plasmin can also hydrolyze components of the complement system to release chemotactic complement fragments.

[0061] "Plasmin" is a very important enzyme present in blood, which hydrolyzes fibrin clots into fibrin degradation products and D-dimers.

[0062] "Plasminogen" is the proenzyme form of plasmin, and based on the sequence in Swiss prot, the amino acid sequence of natural human plasminogen (SEQ ID NO: 4), including the signal peptide, is calculated to consist of 810 amino acids, has a molecular weight of approximately 90 kD, and is a glycoprotein that is synthesized primarily in the liver and can circulate in the blood; the cDNA sequence encoding this amino acid sequence is shown in SEQ ID NO: 3. Full-length plasminogen contains seven domains: a serine protease domain located at the C-terminus, a Pan Apple (PAp) domain located at the N-terminus, and five Kringle domains (Kringles 1-5). According to the sequence in swissprot, the signal peptide contains residues Met1-Gly19, PAp contains residues Glu20-Val98, Kringle1 contains residues Cys103-Cys181, Kringle2 contains residues Glu184-Cys262, Kringle3 contains residues Cys275-Cys352, Kringle4 contains residues Cys377-Cys454, and Kringle5 contains residues Cys481-Cys560. According to NCBI data, the serine protease domain contains residues Val581-Arg804.

[0063] Glu-plasminogen is natural, full-length plasminogen, consisting of 791 amino acids (excluding the 19-amino acid signal peptide). The cDNA sequence encoding this sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2. Lys-plasminogen, formed in vivo by further hydrolysis of Glu-plasminogen at amino acids 76-77, exists, e.g., as shown in SEQ ID NO: 6, and the cDNA sequence encoding this amino acid sequence is shown in SEQ ID NO: 5. Delta-plasminogen (δ-plasminogen) is a fragment of full-length plasminogen lacking the Kringle 2 to Kringle 5 structures, and contains only Kringle 1 and a serine protease domain (also called the proteolytic domain or plasminogen protease domain). The amino acid sequence of δ-plasminogen (SEQ ID NO: 8) has been reported in a literature, and the cDNA sequence encoding this amino acid sequence is shown, for example, in SEQ ID NO: 7. Mini-plasminogen consists of Kringle 5 and serine protease domains, and the literature reports residues Val443-Asn791 (the starting amino acid is the Glu residue of the Glu-plasminogen sequence without the signal peptide). The amino acid sequence is as shown in SEQ ID NO: 10, and the cDNA sequence encoding this amino acid sequence is as shown in SEQ ID NO: 9. However, literature reports that microplasminogen only contains a serine protease domain, and its amino acid sequence is Ala543-Asn791 (the Glu residue in the Glu-plasminogen sequence without the signal peptide is the starting amino acid), and patent document CN102154253A discloses that it contains Lys531-Asn791 (the Glu residue in the Glu-plasminogen sequence without the signal peptide is the starting amino acid). For the sequence of this patent, please refer to patent document CN102154253A, whose amino acid sequence is as shown in SEQ ID NO: 12, and the cDNA sequence encoding this amino acid sequence is as shown in SEQ ID NO: 11.

[0064] In the present invention, "plasmin", "fibrinplasmin", and "fibrous protein plasmin" can be used interchangeably and have the same meaning. "Plasminogen", "fibrinplasmin", and "fibrinplasminogen" can be used interchangeably and have the same meaning.

[0065] In this application, the term "insufficiency" of plasminogen refers to the content or activity of plasminogen in a subject's body being lower than that of a normal person and being low enough to affect the subject's normal physiological functions. The term "deficiency" of plasminogen refers to the content or activity of plasminogen in a subject's body being significantly lower than that of a normal person, with only trace activity or expression, and normal physiological functions being maintained only by external supply.

[0066] Those skilled in the art will understand the following: All technical features of plasminogen in the present invention can be applied to plasmin, and therefore, the technical features described in the present invention cover both plasminogen and plasmin. During the circulation process, plasminogen is in a closed, inactive conformation. Upon binding to a thrombus or cell surface, it becomes active plasmin with an open conformation under the intervention of a plasminogen activator (PA). Active plasmin further hydrolyzes fibrin clots into fibrin degradation products and D-dimers, thereby dissolving the thrombus. The PAp domain of plasminogen is an important epitope that maintains plasminogen in the closed, inactive conformation, while the KR domain can bind to lysine residues on receptors and substrates. Several enzymes are known as plasminogen activators, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hagemann factor).

[0067] The term "active plasminogen fragment" in the present application refers to: 1) an active fragment (also referred to as a lysine-binding fragment) capable of binding to a target sequence of a substrate in a plasminogen protein, for example, a fragment containing Kringle 1, Kringle 2, Kringle 3, Kringle 4, and / or Kringle 5 (for the structure of plasminogen, see the description in Aisina RB, Mukhametova L I. Structure and function of plasminogen / plasmin system [J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6):590-605); 2) an active fragment that exerts a proteolytic function in a plasminogen protein, for example, a fragment having the plasminogen activity (proteolytic function) shown in SEQ ID NO: 14; and 3) a fragment having both the activity of binding to a target sequence of a substrate (lysine-binding activity) and the plasminogen activity (proteolytic function) in a plasminogen protein. In some embodiments of the present application, the plasminogen is a protein comprising a plasminogen active fragment set forth in SEQ ID NO: 14. In some embodiments of the present application, the plasminogen is a protein comprising a lysine-binding fragment of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and / or Kringle 5. In some embodiments, the plasminogen active fragment of the present application includes a protein comprising SEQ ID NO: 14 or comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% homology to SEQ ID NO: 14. Thus, plasminogen according to the present invention includes proteins comprising a plasminogen active fragment and still retaining plasminogen activity.In some embodiments, the plasminogen of the present application comprises Kringle 1, Kringle 2, Kringle 3, Kringle 4, and / or Kringle 5, or a protein having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% homology to Kringle 1, Kringle 2, Kringle 3, Kringle 4, or Kringle 5 and still having lysine-binding activity.

[0068] Currently, methods for measuring plasminogen and its activity in blood include tissue plasminogen activator activity (t-PAA), plasma tissue plasminogen activator antigen (t-PAAg), plasma tissue plasminogen activity (plgA), plasma tissue plasminogen antigen (plgAg), plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator inhibitor antigen, and plasma plasmin-antiplasmin complex (PAP). The most common method is the chromogenic substrate method: streptokinase (SK) and a luminescent substrate are added to the subject's plasma. PLG in the subject's plasma is converted into PLM under the action of SK, which then reacts with the luminescent substrate. The increase in absorbance is then measured using a spectrophotometer; the increase in absorbance is directly proportional to the activity of plasminogen. In addition, plasminogen activity in blood can be measured using immunochemical methods, gel electrophoresis, immunoturbidimetry, radial immunodiffusion, and the like.

[0069] "Ortholog" refers to a homologue between different species, including both protein and DNA homologues, also known as a direct gene. It specifically refers to a protein or gene evolved from the same ancestral gene between different species. The plasminogen of the present invention includes human native plasminogen, and further includes orthologs or orthologs of plasminogens with plasminogen activity derived from different species.

[0070] "Conservative substitution variants" refer to polypeptides or enzymes in which a single, specific amino acid residue has been altered without altering the overall conformation and function of the protein or enzyme. This includes, but is not limited to, substitutions of amino acids with similar properties (e.g., acidic, alkaline, hydrophobic, etc.) for amino acids in the amino acid sequence of the parent protein. Amino acids with similar properties are known. For example, arginine, histidine, and lysine are hydrophilic, alkaline amino acids and can be substituted for each other. Similarly, isoleucine is a hydrophobic amino acid and can be substituted by leucine, methionine, or valine. Therefore, the similarity between two functionally similar proteins or amino acid sequences may vary. For example, they may have 70% to 99% similarity (identity) based on the MEGALIGN algorithm. "Conservative substitution variants" also include polypeptides or enzymes with 60% or more amino acid identity based on the BLAST or FASTA algorithm, preferably 75% or more, most preferably 85% or more, and even more preferably 90% or more, and still have the same or essentially similar properties or functions as the native or parent protein or enzyme.

[0071] "Pathological TDP-43 protein" is a relative term to "physiologically functional TDP-43 protein." A physiologically functional TDP-43 protein refers to a TDP-43 protein that is capable of performing its desired function in a cellular environment in a living organism. On the other hand, a "pathological TDP-43 protein" refers to a TDP-43 protein that is unable to perform its desired function in a cellular environment in a living organism. Examples of pathological TDP-43 proteins include, but are not limited to, mutated TDP-43 proteins that have lost their physiological function (e.g., more than 50%, 60%, 70%, 80%, or 90% of their relevant physiological function), TDP-43 proteins that form protein aggregates, misfolded TDP-43 proteins, abnormally modified TDP-43 proteins (including hyperphosphorylated, ubiquitinated, acetylated, and proteolytically cleaved C-terminal fragments), and TDP-43 proteins that have undergone proteolysis. Another characteristic of pathological states of TDP-43 is that TDP-43 redistributes and accumulates from the nucleus to the cytoplasm, and such proteins are also included within the scope of pathological TDP-43 proteins of the present application. In the present application, when plasminogen "promotes the degradation of TDP-43 (protein)," it means that plasminogen promotes the degradation of pathological TDP-43 (protein).

