Methods and agents for increasing NGF levels

Abstract

The present invention relates to methods for promoting the formation of mature NGF and increasing NGF expression, comprising administering to a subject a therapeutically effective amount of a plasminogen pathway activator. The present invention also provides pharmaceutical compositions, products, and kits containing the plasminogen pathway activator for promoting the formation of NGF and increasing NGF expression.

Description

Technical Field

[0001] The present invention relates to a method for promoting the formation of mature NGF and increasing the level of NGF, which includes administering an effective amount of a component of the plasminogen activation pathway or a related compound thereof, such as plasminogen, to a subject in need thereof for repairing damaged nerves.

Background Art

[0002] Nerve growth factor (NGF) is the most important class among the neurotrophic factor family. The neurotrophic factor family includes nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4 / 5, neurotrophin-6, and neurotrophin-7, etc., mainly including the first four. NGF mainly exists in the form of a precursor in tissues and is processed in the submandibular gland to form mature NGF.

[0003] The biological effects of NGF include the effects of nourishing nerves, protecting nerves, promoting nerve regeneration, and other effects. During a specific period of embryonic development, NGF is necessary for the survival of effector neurons. NGF and its receptors are widely distributed in the central nervous system, and NGF produced in the hippocampus and cerebral cortex is retrogradely transported to the basal ganglia of the forebrain via cholinergic nerves to maintain the survival and function of cholinergic neurons. In early embryonic development, the content of central NGF determines the density of cholinergic nerves.

[0004] When the effector neurons of NGF are damaged by trauma, drug injury, ischemia, hypoxia, etc., the neurons will undergo a series of pathological changes including death. The possible mechanisms of the effect of NGF on inhibiting the above nerve damage are as follows: (1) inhibiting the release of toxic amino acids; (2) inhibiting calcium ion overload; (3) inhibiting the release of superoxide free radicals; (4) inhibiting apoptosis and other mechanisms to significantly reduce or prevent the occurrence of these secondary pathological damages.

[0005] Administration of NGF after axotomy will reduce the degeneration and death of some neurons, which will undoubtedly help to enhance the possibility of axonal regeneration. At the same time, it also affects the onset time of axonal regeneration, the number of neurons involved in regeneration, and the quality and speed of the regenerated nerves.

[0006] Furthermore, NGF has other functions. For example, NGF can affect the activities of immune cells, thereby regulating the functions of the immune system. NGF inhibits the mitosis of some tumors and promotes benign differentiation. NGF can also promote the repair response of wound tissues and accelerate wound healing. Summary of the Invention

[0007] Through research, the present invention has discovered that plasminogen pathway activators such as plasminogen can significantly promote the formation of mature NGF, increase the level of NGF, and thereby play a role in the survival, differentiation, growth, and development of neurons.

[0008] In one aspect, the present invention relates to the following.

[0009] 1. A method for promoting the formation of mature NGF and increasing the NGF level, comprising administering a therapeutically effective amount of a plasminogen pathway activator to a subject.

[0010] 2. The method according to item 1, wherein the plasminogen pathway activator promotes the formation of mature NGF and the expression of NGF by cleaving Pro-NGF (nerve growth factor precursor) in the nerve tissue of the subject.

[0011] 3. The plasminogen pathway activator promotes the formation of mature NGF in the nerve tissue of a subject with nerve tissue damage and / or increases the expression of NGF, and the nerve tissue damage is 1) One or more infectious diseases selected from meningitis, encephalitis, poliomyelitis, and epidural abscess; 2) one or more vascular diseases selected from stroke, transient ischemic attack (TIA), subarachnoid hemorrhage, subdural hemorrhage and hematoma, and epidural hemorrhage; 3) one or more nerve structure damage diseases selected from brain or spinal cord injury, Bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumor, peripheral neuropathy, and Guillain - Barré syndrome; 4) one or more dysfunctions selected from headache, epilepsy, insomnia, neuralgia, anxiety, and depression; 5) one or more neurodegenerative diseases selected from Alzheimer's disease, Parkinson's disease, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), spinocerebellar ataxia, and Pick's disease; 6) one or more motor neuron diseases selected from spinal muscular atrophy (SMA), progressive bulbar palsy, progressive muscular atrophy, and primary lateral sclerosis; 7) one or more tumors selected from brain tumor and brain cancer The method according to item 1 or 2, which is caused by one or more diseases or conditions selected from

[0012] 4. The method according to item 1, wherein the plasminogen pathway activator promotes the formation of mature NGF and the expression of NGF by cleavage of Pro - NGF in the nerve tissue of a subject having Alzheimer's disease.