[0072] "Isolated" plasminogen refers to plasminogen protein that has been separated and / or recovered from its natural environment. In some embodiments, the plasminogen is (1) purified to greater than 90%, greater than 95%, or greater than 98% purity (by weight), e.g., as determined by the Lowry method, e.g., greater than 99% purity (by weight); (2) purified to the extent that at least 15 residues of N-terminal or internal amino acid sequence are obtained using a spinning cup sequencer; or (3) purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions with Coomassie brilliant blue or silver staining. Isolated plasminogen includes plasminogen produced from recombinant cells by bioengineering techniques and separated by at least one further purification step.

[0073] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term also encompasses fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions containing leader sequences of heterologous and homologous origin (with or without an N-terminal methionine residue); and the like.

[0074] The "percentage (%) amino acid sequence identity" of a reference peptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and excluding any conservative substitutions that are not considered part of the sequence identity. Alignment for purposes of determining percentage amino acid sequence identity can be accomplished by several means within the skill of the art, including publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm that achieves the maximum comparison required for the full length of the sequences being compared. However, for purposes of the present invention, the percentage amino acid sequence identity is determined by the sequence comparison computer program ALIGN-2.

[0075] When comparing amino acid sequences by using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (also referred to as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or for a given amino acid sequence B) is calculated as follows: Fraction X / Y×100

[0076] where X is the number of amino acid residues that the sequence alignment program ALIGN-2 evaluates as identical and matching in its alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood as follows: if the lengths of amino acid sequence A and amino acid sequence B are not equal, the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are as described in the preceding paragraph and are generated by the ALIGN-2 computer program.

[0077] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0078] A "therapeutically effective amount" or "effective amount" is an amount of plasminogen that, when administered to a mammal or other subject to treat a disease, can prevent and / or treat the disease. The "therapeutically effective amount" varies depending on the plasminogen used, the severity of the disease and / or symptoms, and the age, weight, etc., of the subject to be treated.

[0079] The term "treatment" of a disease state includes inhibiting or arresting the progression of said disease state or its clinical symptoms, or alleviating said disease state or symptoms such that said disease state or its clinical symptoms are diminished, either temporarily or permanently.

[0080] Preparation of the Plasminogen of the Present Invention Plasminogen may be isolated and purified from nature for further therapeutic use, or it may be synthesized by standard chemical peptide synthesis techniques. When polypeptides are synthesized by chemical methods, synthesis can be performed in either liquid or solid phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support and the remaining amino acids in the sequence are sequentially coupled, is suitable for chemically synthesizing plasminogen. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen. The techniques used in solid-phase synthesis are described in Barany and Solid-Phase Peptide Synthesis; p. 3-284; The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al., J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6: 3-10 and Camarero JA et al., 2005 Protein Pept Lett. 12: 723-8. Briefly, small insoluble porous beads are prepared with functional units onto which peptide chains are constructed. After repeated coupling / deprotection cycles, a single N-protected amino acid unit is coupled to the free N-terminal amine of the attached solid phase. The unit is then deprotected, revealing a new N-terminal amine for coupling with another amino acid. The peptide remains immobilized on the solid phase, and is then cleaved.

[0081] The plasminogen of the present invention is produced by standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector and operably linked to control sequences in the expression vector. Expression control sequences include, but are not limited to, a promoter (e.g., a naturally associated promoter or a heterologous promoter), a signal sequence, an enhancer element, and a transcription termination sequence. Expression can be controlled by a eukaryotic promoter system in a vector that is transformed or transfected into eukaryotic host cells (e.g., COS or CHO cells). Once the vector is introduced into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for the collection and purification of the plasminogen.

[0082] Suitable expression vectors typically replicate in the host either episomes or as an integral part of the host chromosomal DNA. Expression vectors usually contain selectable markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline-resistance, kanamycin-resistance, or neomycin-resistance) that are useful for detecting in vitro those cells transformed with the desired DNA sequences.

[0083] Escherichia coli is an example of a prokaryotic host cell that can be used to clone a polynucleotide encoding plasminogen. Other suitable microbial hosts include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors can be generated in these prokaryotic hosts and typically contain expression control sequences (e.g., an origin of replication) compatible with the host cell. Many known promoters are available, including the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, and the phage lambda-derived promoter system. Promoters generally control expression and, if necessary, may contain ribosome binding sequences to initiate transcription and translation of the gene sequence.

[0084] Other microorganisms, such as yeast, can also be used for expression. Yeast (e.g., Saccharomyces (S. cerevisiae) and Pichia are examples of suitable yeast host cells, in which suitable carriers include expression control sequences (e.g., promoters), origins of replication, termination sequences, etc., as needed. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters for alcohol dehydrogenase, isocytochrome C, and enzymes for maltose and galactose utilization.

[0085] In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in in vitro cell culture) can also be used to express and produce the plasminogen of the present invention (e.g., a polynucleotide encoding plasminogen). See, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for use in these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from the albumin immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0086] Once synthesized (chemically or recombinantly), the plasminogen described in the present invention can be purified by standard procedures in the art, such as ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, etc. The plasminogen can be essentially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure or even purer, e.g., free from contaminants, such as cellular debris, large molecules other than plasminogen, etc.

[0087] Drug combinations Plasminogen of the desired purity is mixed with pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)) as needed to form a lyophilized preparation or aqueous solution to obtain a therapeutic formulation. Acceptable carriers, excipients, and stabilizers are non-toxic to subjects at the required doses and concentrations, and further include buffers such as phosphates, citrates, and other organic acids. Antioxidants include ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethylenediamine chloride; benzalkonium chloride). chloride), benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parahydroxybenzoate esters, such as methyl or propyl parahydroxybenzoate ester; pyrocatechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptides (fewer than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, fucose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants, such as TWEENT™, PLURONICS™, or polyethylene glycol (PEG).

[0088] The combination preparation of the present invention may contain one or more active compounds required for the specific symptoms requiring treatment, preferably compounds with complementary activities and no adverse effects, such as antihypertensives, antiarrhythmics, and antidiabetic drugs.

[0089] The plasminogen of the present invention can be encapsulated in microcapsules made, for example, by aggregation techniques or interfacial polymerization, and can be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into hydroxymethylcellulose or gel microcapsules and poly(methyl methacrylate) microcapsules in coarse emulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0090] The plasminogen of the present invention to be administered internally must be sterile, which is readily accomplished by filtration through sterile filtration membranes, prior to or after lyophilization and recombination.

[0091] The plasminogen of the present invention can be prepared in sustained-release preparations. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers having defined shapes and containing the glycoprotein, such as membranes or microcapsules. Examples of buffer matrices include polyesters, aqueous gels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981); Langer, Chem. Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S. Patent 3,773,919, EP 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer et al., supra), or degradable lactic acid-hydroxyacetic acid copolymers, e.g., Lupron. These include Depot™ (injectable microspheres composed of lactic acid-hydroxyacetic acid copolymer and leuprolide acetate), and poly(D-(-)-3-hydroxybutyrate). Polymers such as ethylene-ethyl acetate and lactic acid-hydroxyacetic acid can release molecules sustainedly for over 100 days, whereas some aqueous gels release proteins for a relatively short period of time. Rational strategies for protein stabilization can be designed depending on the mechanism involved. For example, if the aggregation mechanism involves the exchange of sulfur disulfide bonds to form intermolecular disulfide bonds, stabilization can be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions.

[0092] Dosage and dosage The administration of the pharmaceutical compositions of the present invention can be achieved by different ways, such as intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (eg, via the carotid artery), intramuscular administration.

[0093] Preparations used for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte supplements, and the like. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, inert gases, and the like, may also be present.

[0094] A medical professional can determine dosage recommendations based on various clinical factors. For example, as is well known in the medical arts, the dosage for any given patient depends on several factors, including the patient's body size, body surface area, age, the specific compound being administered, sex, frequency and route of administration, overall health, and other concurrently administered medications. The dosage range for the plasminogen-containing pharmaceutical compositions of the present invention can be about 0.0001 to 2000 mg / kg of subject body weight daily, or about 0.001 to 500 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 10 mg / kg, 50 mg / kg, etc.). For example, dosages can be 1 mg / kg body weight or 50 mg / kg body weight, or in the range of 1-50 mg / kg, or at least 1 mg / kg. Doses higher or lower than this exemplary range are also encompassed, particularly when the aforementioned factors are taken into consideration. Intermediate doses within the above range are also within the scope of the present invention. Subjects can receive such doses daily, every other day, weekly, or according to any schedule determined by empirical analysis. An exemplary dosing schedule includes administration of 1-10 mg / kg for several consecutive days. During the administration of the agents of the present invention, real-time evaluation of therapeutic efficacy and safety is required.

[0095] Product or Kit One embodiment of the present invention relates to a product or kit containing the plasminogen or plasmin of the present invention, which can be used to treat cardiovascular disease and related diseases caused by diabetes. The product preferably includes a container, label, or protocol. Suitable containers include bottles, vials, syringes, etc. The container can be made of various materials, such as glass or plastic. The container contains a composition that effectively treats the disease or condition of the present invention and has a sterile access point (e.g., the container is an intravenous infusion pack or vial and includes a stopper that can be pierced by a hypodermic needle). At least one activator in the composition is plasminogen or plasmin. A label on or attached to the container explains that the composition is used to treat cardiovascular disease and related diseases caused by diabetes according to the present invention. The product may further include a second container containing a pharmaceutical buffer, such as phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials required from a commercial and user perspective, such as other buffers, diluents, filters, needles, and syringes. The product also includes a protocol with instructions for use, for example, instructing a user of the composition to administer the plasminogen composition and other agents to a patient for the treatment of the associated disorder.