[0013] 5. The method according to item 1, wherein the plasminogen pathway activator promotes an increase in NGF level.

[0014] In some embodiments, the plasminogen pathway activator increases the expression and levels of other neurotrophic factors in the damaged nerve tissue of a subject. In some embodiments, the neurotrophic factors include brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), insulin like-growth factor-1 (IGF-1), transforming growth factor (TGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), or platelet-derived growth factor (PDGF).

[0015] In some embodiments, the present application also relates to a method for preventing or treating an NGF-related disease or condition, comprising administering to a subject a therapeutically effective amount of a plasminogen pathway activator.

[0016] In some embodiments, the NGF-related disease or condition is 1) an infectious disease selected from any of meningitis, encephalitis, poliomyelitis, and epidural abscess; 2) a vascular disease selected from any of stroke, transient ischemic attack (TIA), subarachnoid hemorrhage, subdural hemorrhage and hematoma, and epidural hemorrhage; 3) a nerve structure damage disease selected from any of brain or spinal cord injury, Bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumor, peripheral neuropathy, and Guillain-Barré syndrome; 4) a functional disorder selected from any of headache, epilepsy, insomnia, neuralgia, anxiety disorder, and depression; 5) A neurodegenerative disease selected from any of Alzheimer's disease, Parkinson's disease, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), spinocerebellar ataxia, and Pick's disease; 6) A motor neuron disease selected from any of spinal muscular atrophy (SMA), progressive bulbar palsy, progressive muscular atrophy, and primary lateral sclerosis; 7) A tumor selected from any of brain tumors and brain cancers; 8) Any one selected from the group consisting of peripheral neuropathy, nerve injury due to trauma, optic nerve disorder, polyneuritis, herpes zoster, facial nerve paralysis, burn, bedsores, corneal ulcer, and side effects of radiotherapy or chemotherapy is included.

[0017] In all of the above technical solutions, the plasminogen pathway activator increases the plasminogen level in the nerve tissue of the subject.

[0018] 6. The method according to any one of items 1 to 5, wherein the plasminogen pathway activator is used in combination with one or more other drugs or treatment methods.

[0019] 7. The method according to any one of items 1 to 6, wherein the plasminogen pathway activator is administered intravenously, intramuscularly, intrathecally, by nasal inhalation, aerosol inhalation, nasal drops, or eye drops.

[0020] 8. The method according to any one of items 1 to 7, wherein the plasminogen pathway activator is a component of the plasminogen activation pathway, preferably plasminogen.

[0021] 9. The method according to any one of items 1 to 8, wherein the plasminogen activator has one or more uses or activities selected from the group consisting of nourishing nerves, protecting nerves, promoting nerve regeneration, inhibiting nerve damage, promoting repair of wound tissues, and promoting wound healing.

[0022] 10. The method according to any one of items 1 to 9, wherein the plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12 and has plasminogen activity.

[0023] 11. The method according to any one of items 1 to 9, wherein the plasminogen is a plasminogen active fragment and is a protein having the proteolytic activity or lysine-binding activity of plasminogen.

[0024] 12. The method according to any one of items 1 to 9, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogen, or mutants thereof that retain plasminogen activity.

[0025] 13. The method according to any one of items 1 to 9, wherein the plasminogen contains the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12.

[0026] In the above technical solution, the plasminogen pathway activator is one or more selected from components of the plasminogen activation pathway, a compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway, a compound mimicking the activity of plasminogen or plasmin, a compound capable of up-regulating the expression of plasminogen or plasminogen activator, a plasminogen analog, a plasmin analog, a tPA or uPA analog, and an antagonist of a fibrinolytic inhibitor.

[0027] In some specific embodiments, the components of the plasminogen activation pathway are selected from natural or recombinant plasminogen, human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin mutants and analogs containing one or more kringle domains and / or protease domains of plasminogen, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activator, tPA, and uPA. In some specific embodiments, the antagonist of the fibrinolytic inhibitor is an inhibitor of natural or recombinant PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, for example, an antibody against PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0028] In some specific embodiments, the plasminogen pathway activator is administered in combination with one or more other agents and / or treatment methods. Preferably, the treatment methods include cell therapy (such as stem cell therapy) and gene therapy (such as antisense RNA, small molecule splicing modifiers).

[0029] In some specific embodiments, the plasminogen pathway activator is a component of the plasminogen activation pathway, such as plasminogen. In some specific embodiments, the plasminogen comprises or has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12, and has plasminogen activity and / or lysine binding activity. 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 an active fragment of plasminogen 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 active fragment of the plasminogen comprises or has a plasminogen serine protease domain or a plasminogen protease domain. In some specific embodiments, the amino acid sequence of the active fragment of the plasminogen is shown in SEQ ID NO: 14. In some specific embodiments, the plasminogen is selected from Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (human full-length plasminogen cleaved between amino acids 76 and 77), miniplasminogen (comprising kringle 5 (K5) and serine protease domain), microplasminogen (comprising serine protease domain), delta-plasminogen (comprising kringle 1 and serine protease domain), or variants thereof that retain plasminogen activity.In some embodiments, the plasminogen is human full-length plasminogen, or a variant 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 variant or fragment thereof that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen comprises the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human native plasminogen.

[0030] In some embodiments, the present invention also relates to the following.

[0031] 1. In one aspect, the present application relates to a plasminogen pathway activator, or a pharmaceutical composition comprising a plasminogen pathway activator, for promoting the formation of mature NGF and / or increasing the level of NGF in the nerve tissue of a subject. In one aspect, the present application also relates to the use of a plasminogen pathway activator in the preparation of a medicament for promoting the formation of mature NGF and / or increasing the level of NGF in the nerve tissue of a subject.

[0032] 2. The plasminogen pathway activator according to item 1, or a pharmaceutical composition or use comprising a plasminogen pathway activator, wherein the plasminogen pathway activator promotes the formation of mature NGF in the nerve tissue of a subject and / or increases the expression of NGF.

[0033] 3. The plasminogen pathway activator according to item 1 or 2, or a pharmaceutical composition or use comprising a plasminogen pathway activator, wherein the plasminogen pathway activator increases the expression and level of other neurotrophic factors in the nerve tissue of a subject.

[0034] 4. The plasminogen pathway activator according to item 3, or a pharmaceutical composition or use comprising the plasminogen pathway activator, wherein the neurotrophic factor comprises one or more selected from brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), insulin like-growth factor-1 (IGF-1), transforming growth factor (TGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), or platelet-derived growth factor (PDGF).

[0035] 5. The plasminogen pathway activator according to any one of items 1 to 4, or a pharmaceutical composition or use comprising the plasminogen pathway activator, which has one or more uses or activities selected from providing nutrition to nerves, protecting nerves, promoting nerve regeneration, inhibiting nerve damage, promoting repair of wound tissues, and promoting wound healing.

[0036] 6. The subject is a subject suffering from damage to nerves or brain tissue, and the damage to the nerves or brain tissue is 1) one or more infectious diseases selected from meningitis, encephalitis, poliomyelitis, and epidural abscess; 2) one or more vascular diseases selected from stroke, transient ischemic attack (TIA), subarachnoid hemorrhage, subdural hemorrhage and hematoma, and epidural hemorrhage; 3) One or more nerve structure damage diseases selected from meningitis, encephalitis, poliomyelitis, and epidural abscess; 4) One or more functional disorders selected from headache, epilepsy, insomnia, neuralgia, anxiety disorder, and depression; 5) One or more neurodegenerative diseases selected from Alzheimer's disease, Parkinson's disease, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), spinocerebellar ataxia, and Pick's disease; 6) One or more motor neuron diseases selected from spinal muscular atrophy (SMA), progressive bulbar palsy, progressive muscular atrophy, and primary lateral sclerosis; 7) One or more tumors selected from brain tumor and brain cancer The plasminogen pathway activator according to any one of items 1 to 5, or a pharmaceutical composition containing the plasminogen pathway activator, or the use, caused by one or more diseases or conditions selected from the above.

[0037] 7. A plasminogen pathway activator for preventing or treating NGF-related diseases or conditions, or a pharmaceutical composition containing the plasminogen pathway activator, or the use of the plasminogen pathway activator in the preparation of a medicament for preventing or treating NGF-related diseases or conditions.

[0038] 8. The NGF-related disease or condition is 1) An infectious disease selected from any one of meningitis, encephalitis, poliomyelitis, and epidural abscess; 2) A vascular disease selected from any one of stroke, transient ischemic attack (TIA), subarachnoid hemorrhage, subdural hemorrhage and hematoma, and epidural hemorrhage; 3) A nerve structure damage disease selected from any one of brain or spinal cord injury, Bell's palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumor, peripheral neuropathy, and Guillain-Barré syndrome; 4) A dysfunction selected from any one of headache, epilepsy, insomnia, neuralgia, anxiety disorder, and depression; 5) A neurodegenerative disease selected from any one of Alzheimer's disease, Parkinson's disease, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), spinocerebellar ataxia, and Pick's disease; 6) A motor neuron disease selected from any one of spinal muscular atrophy (SMA), progressive bulbar palsy, progressive muscular atrophy, and primary lateral sclerosis; 7) A tumor selected from any one of brain tumor and brain cancer; 8) Any one selected from the group consisting of peripheral neuropathy, nerve injury due to trauma, optic nerve disorder, polyneuritis, herpes zoster, facial nerve paralysis, burn, bedsores, corneal ulcer, side effects of radiotherapy or chemotherapy The plasminogen pathway activator, pharmaceutical composition, or use according to item 7, comprising any one selected from the above group.