[0096] In this application, "muscle atrophy" refers to a decrease in the amount, structural abnormality or loss, and / or functional abnormality or weakness or loss of muscle tissue caused by various causes. The causes of muscle atrophy mainly include various muscle diseases or trauma, such as syringomyelia, myelitis, cervical spondylotic radiculopathy, basilar arachnoiditis, brainstem lesions, and brain and spinal nerve lesions. [Example]

[0097] The human plasminogen used in the following examples was derived from donor plasma and has been described in the following literature: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry, 1965, 240(1):541-550; Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J Biol Chem. 1976 Jun 25; 251(12):3693-9; Hagan JJ, Ablondi F, De Renzo EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 Based on the method described in [1-3], the process was optimized and purified from human donor plasma, resulting in over 98% human Lys-plasminogen (Lys-plasminogen) and Glu-plasminogen (Glu-plasminogen).

[0098] Example Example 1 Plasminogen promotes cleavage of pathological TDP-43 protein in normal mouse brain homogenate Four C57BL / 6J male mice, 11–12 weeks old and weighing 18–25 g, were sacrificed to collect whole brain tissue, which was then weighed and homogenized at 4°C (1 min, 3–4 times) in 1x PBS (Thermo Fisher, pH 7.4; 10010-031) at 150 mg tissue / mL PBS. After homogenization, the homogenized tissue was centrifuged at 4°C (12,000 rpm, 20 min), and the supernatant (i.e., brain tissue homogenate) was collected and transferred to a new EP tube.

[0099] Eppendorf (EP) tubes were taken and five parallel tubes were set up for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL of saline, 4.6 μL of solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL of mouse brain homogenate; the blank control group was added with 21.5 μL of saline, 2.3 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate; the solvent control group was added with 20.5 μL TDP-43 (GenScript Biotech Corporation, custom-expressed human TDP-43, C134WHE160-2 / P5HF001, 1.05 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate; the plasminogen group was added with 20.5 μL TDP-43 (1.05 mg / mL), 2.3 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to each group. After adding the samples to each group, the mixture was incubated at 37°C for 3 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction.

[0100] A 12% gel was prepared according to the SDS-PAGE gel preparation method. Each sample was mixed uniformly with 4x loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated to 100°C for 5 minutes, cooled, centrifuged for 2 minutes, and then 20 μL was loaded. Electrophoresis was performed at 30 V for 45 minutes, followed by electrophoresis at 100 V until the gel bottom was reached. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753). Electrophoresis was performed at 15 V for 2.5 hours. The transferred PVDF membrane was immersed in blocking solution (5% skim milk) and blocked overnight in a 4°C refrigerator. After washing four times with TBST (0.01M Tris-NaCl, pH 7.6 buffer), rabbit anti-human TDP-43 antibody (Proteintech (China), 12892-1-AP) and cyclophilin antibody were added and incubated at room temperature for 1.5 hours. After washing four times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated at room temperature for 1 hour. After washing four times with TBST, the PVDF membrane was placed on a clean imaging plate and developed with Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100). Images were captured using a biomolecular imaging system and quantitative analysis was performed using Image J.

[0101] The results showed that the molecular weight of the recombinant TDP-43 protein monomer was approximately 43 kDa, the molecular weight of the high-molecular-weight TDP-43 protein (HMW) was greater than 55 kDa, and the molecular weight of the low-molecular-weight TDP-43 fragment (LMW) was less than 40 kDa. Furthermore, in brain homogenates from normal mice, the amounts of TDP-43 monomer, HMW, and LMW in the plasminogen group were significantly lower than those in the solvent control group, and the differences were highly significant (*** indicates P<0.001, * indicates P<0.05) (Figure 2). This suggests that plasminogen can promote the cleavage of TDP-43 in brain homogenates from normal mice.

[0102] Example 2 Plasminogen promotes cleavage of pathological TDP-43 protein in brain homogenates from amyotrophic lateral sclerosis model mice Four B6.Cg-Tg(SOD1-G93A)1Gur / J transgenic male mice (abbreviated as SOD1-G93A transgenic mice) were sacrificed to collect whole brain tissue, which was then weighed and homogenized at 4°C (1 min, 3–4 times) in 1x PBS (Thermo Fisher, pH 7.4; 10010-031) at 150 mg tissue / mL PBS. After homogenization, the tissue was centrifuged at 4°C (12,000 rpm, 20 min), and the supernatant (i.e., brain tissue homogenate) was collected and transferred to a new EP tube.

[0103] Eppendorf (EP) tubes were taken and five parallel tubes were set up for each group: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. The blank group was added with 21.5 μL of saline, 4.6 μL of solvent solution (10 mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH 7.4), and 23.9 μL of mouse brain homogenate; the blank control group was added with 21.5 μL of saline, 2.3 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate; the solvent control group was added with 20.5 μL TDP-43 (GenScript Biotech Corporation, custom-expressed human TDP-43, C134WHE160-2 / P5HF001, 1.05 mg / mL), 4.6 μL of solvent solution, and 23.9 μL of mouse brain homogenate; the plasminogen group was added with 20.5 μL TDP-43 (1.05 mg / mL), 2.3 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added to each group. After adding the samples to each group, the mixture was incubated at 37°C for 3 hours, and then 50 μL of 0.1% trifluoroacetic acid solution was added to each group to stop the reaction.

[0104] A 12% gel was prepared according to the SDS-PAGE gel preparation method. Each sample was mixed uniformly with 4x loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated to 100°C for 5 minutes, cooled, centrifuged for 2 minutes, and then 20 μL was loaded. Electrophoresis was performed at 30 V for 45 minutes, followed by electrophoresis at 100 V until the gel bottom was reached. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753). Electrophoresis was performed at 15 V for 2.5 hours. The transferred PVDF membrane was immersed in blocking solution (5% skim milk) and blocked overnight in a refrigerator at 4°C. After washing four times with TBST (0.01M Tris-NaCl, pH 7.6 buffer), rabbit anti-human TDP-43 antibody (Proteintech, 12892-1-AP) and cyclophilin antibody were added and incubated for 1.5 hours at room temperature. After washing four times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added and incubated for 1 hour at room temperature. After washing four times with TBST, the PVDF membrane was placed on a clean imaging plate and developed with Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100). Images were captured using a biomolecular imaging system and quantitative analysis was performed using Image J.

[0105] The results showed that the molecular weight of the recombinant TDP-43 protein monomer was approximately 43 kDa, the molecular weight of the high-molecular-weight TDP-43 protein (HMW) was greater than 55 kDa, and the molecular weight of the low-molecular-weight TDP-43 fragment (LMW) was less than 40 kDa. Furthermore, in the brain homogenates of amyotrophic lateral sclerosis (ALS) model mice, the amounts of TDP-43 monomer, HMW, and LMW were significantly lower in the plasminogen group than in the solvent control group, and the differences were highly significant (*** indicates P<0.001, ** indicates P<0.01) (Figure 3). This suggests that plasminogen can promote the cleavage of TDP-43 in the brain homogenates of ALS model mice.

[0106] Example 3 Plasminogen promotes degradation of TDP-43 in spinal cord tissue of amyotrophic lateral sclerosis model mice Six SOD1-G93A mice aged 10–15 weeks were randomly divided into two groups: three in the vehicle control group and three in the drug-treated group. Mice in the vehicle control group were injected with vehicle at 5 ml / kg via the tail vein, while mice in the drug-treated group were injected with plasminogen (10 mg / ml) at 50 ml / kg body weight via the tail vein. Mice were sacrificed 24 hours after injection, and their spinal cords were collected. After homogenization, Western blot detection of TDP-43 was performed.

[0107] The results showed that the amounts of TDP-43 monomer and low-molecular-weight TDP-43 in the spinal cord tissue of mice in the drug-treated group were significantly lower than those in the vehicle group, and the difference was statistically significant (* indicates P<0.05) (Figure 4). This indicates that plasminogen can promote the degradation of TDP-43 in the spinal cord tissue of amyotrophic lateral sclerosis model mice.

[0108] Example 4 Plasminogen promotes degradation of TDP-43 in brain tissue of amyotrophic lateral sclerosis model mice Nine 6-7 week-old C57BL / 6J male mice were weighed before modeling. After weighing, all mice were randomly divided into two groups: a blank control group of three mice and a model group of six mice. After grouping, the sham-operated and model groups were anesthetized with an intraperitoneal injection of 20 mL / kg tribromoethanol. The model group mice were located in the hippocampus using a 3D stereotaxic atlas (based on bregma coordinates: AP -2.54 mm, ML ±2 mm, DV -2.4 mm), and each mouse was slowly microinjected bilaterally. The sham-operated group mice were drilled only 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 μL / min and a volume of 3 μL per side. After injection, the syringe was left in place for 5 minutes, after which it was slowly withdrawn. Three days after the brain-localized injection, all mice were weighed, and the model group mice were intraperitoneally injected with 5 mg / kg LPS solution according to their body weight. The model group mice were then randomly divided into two groups: three mice in the drug-treated group and three mice in the vehicle group. 24 hours after the LPS injection, the sham-operated and vehicle groups were injected with 5 mL / kg of vehicle via the tail vein, while the drug-treated group mice were injected with 50 mg / kg plasminogen via the tail vein for three consecutive days. Two hours after the third injection, the mice were sacrificed, and brain tissue was collected. After homogenization, Western blot detection of TDP-43 was performed.