[0039] 9. The plasminogen pathway activator according to any one of items 1 to 8, or a pharmaceutical composition or use comprising a plasminogen pathway activator, wherein the plasminogen pathway activator increases the plasminogen level in the damaged nerve tissue of a subject.

[0040] 10. The plasminogen pathway activator according to any one of items 1 to 9, or a pharmaceutical composition or use comprising a plasminogen pathway activator, wherein the plasminogen pathway activator is used in combination with one or more other drugs or treatment methods.

[0041] 11. The plasminogen pathway activator according to any one of items 1 to 10, or a pharmaceutical composition or use comprising a plasminogen pathway activator, wherein the plasminogen pathway activator is administered by intravenous, intramuscular, intrathecal, nasal inhalation, aerosol inhalation, nasal drops, or eye drops.

[0042] 12. The plasminogen pathway activator is one or more selected from components of the plasminogen activation pathway, a compound capable of directly activating plasminogen, or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway, a compound mimicking the activity of plasminogen or plasmin, a compound capable of upregulating the expression of plasminogen or a plasminogen activator, a plasminogen analog, a plasmin analog, a tPA or uPA analog, and an antagonist of a fibrinolysis inhibitor, the plasminogen pathway activator according to any one of items 1 to 11, or a pharmaceutical composition or use comprising a plasminogen pathway activator.

[0043] 13. The components of the plasminogen activation pathway are selected from natural or recombinant plasminogen, human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin mutants and analogs containing one or more kringle domains and / or protease domains of plasminogen, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activator, tPA, and uPA, the plasminogen pathway activator according to claim 12, or a pharmaceutical composition or use comprising a plasminogen pathway activator.

[0044] 14. The antagonist of the fibrinolysis inhibitor is a natural or recombinant PAI-1, a complement C1 inhibitor, an antagonist of α2-antiplasmin or α2-macroglobulin, for example, an antibody against PAI-1, a complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, the plasminogen pathway activator according to claim 12, or a pharmaceutical composition or use comprising a plasminogen pathway activator.

[0045] 15. The plasminogen pathway activator according to any one of items 1 to 13, or a pharmaceutical composition containing the plasminogen pathway activator, or use, wherein the plasminogen pathway activator is plasminogen.

[0046] 16. The plasminogen pathway activator according to item 15, or a pharmaceutical composition containing the plasminogen pathway activator, or use, wherein the plasminogen contains the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12, or contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12, and has the proteolytic activity and / or lysine-binding activity of plasminogen.

[0047] 17. The plasminogen has 1) a serine protease domain having the one shown in SEQ ID NO: 14; 2) a serine protease domain having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 14 and retaining proteolytic activity; 3) one or more kringle domains selected from 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%, 99% identity with one or more of kringle 1, kringle 2, kringle 3, kringle 4 and kringle 5 and retaining lysine-binding activity The plasminogen pathway activator according to item 15, or a pharmaceutical composition containing the plasminogen pathway activator, or use, which comprises one or more selected from the above.

[0048] 18. The plasminogen activator according to item 15, or a pharmaceutical composition or use comprising the plasminogen activator, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogen, or mutants thereof that retain the proteolytic activity of plasminogen.

[0049] In some specific embodiments, the plasminogen activator is administered systemically or locally, for example, by intravenous, intramuscular, intrathecal, nasal inhalation, aerosol inhalation, nasal drops, or eye drops. In some embodiments, the subject is a human. In some embodiments, the subject has a deficiency or lack of plasminogen. In some embodiments, the deficiency or lack is congenital, secondary, and / or local. In some embodiments, the plasminogen is administered daily, every other day, or every three days at a dose of 0.0001 - 2000 mg / kg, 0.001 - 800 mg / kg, 0.01 - 600 mg / kg, 0.1 - 400 mg / kg, 1 - 200 mg / kg, 1 - 100 mg / kg, 10 - 100 mg / kg (calculated per kilogram of body weight) or 0.0001 - 2000 mg / cm 2 、0.001 - 800 mg / cm 2 、0.01 - 600 mg / cm 2 、0.1 - 400 mg / cm 2 、1 - 200 mg / cm 2 、1 - 100 mg / cm 2 、10 - 100 mg / cm 2 (calculated per square centimeter of body surface area) and is administered continuously.

[0050] In one aspect, the present application also relates to pharmaceutical compositions, drugs, formulations, kits, and products used in the above methods, including the above plasminogen activators such as the above plasminogen.

[0051] In some embodiments, the pharmaceutical composition, agent, or formulation comprises a pharmaceutically acceptable carrier and a plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as plasminogen. In some embodiments, the kit and product comprise one or more containers containing the pharmaceutical composition, agent, or formulation. In some embodiments, the kit or product further comprises a label or protocol instructing the use of a plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as plasminogen, in the above methods. In some embodiments, the kit or product further comprises one or more other containers containing one or more other agents.

[0052] In one aspect, the present application also relates to plasminogen pathway activators for use in the above applications, such as the above plasminogen.

[0053] In one aspect, the present application also relates to the use of a therapeutically effective amount of the above plasminogen pathway activator in the preparation of a pharmaceutical composition, agent, formulation, kit, and product for use in the methods described above.

[0054] In some embodiments, the plasminogen pathway activator is one or more selected from a compound that can directly activate a component of the plasminogen activation pathway, plasminogen, or can indirectly activate plasminogen 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 a plasminogen activator, a plasminogen analog, a plasmin analog, a tPA or uPA analog, and an antagonist of a fibrinolytic inhibitor.

[0055] In some specific embodiments, the components of the plasminogen activation pathway are 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, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activator, tPA, and uPA. In some specific embodiments, the antagonist of the fibrinolytic inhibitor is an inhibitor of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, for example, an antibody against PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0056] In some specific embodiments, the plasminogen pathway activator is administered in combination with one or more other agents and / or treatment methods, preferably, the treatment methods include cell therapy (e.g., stem cell therapy) and gene therapy (e.g., antisense RNA, small molecule splicing modifiers).

[0057] In some specific embodiments, the plasminogen pathway activator is a component of the plasminogen activation pathway, such as plasminogen. In some specific embodiments, the plasminogen comprises or has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12, and has plasminogen activity and / or lysine binding activity. 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 that comprises an active fragment of plasminogen and has 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 active fragment of the plasminogen comprises or has a plasminogen serine protease domain or a plasminogen protease domain. In some specific embodiments, the amino acid sequence of the active fragment of the plasminogen is shown in SEQ ID NO: 14. In some specific embodiments, the plasminogen is selected from Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (human full-length plasminogen cleaved 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 variants thereof that retain plasminogen activity.In some embodiments, the plasminogen is human full-length plasminogen, or a variant 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 variant or fragment thereof that still retains plasminogen activity and / or lysine-binding activity. In some embodiments, the plasminogen comprises the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is human native plasminogen.

[0058] In some embodiments, the plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as plasminogen, is used in combination with one or more other agents and / or treatment methods. In some particular embodiments, the plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as plasminogen, is administered by intravenous, intramuscular, intrathecal, nasal inhalation, aerosol inhalation, nasal drops, or eye drops.

[0059] In some embodiments, the pharmaceutical composition, agent, formulation comprises a pharmaceutically acceptable carrier and a plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as plasminogen. In some embodiments, the kit and product comprise one or more containers containing the pharmaceutical composition, agent, or formulation. In some embodiments, the kit or product further comprises a label or protocol indicating the use of the plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as plasminogen, for the above uses.

[0060] In some embodiments, the kit or product further comprises one or more other containers containing one or more other agents.

[0061] The present invention clearly covers all combinations of technical features belonging to the embodiments of the present invention, and the technical configurations after these combinations are clearly disclosed in this application in the same manner as the above technical configurations are separately and clearly disclosed. Furthermore, the present invention also clearly covers the combinations between each embodiment and their elements, and the technical configurations after the combinations are clearly disclosed in this specification.

Brief Description of the Drawings

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[0063] The fibrinolytic system, also known as the fibrinolysis system, is a system composed of a series of chemical substances involved in the process of fibrinolytic (fibrinolysis). It mainly includes plasminogen (PLG), plasmin, plasminogen activator, and fibrinolytic inhibitor. 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, and this process mainly 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 the need for fibrin as a cofactor. Plasminogen (PLG) is synthesized in the liver. When blood coagulates, PLG is adsorbed in large amounts on the fibrin net and is activated to plasmin by the action of t-PA or u-PA, promoting fibrinolysis. Plasminase (PL) is a serine protease that decomposes fibrin and fibrinogen, hydrolyzes various coagulation factors V, VIII, X, VII, XI, II, etc., converts plasminogen to plasmin, and hydrolyzes complement. Fibrinolytic inhibitors include plasminogen activator inhibitor (PAI) and α2-antiplasmin (α2-AP). PAI mainly has two forms, PAI-1 and PAI-2, which can specifically bind to t-PA in a 1:1 ratio to inactivate t-PA and at the same time activate PLG. α2-AP is synthesized in the liver, binds to PL in a 1:1 ratio to form a complex, thereby inhibiting PL activity. FXIII covalently binds α2-AP to fibrin, thereby weakening the sensitivity of fibrin to PL. Substances that inhibit the activity of the fibrinolytic system in vivo include PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin.