[0109] The results showed that the amounts of TDP-43 monomer 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, and the difference was statistically significant (* indicates P<0.05) (Figure 5). This indicates that plasminogen can promote the degradation of TDP-43 in the brain tissue of amyotrophic lateral sclerosis model mice.

[0110] Example 5 Plasminogen is concentrated in the spinal cord tissue of amyotrophic lateral sclerosis model mice and co-localizes with TDP-43 intracellularly. Five wild-type male mice and nine SOD1-G93A male mice of similar age were used. Wild-type mice served as a blank control group, while SOD1-G93A mice were observed and recorded from the time of hindlimb tremors at 14 weeks of onset. The time of onset for each mouse was recorded, and drug administration began 14 days after onset. All mice were randomly assigned to a vehicle or drug-treatment group based on the onset of symptoms. Five mice in the vehicle group received daily injections of 0.1 ml of vehicle (sodium citrate buffer) via the tail vein, while four mice in the drug-treatment group received daily injections of 1 mg of plasminogen per 0.1 ml via the tail vein. These mice were continuously administered in an SPF environment, and samples were collected near the end of life. The maximum treatment period was 61 days. Spinal cord tissue was fixed in formalin. Fixed tissue was dehydrated through an alcohol gradient, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, rehydrated, and then washed once with water. The sections were immersed in an antigen retrieval solution (0.01 M sodium citrate buffer) and microwaved for 5 minutes (preheating), 2 minutes on high heat, and 15 minutes on low heat. The tissue was circled with a PAP marker and incubated with 3% hydrogen peroxide for 15 minutes, followed by two washes with 0.01 M PBS, each for 5 minutes. The sections were blocked with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 minutes. The serum was discarded at the end of the blockade period. Self-prepared anti-plasminogen antibody was added dropwise and incubated overnight at 4°C, followed by two washes with 0.01 M PBS, each for 5 minutes. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added for 1 hour at room temperature and then washed twice with 0.01 M PBS, each for 5 minutes. Green fluorescence staining of the corresponding anti-plasminogen secondary antibody was performed according to the protocol of the XTSA520 IHC kit (Alpha X Biotech, AXT6202500). After washing three times with PBS for 5 minutes each time, the above antigen retrieval and blocking procedures were repeated, followed by staining with anti-TDP-43 antibody (Proteintech, 12892-1-AP) and incubation at 37°C for 1 hour. After washing three times with PBS for 5 minutes each time, the cells were stained with anti-TDP-43 antibody (Proteintech, 12892-1-AP).The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. Red fluorescence staining with the corresponding anti-TDP-43 secondary antibody was performed according to the protocol of the XTSA520 IHC kit (Alpha X Biotech, AXT6202500). The sections were washed three times with PBS for 5 minutes each time. Nuclear staining was performed with DAPI (BOSTER, 11K16B77). The sections were dehydrated through an alcohol gradient, cleared with xylene, and mounted in neutral rubber. Sections were observed and photographed under a light microscope at 400x magnification.

[0111] JPEG2025539229000003.jpg53129

[0112] Example 6 Plasminogen is concentrated in muscle tissue of amyotrophic lateral sclerosis model mice and co-localizes with TDP-43 in cells Five wild-type male mice and nine SOD1-G93A male mice of similar age were used. Wild-type mice served as a blank control group, while SOD1-G93A mice were observed and recorded from the time of hindlimb tremors at week 14 of onset. The time of onset for each mouse was recorded, and drug administration began 14 days after onset. All mice were randomly assigned to a vehicle or drug-treatment group based on onset status. Five mice in the vehicle group received daily injections of 0.1 ml of vehicle (sodium citrate buffer) via the tail vein, while four mice in the drug-treatment group received daily injections of 1 mg of plasminogen at 0.1 ml per mouse via the tail vein. These mice were continuously administered in an SPF environment, and samples were collected near the end of life. The maximum treatment period was 61 days. Gluteal muscle tissue was fixed in formalin. Fixed tissue was dehydrated through an alcohol gradient, permeabilized in xylene, and embedded in paraffin. Tissue sections were 3 μm thick. They were deparaffinized, rehydrated, and then washed once with water. The sections were immersed in an antigen retrieval solution (0.01 M sodium citrate buffer) and microwaved for 5 minutes, followed by 2 minutes on high heat and 15 minutes on low heat. The tissue was circled with a PAP marker and incubated with 3% hydrogen peroxide for 15 minutes, followed by two 5-minute washes with 0.01 M PBS. The sections were then blocked with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 minutes. The serum was then discarded, and autologous anti-plasminogen antibody (autologous) was added and incubated overnight at 4°C. The sections were then incubated with a goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two 5-minute washes with 0.01 M PBS. Green fluorescence staining of the corresponding anti-plasminogen secondary antibody was performed according to the protocol of the XTSA520 IHC kit (Alpha X Biotech, AXT6202500). After washing three times with PBS for 5 minutes each time, the above antigen retrieval and blocking procedures were repeated, followed by staining with anti-TDP-43 antibody (Proteintech, 12892-1-AP) and incubation at 37°C for 1 hour. After washing three times with PBS for 5 minutes each time, the cells were stained with anti-TDP-43 antibody (Proteintech, 12892-1-AP).The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. The sections were incubated with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody for 1 hour at room temperature, followed by two washes with 0.01M PBS for 5 minutes each time. Red fluorescence development with the corresponding anti-TDP-43 secondary antibody was performed according to the protocol of the XTSA520 IHC kit (Alpha X Biotech, AXT6202500). The sections were washed three times with PBS for 5 minutes each time. Nuclear staining was performed with DAPI (BOSTER, 11K16B77). The sections were dehydrated through an alcohol gradient, cleared with xylene, and mounted in neutral rubber. Sections were observed and photographed under a light microscope at 400x magnification.

[0113] JPEG2025539229000004.jpg37127

[0114] Example 7 Plasminogen promotes degradation of TDP-43 in brain tissue of okadaic acid-induced dementia model mice Ten 30-32 week-old B6SJL-Tg(APPSwFlLon, PSEN1*M146L*L286V) 6799Vas / Mmjax (hereafter referred to as FAD) female mice (purchased from Jackson Lab, stock number: 034840) were weighed before modeling and used as the model group. Five 6-7 week-old C57 female mice were used as the blank group. After grouping, the blank and model groups were anesthetized with an intraperitoneal injection of 20 mL / kg tribromoethanol. The model group mice were positioned in the basolateral amygdala using a mouse stereotaxic atlas (based on bregma coordinates: AP -1.94 mm, ML ±3.15 mm, DV -4.5 mm) and slowly microinjected bilaterally. The blank group mice were drilled only at the coordinate positioning points and no injections were performed [5]. Model mice were injected with 50 ng / μL okadaic acid (manufacturer: Shanghai Yuanye Bio-Technology Co., Ltd., catalog number S30686-25μg) solution at a rate of 0.5 μL / min in a volume of 2 μL. After injection, the syringe was left in place for 6 minutes and then slowly withdrawn. Three days after the brain-localized injection, all mice were weighed. Based on their weight, the model mice were randomly divided into two groups: five drug-treated and five vehicle-treated. The blank and vehicle-treated mice were injected with 5 mL / kg vehicle via the tail vein, while the drug-treated mice were injected with 50 mg / kg plasminogen via the tail vein. Six hours after the single injection, the mice were sacrificed and brain tissue was collected. After homogenization, Western blot detection of TDP-43 was performed.

[0115] The results showed that the levels of TDP-43 monomer and low-molecular-weight TDP-43 in the brain tissue of mice treated with the drug were significantly lower than those in the vehicle group (Figure 8), indicating that plasminogen can promote the degradation of TDP-43 in the brain tissue of okadaic acid-induced dementia model mice.

[0116] Example 8 Plasminogen promotes degradation of TDP-43 in kidney nuclei of amyotrophic lateral sclerosis model mice Nine to ten-week-old SOD1-G93A mice (Jackson Laboratory, stock number: 004435) and C57BL / 6J mice were obtained. The SOD1-G93A mice were randomly divided into two groups (vehicle group and drug-treated group), with three C57BL / 6J mice as normal controls. Mice in the vehicle group received daily injections of 5 mL / kg of vehicle (10 mM citric acid sodium citrate solution, pH 7.4) via the tail vein, while mice in the drug-treated group received daily injections of 50 mg / kg of plasminogen via the tail vein. Control mice received no drug. After 7 days, the mice were sacrificed and kidney tissue was collected. The collected kidney tissue was placed in pre-chilled RPMI-1640 (Sigma-Aldrich) medium on ice. After rinsing with PBS, the kidney tissue was minced and digested with 0.25% trypsin at 37°C for 10 minutes, with constant shaking every 2 minutes. The digestion was stopped by adding DMEM medium containing 10% fetal bovine serum. After centrifugation (1500 rpm, 5 minutes), the supernatant was removed to obtain a single cell pellet. For every 20 μL of cell pellet, 200 μL of plasma protein extraction reagent (2 × 10 6The volume of each cell pellet was approximately 20 μL (or 40 mg) (Solarbio, R0050). The cell pellet was completely dispersed into a single cell suspension by pipetting using a pipette or vortexing at high speed for 15 seconds. The mixture was then placed in an ice bath for 10 minutes. The mixture was then vortexed vigorously at maximum speed for 10 seconds and centrifuged at 12,000–16,000 g for 10 minutes at 4°C. The supernatant, which represents the extracted cytoplasmic proteins, was immediately aspirated into a pre-chilled sample tube for later use. The precipitate, representing the cell nuclei, was then added to the remaining supernatant (to avoid contamination with cytoplasmic proteins). The remaining supernatant was then completely aspirated (to avoid contamination with cytoplasmic proteins) and 50–100 μL of nuclear protein extraction reagent was added. The precipitate was completely dispersed by pipetting using a pipette or vortexing for 15 seconds (optionally extended), and then placed in an ice bath for 10 minutes. After vigorously vortexing at maximum speed for 10 seconds, the mixture was centrifuged at 12,000-16,000 g for 10 minutes at 4°C. The supernatant was immediately aspirated and placed in a pre-chilled sample tube. This represents the extracted nuclear proteins. The extracted nuclear proteins were detected by TDP-43 Western blotting.