[0064] As used herein, the term "plasminogen pathway activator" or "PLG pathway activator" covers components of the plasminogen activation pathway, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can up-regulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.

[0065] As used herein, the term "component of the plasminogen activation pathway" or "component of the PLG activation pathway" refers to 1. plasminogen, Lys-plasminogen, Glu-plasminogen, micro-plasminogen, delta-plasminogen, their variants or analogs; 2. plasmin and their variants or analogs; and 3. plasminogen activators, such as tPA and uPA, and tPA or uPA variants and analogs containing one or more domains of tPA or uPA (such as one or more kringle domains and protease domains).

[0066] The term "antagonist of fibrinolytic inhibitor" covers antagonists of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, such as antibodies against PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0067] The "variants" of the above plasminogen, plasmin, tPA and uPA include all naturally occurring human genetic variants and other mammalian forms of these proteins, and proteins in which, 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 has been added, deleted, and / or substituted and which still have plasminogen activity, plasmin activity, tPA or uPA activity. For example, the "variants" of plasminogen, plasmin, tPA or uPA include mutants of these proteins obtained by substitution with, 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 conservative amino acid.

[0068] The "plasminogen variant" of the present invention covers a protein that contains or has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12, and has plasminogen activity and / or lysine binding activity. For example, the "plasminogen variant" of the present invention may be a protein in which, based on SEQ ID NO: 2, 6, 8, 10 or 12, 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, and still has plasminogen activity and / or lysine binding activity. Specifically, the plasminogen variants of the present invention include all naturally occurring human genetic variants and other mammalian forms of these proteins, and, for example, mutants of these proteins obtained by conservative 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 amino acid.

[0069] The plasminogen of the present invention can be an ortholog of human plasminogen derived from a primate or rodent, or a variant that still retains plasminogen activity and / or lysine binding activity, for example, the plasminogen shown in SEQ ID NO: 2, 6, 8, 10 or 12, for example, human native plasminogen shown in SEQ ID NO: 2.

[0070] The "analogs" of the above plasminogen, plasmin, tPA and uPA each include a compound that gives substantially the same effect as plasminogen, plasmin, tPA or uPA.

[0071] The "variants" and "analogs" of the above plasminogen, plasmin, tPA, and uPA cover the "variants" and "analogs" of plasminogen, plasmin, tPA, and uPA that contain one or more domains (e.g., one or more kringle domains and protease domains). For example, the "variants" and "analogs" of plasminogen cover plasminogen variants and analogs that contain one or more plasminogen domains (e.g., one or more kringle (k) domains and protease domains (or serine protease domains, or plasminogen protease domains), such as mini-plasminogen. The "variants" and "analogs" of plasmin cover the "variants" and "analogs" of plasmin such as mini-plasmin and delta-plasmin that contain one or more plasmin domains (e.g., one or more kringle domains and protease domains).

[0072] Whether the "variant" or "analog" of the above plasminogen, plasmin, tPA or uPA has the activity of plasminogen, plasmin, tPA or uPA respectively, or whether they give substantially the same effects as plasminogen, plasmin, tPA or uPA respectively can be measured by the levels of activated plasmin activity using methods known in the art, such as enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS (fluorescence-activated cell sorting method). For example, it can be measured with reference to the methods described in the following documents.Ny, A., Leonardsson, G., Hagglund, A. C, Hagglof, P., Ploplis, V. A., Carmeliet, P. and Ny, T. (1999). Ovulation in plasminogen - deficient mice. Endocrinology 140, 5030 - 5035; Silverstein RL, Leung LL, Harpel PC, Nachman RL (November 1984). “Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator”. J. Clin. Invest. 74 (5): 1625 - 33; Gravanis I, Tsirka SE (February 2008). “Tissue - type plasminogen activator as a therapeutic target in stroke”. Expert Opinion on Therapeutic Targets. 12 (2): 159 - 70; Geiger M, Huber K, Wojta J, Stingl L, Espana F, Griffin JH, Binder BR (Aug 1989). “Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo”. Blood. 74 (2): 722 - 8。

[0073] 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, δ-plasminogen, or mutants retaining their 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 an amino acid sequence as shown 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 the human full-length plasminogen shown in SEQ ID NO: 2.

[0074] The "compound that can directly activate plasminogen or indirectly activate plasminogen by activating an upstream component of the plasminogen activation pathway" refers to any compound that can directly activate plasminogen or indirectly activate plasminogen by activating an upstream component of the plasminogen activation pathway, and examples thereof include tPA, uPA, streptokinase, saruplase, alteplase, reteplase, tenecteplase, anisstreplase, monteplase, lanoteplase, pamiteplase, and staphylokinase.

[0075] The "antagonist of fibrinolytic inhibitor" of the present invention is a compound that antagonizes the action of a fibrinolytic inhibitor, weakens, blocks, and inhibits its action. The fibrinolytic inhibitor is, for example, PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin. The antagonist is an antibody against PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or an antisense RNA or microRNA that blocks or downregulates the expression of, for example, PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or a compound that occupies the binding site of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin but does not have the function of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or a compound that blocks the binding domain and / or active domain of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0076] Plasmin is an important component of the plasminogen activation system (PA system). It is a broad-spectrum protease that can hydrolyze several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycan. Plasmin can also activate some pro-matrix metalloproteinases (pro-MMPs) to active matrix metalloproteinases (MMPs). Therefore, plasmin is an important upstream regulator of extracellular proteolysis. Plasmin is formed by the proteolysis of plasminogen by two types of physiological PAs: tissue-type plasminogen activator (tPA) or urokinase plasminogen activator (uPA). Plasminogen has relatively high levels in plasma and other body fluids, and conventionally, the regulation of the PA system is considered to be mainly achieved by the synthesis and activity levels of PAs. The synthesis of PA system components is strictly regulated by different 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) of uPA and tPA, and is regulated by plasminogen activator inhibitor-2 (PAI-2) that mainly inhibits uPA. There is a uPA-specific cell surface receptor (uPAR) with direct hydrolyzing activity on the surface of some cells.

[0077] Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. Plasminogen is mainly synthesized in the liver and is present in large amounts in the extracellular fluid. The content of plasminogen contained in plasma is approximately 2 μM. Therefore, plasminogen is a major potential source of proteolytic activity in tissues and body fluids. There are two molecular forms of plasminogen: glutamic acid-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). Naturally secreted and undegraded plasminogen has a single amino group terminal (N-terminal) glutamic acid and is therefore called glutamic acid-plasminogen. However, in the presence of plasmin, glutamic acid-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys77. Compared with glutamic acid-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate. The Arg560-Val561 peptide bond of these two forms of plasminogen is cleaved by uPA or tPA, resulting in the formation of the double-chain protease plasmin linked by a disulfide bond. The amino-terminal portion of plasminogen contains five homologous tricycles, namely the so-called kringle, and the carboxyl-terminal portion contains a protease domain. Some kringles contain lysine-binding sites that mediate specific interactions between plasminogen and fibrin and its inhibitor α2-AP. The most recently discovered plasminogen is a 38 kDa fragment containing kringle l-4 and is an effective inhibitor of angiogenesis. This fragment is named angiostatin and is generated by hydrolyzing plasminogen with several proteases.

[0078] The main substrate of plasmin is fibrin, and the lysis of fibrin is a key point in preventing the formation of pathological thrombi. Plasmin further has substrate specificity for several components of the ECM, which include laminin, fibronectin, proteoglycan, and gelatin, indicating that plasmin has an important role in ECM reconstruction. Indirectly, plasmin further degrades other components of the ECM by converting several protease precursors, including MMP-1, MMP-2, MMP-3, and MMP-9, into active proteases. Therefore, it has been proposed that plasmin is an important upstream regulator of extracellular proteolysis. In addition, plasmin has the ability to activate several potential forms of growth factors. In vitro, plasmin can further hydrolyze components of the complement system to release chemotactic complement fragments.

[0079] "Plasmin" is a very important enzyme present in the blood that hydrolyzes fibrin clots into fibrin degradation products and D-dimers.

[0080] "Plasminogen" is in the form of an enzyme precursor of plasmin. Based on the sequence in Swiss-Prot, it consists of 810 amino acids when calculated as the amino acid sequence of native human-derived plasminogen (Sequence 4) including the signal peptide, with a molecular weight of approximately 90 kDa. It is a glycoprotein mainly synthesized in the liver and capable of circulating in the blood, and the cDNA sequence encoding this amino acid sequence is as shown in Sequence 3. Full-size 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 (kringle 1-5). Referring to the sequence in Swiss-Prot, its signal peptide contains residues Met1-Gly19, PAp contains residues Glu20-Val98, kringle 1 contains residues Cys103-Cys181, kringle 2 contains residues Glu184-Cys262, kringle 3 contains residues Cys275-Cys352, kringle 4 contains residues Cys377-Cys454, and kringle 5 contains residues Cys481-Cys560. According to NCBI data, the serine protease domain contains residues Val581-Arg804.