[0117] The results show that the TDP-43 levels in the kidney cell nuclei of the drug-treated group were significantly lower than those of the vehicle-treated group (Figure 9A-B), suggesting that plasminogen can promote the degradation of TDP-43 in the kidney cell nuclei.

[0118] Example 9 Plasminogen promotes degradation of TDP-43 in the cytoplasm and nucleus of okadaic acid-treated NSC34 cells 10 6 NSC34 cells (Otwo Biotech, HTX1846) were cultured in a 9cm 2The cells were seeded in petri dishes and cultured in DMEM medium (Gibco, 11965092) containing 10% fetal bovine serum (EVERY GREEN, 11011-8611), then placed in a carbon dioxide incubator at 37.0°C with 5% CO2. After the cells had grown for 48 hours and reached approximately 80%-90% confluence, the medium was replaced and the next experiment was performed. The cells were divided into four groups: a blank control group, a vehicle group, a drug-treated group, and a drug + EACA group. The blank control group was not treated after the medium change. The vehicle, drug-treated, and drug + EACA groups were exposed to 2.5 ng / μL okadaic acid (OA) (Shanghai Yuanye Bio-Technology, S30686-25μg). After 24 hours of stimulation with okadaic acid, the vehicle was added to the cell culture medium of the vehicle group, plasminogen (0.5 mg / mL) was added to the cell culture medium of the drug-treated group, and plasminogen (final concentration 0.5 mg / mL) and aminocaproic acid (EACA) (20 mM) were added to the cell culture medium of the drug + EACA group. After 24 hours of incubation with plasminogen, the cells were harvested. The culture supernatant was aspirated, washed with 1x PBS, and digested with 1 mL of 0.25% trypsin for 2–3 minutes. When cells were clearly detached, the digestion was stopped with 5–6 mL of DMEM complete medium. The cells were gently pipetted, the suspension was collected in a centrifuge tube, and centrifuged at 1500 rpm for 5 minutes to remove the supernatant. The cells were resuspended in pre-chilled 1x PBS, and then counted. For every 20 μL of cell pellet, 200 μL of plasma protein extraction reagent (2 x 10 6The volume of each cell pellet was approximately 20 μL (or 40 mg) of 1000-μL nuclear protein extraction reagent (Solarbio, R0050) was added. The cell pellet was completely dispersed into a single cell suspension by pipetting using a pipette or vortexing at high speed for 15 seconds. The mixture was then placed in an ice bath for 10 minutes. The mixture was then vortexed vigorously at maximum speed for 10 seconds and centrifuged at 12,000–16,000 g for 10 minutes at 4°C. The supernatant, which contains the extracted cytoplasmic proteins, was immediately pipetted into a pre-chilled sample tube for later use. The precipitate, which contains the cell nuclei, was then added to the remaining supernatant by aspirating (to avoid contamination with cytoplasmic proteins). The precipitate was completely dispersed by pipetting using a pipette or vortexing at high speed for 15 seconds (this can be extended as needed), and the mixture was then placed in an ice bath for 10 minutes. After vigorously vortexing at maximum speed for 10 seconds, the mixture was centrifuged at 12,000-16,000 g for 10 minutes at 4°C. The supernatant was immediately aspirated and placed in a pre-chilled sample tube. This represents the extracted nuclear proteins. The extracted nuclear proteins were detected by TDP-43 Western blotting.

[0119] Aminocaproic acid (EACA) is a lysine analog that blocks the high-affinity lysine binding site on plasminogen [6].

[0120] The results show that the TDP-43 levels in the cytoplasm and nucleus of the drug-treated group were significantly lower than those in the nucleus of the vehicle group, indicating that the addition of EACA completely inhibited the effect of plasminogen on TDP-43 (Figure 10A-D). These results suggest that plasminogen can promote the degradation of TDP-43 in the cytoplasm and nucleus, and that this effect of plasminogen is closely related to the lysine-binding site in its structure.

[0121] Example 10 Plasminogen promotes increased levels of plasminogen and plasmin activity in the cytoplasm and nucleus of okadaic acid-treated NSC34 cells 10 6NSC34 cells (Otwo Biotech, HTX1846) were cultured in a 9cm 2 The cells were seeded in petri dishes and cultured in DMEM medium (Gibco, 11965092) containing 10% fetal bovine serum (EVERY GREEN, 11011-8611), then placed in a carbon dioxide incubator at 37.0°C with 5% CO2. After the cells had grown for 48 hours and reached approximately 80%-90% confluence, the medium was replaced and the next experiment was performed. The cells were divided into three groups: vehicle, drug-treated, and drug + EACA. The vehicle, drug-treated, and drug + EACA groups were exposed to 2.5 ng / μL okadaic acid (OA) (Shanghai Yuanye Bio-Technology, S30686-25μg). After 24 hours of stimulation with okadaic acid, the vehicle was added to the cell culture medium of the vehicle group, plasminogen (0.5 mg / mL) was added to the cell culture medium of the drug-treated group, and plasminogen (final concentration 0.5 mg / mL) and aminocaproic acid (EACA) (20 mM) were added to the cell culture medium of the drug + EACA group. After 24 hours of incubation with plasminogen, the cells were harvested. The culture supernatant was aspirated, washed with 1x PBS, and digested with 1 mL of 0.25% trypsin for 2–3 minutes. When cells were clearly detached, the digestion was stopped with 5–6 mL of DMEM complete medium. The cells were gently pipetted, the suspension was collected in a centrifuge tube, and centrifuged at 1500 rpm for 5 minutes to remove the supernatant. The cells were resuspended in pre-chilled 1x PBS, and then counted. For every 20 μL of cell pellet, 200 μL of plasma protein extraction reagent (2 x 10 6The volume of each cell pellet was approximately 20 μL (or 40 mg) of 1000 μL of nuclear protein extraction reagent (Solarbio, R0050) was added. The cell pellet was completely dispersed into a single cell suspension by pipetting using a pipette or vortexing at high speed for 15 seconds. The mixture was then placed in an ice bath for 10 minutes. The mixture was then vortexed vigorously at maximum speed for 10 seconds and centrifuged at 12,000–16,000 g for 10 minutes at 4°C. The supernatant, which contains the extracted cytoplasmic proteins, was immediately pipetted into a pre-chilled sample tube for later use. The precipitate, which contains the cell nuclei, was then added to the remaining supernatant by aspirating (to avoid contamination with cytoplasmic proteins). The precipitate was completely dispersed by pipetting using a pipette or vortexing at high speed for 15 seconds (optional), and then placed in an ice bath for 10 minutes. After vigorously vortexing at maximum speed for 10 seconds, the mixture was centrifuged at 12,000-16,000 g for 10 minutes at 4°C. The supernatant was immediately aspirated and placed in a pre-chilled sample tube. This represents the extracted nuclear protein.

[0122] After cell lysis, detection was performed according to the protocol of the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using the human plasminogen standard included in the kit as an internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per unit of total protein in each sample, and statistical analysis was performed.

[0123] Plasmin activity was detected by enzyme substrate kinetics. Seven different concentrations of standard solutions, blanks, and samples were added sequentially to an ELISA plate at 85 μL per well. Then, 15 μL of 20 mM S-2251 solution (Chromogenix, 82033239) was added to each well and incubated at 37°C. Starting at 0 min, the A405 absorbance values ​​were read every 5 min using a multi-function microplate reader until 90 min. All reactions were fitted with a linear curve using the absorbance values ​​versus time, and the slope of the line was the reaction rate (ΔA405 / min) of the standard / sample. Finally, the potency of the measured samples was calculated using the standard potency values ​​and ΔA405 / min as a standard curve.

[0124] The results showed that the levels of human plasminogen and plasmin activity in the cytoplasm and nucleus of the drug-treated group were significantly higher than those of the vehicle group, with a statistically significant difference. The addition of EACA completely inhibited these effects of plasminogen (Figures 11A-D). This suggests that plasminogen can penetrate into cells and the nucleus to enhance plasmin activity, and that the entry of plasminogen into cells and the nucleus is closely related to its lysine-binding activity.

[0125] Example 11 Administration of plasminogen promotes an increase in blood plasminogen levels in SOD1-G93A mice Twenty-seven 10- to 15-week-old B6.Cg-Tg(SOD1-G93A)1Gur / J(SOD1-G93A) mice (pedigree number: 004435) (abbreviated as SOD1-G93A mice) (breeding mice purchased from Jackson Laboratory, USA) were randomly divided into three groups: 3 mice in the vehicle control group, 12 mice in the 6 mg / kg plasminogen group, and 12 mice in the 50 mg / kg plasminogen group. Mice in the vehicle control group were injected with 5 ml / kg of vehicle via the tail vein. Mice in the 6 mg / kg plasminogen group were injected with plasminogen (1.2 mg / ml) at 6 mg / kg body weight via the tail vein. Mice in the 50 mg / kg plasminogen group were injected with plasminogen (10 mg / ml) at 6 mg / kg body weight via the tail vein. Mice in the vehicle control group were sacrificed 2 hours after administration, and blood samples were collected. Three mice each in the 6 mg / kg plasminogen group and the 50 mg / kg plasminogen group were sacrificed 2, 6, 12, and 24 hours after administration, and blood samples were collected. After centrifugation (3500 rpm, 10 minutes, 4°C), the supernatant was collected and detected using the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1) according to the protocol. The concentration of each sample was calibrated using a human plasminogen standard as an 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 statistical analysis was performed.

[0126] ELISA results for SOD1-G93A mice showed that plasma plasminogen levels increased significantly after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen level in the 50 mg / kg group being significantly higher than that in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after administration and were almost completely metabolized within 12 to 24 hours (Figure 12).

[0127] These results indicate that (1) plasma plasminogen levels exhibit a dose-dependent effect, with the higher the administered plasminogen concentration, the higher the plasma plasminogen level; and (2) plasma plasminogen levels exhibit a time-dependent effect, with an initial increase followed by a gradual decrease over the course of 2 to 12 hours.

[0128] Example 12 Administration of plasminogen promotes an increase in plasminogen levels in brain tissue of SOD1-G93A mice Brain tissues were collected from the mice sacrificed in Example 11 and homogenized, followed by detection according to the protocol for the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using the human plasminogen standard included in the kit as an internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per total protein unit in each sample, and statistical analysis was performed.

[0129] Brain ELISA results for SOD1-G93A mice showed that plasminogen levels in brain tissue increased significantly after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen level in the 50 mg / kg group being significantly higher than that in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after administration and were almost completely metabolized between 12 and 24 hours (Figure 13A). The ratios of brain tissue plasminogen levels to blood plasminogen levels were 3.47%, 4.94%, and 6.79% at 2, 6, and 12 hours after plasminogen administration, respectively (Figure 13B). These results indicate that (1) plasminogen administration under physiological and pathological conditions can promote plasminogen crossing the blood-brain barrier and its concentration in brain tissue, (2) intravenous injection of plasminogen into mice significantly increases plasminogen levels in brain tissue, (3) plasminogen concentration in brain tissue exhibits a time-dependent effect, initially increasing, gradually decreasing over 2 to 12 hours, and being almost completely metabolized over 12 to 24 hours, and (4) plasminogen concentration in brain tissue exhibits a dose-dependent effect, with the higher the dose, the higher the plasminogen level in brain tissue.

[0130] Example 13 Administration of plasminogen promotes an increase in plasminogen levels in spinal cord tissue of SOD1-G93A mice Spinal cord tissues were collected from the mice sacrificed in Example 11 and homogenized, followed by detection according to the protocol for the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using the human plasminogen standard included in the kit as an internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per total protein unit in each sample, and statistical analysis was performed.

[0131] The spinal cord ELISA results of SOD1-G93A mice showed that the spinal cord plasminogen levels of SOD1-G93A mice increased significantly after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen level in the 50 mg / kg group being significantly higher than that in the 6 mg / kg group. The plasminogen level gradually decreased 2 hours after administration and was almost completely metabolized within 12 to 24 hours (Figure 14).

[0132] These results indicate that (1) plasminogen administered at physiological doses can cross the blood-brain barrier and concentrate in the spinal cord tissue of SOD1-G93A mice, (2) plasminogen concentration in the spinal cord exhibits a dose-dependent effect, with the higher the administered dose of plasminogen, the greater the concentration, and (3) plasminogen concentration in the spinal cord exhibits a time-dependent effect, initially increasing, gradually decreasing over 2 to 12 hours, and being almost completely metabolized over 12 to 24 hours.

[0133] Example 14 Administration of plasminogen promotes an increase in plasminogen and plasmin activity levels in brain tissue of SOD1-G93A mice Eight 22-week-old SOD1-G93A mice were randomly divided into two groups: a vehicle group and a drug-treated group (four mice each). 2.5 mg / mL bacterial lipopolysaccharide (LPS) (Solarbio, L8880) was administered intratracheally to the vehicle and drug-treated mice at a modeling dose of 5 mg / kg. Four other age-matched C57 mice served as normal controls. Drug administration began three days after LPS treatment. Mice in the blank and vehicle groups received 5 mL / kg of vehicle via the tail vein, while mice in the drug-treated group received 50 mg / kg of plasminogen via the tail vein. Six hours after the single administration, the mice were sacrificed, and brain tissue was collected. After homogenization, plasminogen levels were detected by ELISA and plasmin activity enzyme substrate assay.

[0134] The results showed that the plasminogen and plasmin activity levels in brain tissue homogenates of mice in the drug-treated group were significantly higher than those in the vehicle group, and the difference was statistically significant (Figures 15A-D). These results suggest that intravenous administration of plasminogen can promote the increase of plasminogen levels and plasmin activity in brain tissue.

[0135] Example 15 Plasminogen promotes increased plasminogen levels in brain tissue, spinal cord tissue, and kidney nuclei of SOD1-G93 mice Ten 9-week-old SOD1-G93A mice were randomly divided into two groups: a vehicle group and a drug-treated group (five mice each). Five C57 mice of the same age served as normal controls. Mice in the normal and vehicle groups received 5 mL / kg of vehicle via the tail vein, while mice in the drug-treated group received 50 mg / kg of plasminogen via the tail vein daily for seven consecutive days. After seven days, the mice were sacrificed, and brain, spinal cord, and kidney tissues were collected. The tissues were minced and digested with 0.25% trypsin (Beyotime Biotechnology, C0201-500 mL). The cells were then filtered through a 200-mesh cell sieve to obtain a single-cell suspension. For every 20 μL of cell pellet, 200 μL of plasma protein extraction reagent (2 × 10 6The volume of each cell pellet was approximately 20 μL (or 40 mg) (Solarbio, R0050). The cell pellet was completely dispersed into a single cell suspension by pipetting using a pipette or vortexing at high speed for 15 seconds. The mixture was then placed in an ice bath for 10 minutes. The mixture was then vigorously vortexed at maximum speed for 10 seconds and centrifuged at 12,000–16,000 g for 10 minutes at 4°C. The supernatant, which represents the extracted cytoplasmic proteins, was immediately aspirated into a pre-chilled sample tube for later use. The precipitate, representing the cell nuclei, was then added to the remaining supernatant (to avoid contamination with cytoplasmic proteins). The remaining supernatant was then completely aspirated (to avoid contamination with cytoplasmic proteins). The mixture was then pipetted using a pipette or vortexed for 15 seconds (optionally extended) until the precipitate was completely dispersed, and then placed in an ice bath for 10 minutes. The mixture was then vigorously vortexed at maximum speed for 10 seconds and centrifuged at 12,000–16,000 g for 10 minutes at 4°C. The supernatant was immediately aspirated and placed in a pre-chilled sample tube. This is the extracted nuclear protein. Human plasminogen levels in the extracted nuclear protein were detected by ELISA.

[0136] The results showed that 7 days after plasminogen administration, the human plasminogen levels in the nuclei of the brain, spinal cord, and kidney tissues of the SOD1-G93A mice in the drug-treated group were significantly higher than those in the vehicle group, and the difference was statistically significant (*** indicates P<0.001) (FIG. 16). This suggests that intravenous administration of plasminogen can promote an increase in the human plasminogen levels in the nuclei of the brain, spinal cord, and kidney tissues.

[0137] Example 16: Administration of plasminogen promotes an increase in blood plasminogen levels in Parkinson's disease model mice Eighteen 6-week-old male C57BL / 6J mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) solution administered intraperitoneally at a dose of 35 mg / kg / mouse for five consecutive days to establish a Parkinson's disease model [7]. MPTP solution was prepared by dissolving 45 mg of MPTP (Sigma, M0896) in 9 mL of saline to a final concentration of 5 mg / mL. Mice were randomly divided into two groups based on body weight: the vehicle group (6 mice) and the drug-treated group (12 mice). The start of treatment was recorded as day 1. The drug-treated group received a plasminogen solution at 50 mg / kg body weight via the tail vein, while the vehicle group received a vehicle solution (10 mM citric acid + sodium citrate solution, pH 7.4) at 5 mL / kg body weight via the tail vein. Three mice from the vehicle group were sacrificed at 2 and 24 hours after treatment, and blood samples were collected. Mice in the drug-treated groups were sacrificed 2, 6, 12, and 24 hours after administration, and blood was collected. The blood was centrifuged (3500 rpm, 10 minutes, 4°C), and the supernatant was collected. Detection was performed according to the protocol of the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using a human plasminogen standard as an internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per total protein unit in each sample, and statistical analysis was performed.

[0138] The results showed that the blood plasminogen levels of mice in the drug-treated group were significantly higher than those of mice in the vehicle group, and the plasminogen levels gradually decreased within 2 hours after administration and were essentially completely metabolized within 12 to 24 hours (Figure 17) (*** indicates P<0.001).

[0139] Example 17 Administration of plasminogen promotes an increase in plasminogen levels in brain tissue of Parkinson's disease model mice Brain tissues were collected from the mice sacrificed in Example 16 and homogenized, followed by detection according to the protocol for the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using a human plasminogen standard as an internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per total protein unit in each sample, and statistical analysis was performed.

[0140] The results showed that plasminogen levels in the brain tissue of mice in the drug-treated group were significantly higher than those in the vehicle-treated group, and 2 hours after administration, plasminogen levels gradually decreased, and by 12 to 24 hours, plasminogen levels had essentially completely decreased (Figure 18) (*** indicates P<0.001). These results indicate that plasminogen injected via the tail vein can cross the blood-brain barrier and promote an increase in plasminogen levels in the brain tissue of Parkinson's disease model mice.

[0141] Example 18 Administration of plasminogen promotes an increase in plasminogen levels in spinal cord tissue of Parkinson's disease model mice Spinal cord tissues were collected from the mice sacrificed in Example 16 and homogenized, followed by detection according to the protocol for the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using a human plasminogen standard as an internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per total protein unit in each sample, and statistical analysis was performed.

[0142] The results showed that plasminogen levels in the spinal cord tissue of mice in the drug-treated group were significantly higher than those in the vehicle-treated group, and that plasminogen levels gradually decreased within 2 hours after administration and were essentially completely metabolized within 12 to 24 hours (Figure 19) (** indicates P<0.01, *** indicates P<0.001). These results indicate that plasminogen injected via the tail vein can cross the blood-brain barrier and promote an increase in plasminogen levels in the spinal cord tissue of Parkinson's disease model mice.

[0143] Example 19: Administration of plasminogen promotes an increase in plasminogen levels in the brain and spinal cord tissues of Parkinson's disease model mice Brain and spinal cord tissues were collected from the mice sacrificed in Example 16 and homogenized, followed by detection according to the protocol for the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using a human plasminogen standard as an internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per total protein unit in each sample, and statistical analysis was performed.

[0144] The results showed that the ratios of plasminogen levels in spinal cord tissue to blood were 1.24%, 1.16%, and 1.46%, respectively, 2, 6, and 12 hours after plasminogen administration, and the ratios of plasminogen levels in brain tissue to blood were 3.47%, 4.18%, and 8.51%, respectively, 2, 6, and 12 hours after plasminogen administration (Figure 20) (* indicates P<0.05, ** indicates P<0.01). These results indicate that plasminogen injected via 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 disease model mice.

[0145] Example 20 Plasminogen promotes increased plasminogen activity in brain tissue of Parkinson's disease model mice Fifteen female mice were weighed before modeling. They were randomly divided into two groups based on their weight: a blank control group of five 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, while mice in the blank control group were intraperitoneally injected with 7 mL / kg saline. Modeling was performed at 9:00 AM every day for five consecutive days. 24 hours after the last MPTP injection, all mice were weighed and intraperitoneally injected with 5 mg / kg LPS solution. 24 hours after the intraperitoneal LPS injection, the model group mice were randomly divided into two groups based on their weight: a drug-treated group of five mice and a vehicle group of five mice. The vehicle group mice were injected with vehicle via the tail vein, and the drug-treated group mice were injected with 50 mg / kg plasminogen via the tail vein. Mice were administered a single dose, and samples were collected by autopsy 2 hours after administration. Plasmin activity in brain tissue homogenates was detected by enzyme substrate kinetics. Plasmin activity was detected by enzyme substrate kinetics. Seven different concentrations of standard solutions, blanks, and samples were added sequentially to an ELISA plate at 85 μL per well. Then, 15 μL of 20 mM S-2251 solution (Chromogenix, 82033239) was added to each well and incubated at 37°C. Starting at 0 min, the A405 absorbance values ​​were read every 5 min up to 90 min using a multi-function microplate reader. All reactions were fitted linearly using the time and absorbance values, and the slope of the line was the reaction rate (ΔA405 / min) of the standard / sample. Finally, the potency of the measured samples was calculated using the standard potency values ​​and ΔA405 / min as a standard curve (calibration curve).

[0146] The results showed that the plasminogen activity levels in the brain tissue of mice in the drug-treated group were significantly higher than those in the vehicle-treated group, and the difference was statistically significant (Figure 21) (* indicates P<0.05). These results indicate that plasminogen injected via the tail vein can cross the blood-brain barrier and promote an increase in plasminogen activity levels in the brain tissue of Parkinson's disease model mice.

[0147] Example 21 Administration of plasminogen promotes an increase in blood plasminogen levels in Alzheimer's disease model mice Twenty-seven 16-week-old B6SJL-Tg(APPSwFlLon, PSEN1*M146L*L286V)6799Vas Mmjax (stock number: 034840) (abbreviated FAD mice) (breeding mice purchased from Jackson Laboratory, USA) were randomly divided into three groups: 3 mice in the vehicle control group, 12 mice in the 6 mg / kg plasminogen group, and 12 mice in the 50 mg / kg plasminogen group. Mice in the vehicle control group were injected with 5 ml / kg of vehicle via the tail vein. Mice in the 6 mg / kg plasminogen group were injected with plasminogen (1.2 mg / ml) at 6 mg / kg body weight via the tail vein. Mice in the 50 mg / kg plasminogen group were injected with plasminogen (10 mg / ml) at 50 mg / kg body weight via the tail vein. Mice in the vehicle control group were sacrificed 2 hours after administration, and blood samples were collected. Three mice each in the 6 mg / kg plasminogen group and the 50 mg / kg plasminogen group were sacrificed 2, 6, 12, and 24 hours after administration, and blood samples were collected. After centrifugation (3500 rpm, 10 minutes, 4°C), the supernatant was collected and detected using the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1) according to the protocol. The concentration of each sample was calibrated using a human plasminogen standard as an 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 statistical analysis was performed.

[0148] ELISA results showed that plasma plasminogen levels in FAD mice were significantly increased after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen level in the 50 mg / kg group being significantly higher than that in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after administration and were almost completely metabolized within 12 to 24 hours (Figure 22).

[0149] The results show that (1) plasma plasminogen levels exhibit a dose-dependent effect, with higher concentrations of administered plasminogen resulting in more aggregation, and (3) plasma plasminogen levels exhibit a time-dependent effect, increasing initially and gradually decreasing over 2 to 12 hours.

[0150] Example 22 Administration of plasminogen promotes an increase in plasminogen levels in brain tissue of Alzheimer's disease model mice Brain tissues were collected from the mice sacrificed in Example 21 and homogenized, followed by detection according to the protocol for the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using a human plasminogen standard as an 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 statistical analysis was performed.

[0151] Brain ELISA assays of FAD mice showed that plasminogen levels in the brain tissue of FAD mice were significantly increased after intravenous injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen levels in the 50 mg / kg group being significantly higher than those in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after injection and were almost completely metabolized within 12 to 24 hours (Figure 23A). The ratios of plasminogen in brain tissue to plasma plasminogen in the 6 mg / kg plasminogen-treated mice were 3.59% and 4.23%, respectively, 2 and 6 hours after plasminogen administration. The ratios of plasminogen in brain tissue to plasma plasminogen in the 50 mg / kg plasminogen-treated mice were 2.49%, 2.31%, and 3.32%, respectively, 2, 6, and 12 hours after plasminogen administration (Figure 23B).

[0152] These results indicate that (1) plasminogen administration under physiological and pathological conditions can promote plasminogen crossing the blood-brain barrier and its concentration in brain tissue, (2) intravenous injection of plasminogen into FAD mice significantly increased plasminogen levels, (3) plasminogen concentration in brain tissue exhibited a time-dependent effect, initially increasing, gradually decreasing over 2 to 12 hours, and being almost completely metabolized over 12 to 24 hours, and (4) plasminogen concentration in brain tissue exhibited a dose-dependent effect, with the higher the dose, the higher the plasminogen level in brain tissue.

[0153] Example 23: Administration of plasminogen promotes an increase in plasminogen levels in brain tissue of Alzheimer's disease model mice Brain tissues were collected from mice sacrificed in Example 21 and homogenized. Plasmin activity was detected by enzyme substrate kinetics. Seven different concentrations of standard solutions, blanks, and samples were added sequentially to an ELISA plate (manufacturer: NUNC, catalog number: 446469) at 85 μL / well. Then, 15 μL of 20 mM S-2251 solution (manufacturer: Chromogenix, catalog number: 82033239) was added to each well and incubated at 37°C. Starting at 0 minutes, A405 absorbance values ​​were read every 5 minutes up to 90 minutes using a multi-function microplate reader. All reactions were fitted linearly using the time and absorbance values, and the slope of the line was the reaction rate (ΔA405 / min) of the standard / sample. Finally, the potency of the measured samples was calculated using the potency values ​​of the standard and ΔA405 / min as a standard curve. The plasmin activity per total protein unit in each sample was calculated.

[0154] The results showed that plasmin activity in the brain tissue of FAD mice was significantly increased 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 (Figure 24).

[0155] These results show that plasminogen injection into mice significantly increased plasmin levels in brain tissue, and that plasmin activity in brain tissue showed a dose-dependent effect, with higher doses resulting in higher plasminogen levels in brain tissue.

[0156] Example 24 Administration of plasminogen promotes an increase in plasminogen levels in spinal cord tissue of Alzheimer's disease model mice Spinal cord tissues were collected from the mice sacrificed in Example 21 and homogenized, followed by detection according to the protocol for the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). The concentration of each sample was calibrated using a human plasminogen standard as an internal standard. The calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per total protein unit in each sample, and statistical analysis was performed.

[0157] ELISA results for spinal cord plasminogen levels in FAD mice showed a significant increase after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, with the plasminogen levels in the 50 mg / kg group being significantly higher than those in the 6 mg / kg group. Plasminogen levels gradually decreased 2 hours after injection and were almost completely metabolized within 12 to 24 hours (Figure 25A). The ratios of plasminogen in spinal cord tissue to plasma plasminogen in mice treated with 6 mg / kg plasminogen were 0.93% and 1.62%, respectively, 2 and 6 hours after plasminogen administration. The ratios of plasminogen in spinal cord tissue to plasma plasminogen in mice treated with 50 mg / kg plasminogen were 0.33%, 0.40%, and 1.56%, respectively, 2, 6, and 12 hours after plasminogen administration (Figure 25B).

[0158] These results indicate that (1) plasminogen administration under physiological and pathological conditions can promote plasminogen crossing the blood-brain barrier and concentration in spinal cord tissue, (2) intravenous injection of plasminogen into mice significantly increases plasminogen levels in spinal cord tissue, (3) plasminogen concentration in spinal cord tissue exhibits a time-dependent effect, initially increasing, gradually decreasing over 2 to 12 hours, and being almost completely metabolized over 12 to 24 hours, and (4) plasminogen concentration in spinal cord tissue exhibits a dose-dependent effect, with the higher the dose, the higher the plasminogen level in spinal cord tissue.

[0159] Example 25 Plasminogen improves symptoms in patients with amyotrophic lateral sclerosis Nine patients with amyotrophic lateral sclerosis (ALS), aged 39 to 60 years, were recruited for this study. Of the nine patients, eight had limb-type ALS and one had bulbar-type ALS, one of whom had a FUS gene mutation. The treatment was approved by the hospital ethics committee. All patients signed informed consent.

[0160] Lyophilized human plasminogen powder was dissolved in sterile water at a concentration of 5 mg / ml and administered to patients by intravenous injection or nebulization. Basic information on the patients and the method of use of plasminogen are shown in Table 1. Table 1. Basic information on ALS patients and plasminogen usage JPEG2025539229000005.jpg147139

[0161] Results: Plasminogen improves motor function in ALS patients The 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].

[0162] The ALSFRS-R scores of nine patients were 20.22 ± 10.01 before treatment, but increased to 23.13 ± 10.82 between 0.5 and 4 months after plasminogen administration, an increase of 4.11 ± 5.30 points. Furthermore, patient 8, a patient with bulbar ALS, experienced a rapid increase in his ALSFRS-R score from 20 to 36 just two weeks after starting plasminogen administration. Patient 6, who had a FUS gene mutation, experienced an increase in his ALSFRS-R score from 27 to 29 just 12 days after starting plasminogen administration. Patient 5's ALSFRS-R score decreased from 29 to 25 eight weeks after the end of the first treatment course, but during the second treatment course, his score increased to 29 just four weeks after starting plasminogen nebulization. Furthermore, during the second treatment course, Patient 5's maximum number of steps increased from just over 40 to just over 200 (Figures 26A-B).

[0163] Furthermore, after plasminogen administration, the patients' respiratory function, writing ability, speech ability, swallowing function, anxiety / depression, and sleep improved significantly (Table 2).

[0164] The therapeutic effect of plasminogen on ALS was compared with that of riluzole and edaravone, two existing drugs approved by the FDA for the treatment of ALS. According to literature reports, after 6 months of riluzole administration, the ALSFRS-R scores of ALS patients decreased by -7.0 ± 7.1 points, and after 6 months of edaravone administration, the ALSFRS-R scores of ALS patients decreased by -5.01 ± 0.64 points [9,10]. After 0.5 months of plasminogen administration, the ALSFRS-R scores of nine ALS patients increased by 4.11 ± 5.30 points, and no adverse events were observed during plasminogen administration (Figure 26C and Table 3).

[0165] These results demonstrate that plasminogen can safely and effectively treat ALS. Table 2 Clinical phenotypes of ALS patients before and after plasminogen administration JPEG2025539229000006.jpg195127JPEG2025539229000007.jpg195128JPEG2025539229000008.jpg195127JPEG2025539229000009.jpg102128Table 3. ALSFRS-R scores before and after administration of plasminogen, riluzole, or edaravone. JPEG2025539229000010.jpg66141References: [1]KENNETH C.ROBBINS,LOUIS SUMMARIA,DAVID ELWYN et al.Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin.Journal of Biological Chemistry,1965,240(1):541-550. [2] Summaria L, Spitz F, Arzadon L et al.Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins.J Biol Chem.1976 Jun 25;251(12):3693-9. [3]HAGAN JJ, ABLONDI FB, DE RENZO EC.Purification and biochemical properties of human plasminogen.J Biol Chem.1960 Apr;235:1005-10. [4]Porta S, Xu Y, Restrepo CR, et al.Patient-derived frontotemporal lobar degeneration brain extracts induce formation and spreading of TDP-43 pathology in vivo[J].Nature Communications,2018,9(1). [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.2002 Apr 29;1596(2):182-92. [7]Ding H,Underwood R,Lavalley N,et al.14-3-3 inhibition promotes dopaminergic neuron loss and 14-3-3θ overexpression 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.1999 Oct 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 19 Study Group.Safety and efficacy of edaravone in well defined patients with amyotrophic lateral sclerosis: a randomized, double-blind, placebo-controlled trial.Lancet Neurol.2017 Jul;16(7):505-512. Sequence Listing Sequence 1 (nucleic acid sequence of native plasminogen (Glu-PLG, Glu-plasminogen) without signal peptide): Sequence 2 (nucleic acid sequence of native plasminogen (Glu-PLG, Glu-plasminogen) without the signal peptide): EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHA HGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKCCQSWSSMTPHRHQKTPE NYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLI SPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Sequence 3 (nucleic acid sequence of native plasminogen (from Swiss Prot) including signal peptide): Sequence 4 (amino acid sequence of native plasminogen (from Swiss Prot) including signal peptide): MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMS GLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKCCQSWSSMT PHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFC GGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Sequence 5 (LYS77-PLG (Lys-plasminogen) nucleic acid sequence): Sequence 6 (LYS77-PLG (Lys-plasminogen) amino acid sequence): KVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQ CLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKK CSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPS SYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Sequence 7 (delta-plg (delta-plasminogen) nucleic acid sequence): Sequence 8 (delta-plg (delta-plasminogen) amino acid sequence): EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGI TCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFG MHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Sequence 9 (miniplg (miniplasminogen) nucleic acid sequence): Sequence 10 (Mini-plg (miniplasminogen) amino acid sequence): VRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEK SPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Sequence 11 (Micro-plg (microplasminogen) nucleic acid sequence): gccccttcatttgattgtgggaagcctcaagtggagccgaagaaatgtcctggaagggttgtaggggggtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcacaccaagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagatattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgctgaccggaccgaatgtttcatcactggctggggagaaacccaaggtacttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgagaaataattaa Sequence 12 (Micro-plg (Microplasminogen) amino acid sequence): APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Array 13 (Nucleic acid sequence of serine protease (structure) domain): gttgtaggggggtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcacaccaagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagatattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgctgaccggaccgaatgtttc360 atcactggctggggagaaacccaaggtacttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgac agttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgaga Sequence 14 (amino acid sequence of the serine protease (structural) domain): VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVA DRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMR

Claims

1. A method for promoting the degradation of pathological TDP-43 protein, comprising administering to a subject a therapeutically effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating an upstream component of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

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

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

4. The method according to any one of claims 1 to 3, wherein the compound has one or more of the following activities: promoting the degradation of pathological TDP-43 protein in nerve tissue; promoting the degradation of pathological TDP-43 protein in muscle tissue; and promoting the degradation of TDP-43 in cells and cell nuclei.

5. 1. A method for treating a pathological TDP-43 protein-associated disease in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds capable of upregulating the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors, wherein the pathological TDP-43 protein-associated disease is selected from amyotrophic lateral sclerosis (ALS), bulbar amyotrophic lateral sclerosis, Fus-mutant amyotrophic lateral sclerosis, Alzheimer's disease, and the like. disease, argyrophilic grain syndrome, ALS-parkinsonism dementia complex of Guam, vascular dementia, frontotemporal dementia (FTD), semantic dementia, dementia with Lewy bodies, Huntington's disease, spinocerebellar degeneration bellarataxia), inclusion body myopathy, inclusion body myositis, and Parkinson's disease.

6. 6. The method of claim 5, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA, and uPA.

7. 6. The method of claim 5, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, alpha 2 antiplasmin or alpha 2 macroglobulin, such as an antibody.

8. The method of any one of claims 1 to 7, wherein the compound is plasminogen or plasmin.

9. The method according to any one of claims 1 to 8, wherein the plasminogen is Glu-plasminogen, Lys-plasminogen, or a conservatively substituted variant thereof.

10. 10. The method of any one of claims 1 to 9, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2 and has the lysine binding activity and / or proteolytic activity of plasminogen.

11. 11. The method of any one of claims 1 to 10, wherein the plasminogen comprises one or more selected from the group consisting of: 1) a serine protease domain as set forth in SEQ ID NO: 14; 2) a serine protease domain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 and retaining proteolytic activity; 3) one or more Kringle domains selected from the group consisting of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5; and 4) A Kringle domain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with one or more selected from the group consisting of Kringle 1, Kringle 2, Kringle 3, Kringle 4 and Kringle 5, and retaining lysine-binding activity.

12. 12. The method of any one of claims 1 to 11, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or variants thereof that retain the proteolytic activity of plasminogen.

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

14. The method of any one of claims 1 to 13, wherein the plasminogen is used in combination with one or more other therapeutic methods or drugs.

15. 15. The method of claim 14, wherein the other treatment modalities include cell therapy (including stem cell therapy), supportive therapy, and physical therapy.

16. 16. The method of any one of claims 1 to 15, wherein the plasminogen is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenously, intraperitoneally, subcutaneously, sublingually, intracranially, intrathecally, intraarterially, or intramuscularly.

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