[0081] Glu-plasminogen is a natural full-size plasminogen, consisting of 791 amino acids (excluding the 19-amino acid signal peptide). The cDNA sequence encoding this sequence is as shown in SEQ ID NO: 1, and its amino acid sequence is as shown in SEQ ID NO: 2. In vivo, Lys-plasminogen, which is formed by hydrolysis at the positions of amino acids 76-77 of Glu-plasminogen, exists, for example, as shown in SEQ ID NO: 6, and the cDNA sequence encoding this amino acid sequence is as shown in SEQ ID NO: 5. Delta-plasminogen is a fragment lacking the kringle 2-kringle 5 structure in the full-size plasminogen, containing only the kringle 1 and serine protease (structure) domains (also called the protease domain or plasminogen protease domain). There is a literature reporting the amino acid sequence of δ-plasminogen (SEQ ID NO: 8), and the cDNA sequence encoding this amino acid sequence is, for example, SEQ ID NO: 7. Mini-plasminogen consists of the kringle 5 and serine protease domains, containing residues Val443-Asn791 (starting with the Glu residue of the Glu-plasminogen sequence excluding the signal peptide), and its amino acid sequence is as shown in SEQ ID NO: 10. There is a literature reporting that the cDNA sequence encoding this amino acid sequence is as shown in SEQ ID NO: 9. However, Micro-plasminogen contains only the serine protease domain, and there is a literature reporting that its amino acid sequence contains residues Ala543-Asn791 (the Glu residue of the Glu-plasminogen sequence excluding the signal peptide is the starting amino acid). Patent Document CN102154253A discloses that its sequence contains residues Lys531-Asn791 (starting with the Glu residue of the Glu-plasminogen sequence excluding the signal peptide). For the sequence of this patent, reference can be made to Patent Document CN102154253A. Its 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.

[0082] In the present invention, "plasmin", "fibrin plasmin", and "fibrous protein plasmin" can be used interchangeably, and their meanings are the same. "Plasminogen", "fibrin plasminogen", and "fibrous protein plasminogen" can be used interchangeably, and their meanings are the same.

[0083] In the present application, the "deficiency" of the plasminogen means that the content or activity of plasminogen in the subject's body is lower than that of a normal person and is low enough to affect the normal physiological functions of the subject. The meaning of the "lack" of the plasminogen is that the content or activity of plasminogen in the subject's body is significantly lower than that of a normal person, the activity or expression is extremely low, and the normal physiological functions can be maintained only by external supply.

[0084] Those skilled in the art can understand as follows. All technical configurations of the plasminogen of the present invention can be applied to plasmin. Therefore, the technical configurations described in the present invention cover plasminogen and plasmin. In the circulation process, plasminogen adopts a closed inactive conformation. However, when it binds to a thrombus or the cell surface, in the presence of a plasminogen activator (PA), it becomes active plasmin with an open conformation. Active plasmin further hydrolyzes fibrin clots into fibrin degradation products and D-dimers, thereby dissolving the thrombus. Among them, the PAp domain of plasminogen contains important epitopes that maintain plasminogen in an inactive closed conformation, but the KR domain can bind to lysine residues on receptors and substrates. Enzymes as plasminogen activators are already known in several types, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hageman factor).

[0085] The "active fragment of plasminogen" in the present application includes: 1) an active fragment in a plasminogen protein that can bind to a target sequence in a substrate, also known as a lysine-binding fragment such as a fragment containing kringle 1, kringle 2, kringle 3, kringle 4, and / or kringle 5 (for the structure of the plasminogen, see Aisina R B, 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 in a plasminogen protein that performs a proteolytic function, such as a fragment containing the plasminogen activity (proteolytic function) shown in SEQ ID NO: 14; 3) a fragment in a plasminogen protein that has both the binding activity (lysine-binding activity) to a target sequence in a substrate and the plasminogen activity (proteolytic function). In some embodiments of the present application, the plasminogen is a protein containing the active fragment of plasminogen shown in SEQ ID NO: 14. In some embodiments of the present application, the plasminogen is a protein containing the 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 having an amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 14. Therefore, the plasminogen of the present invention includes a protein containing the active fragment of the plasminogen and still retaining the activity of the plasminogen.In some embodiments, the plasminogen of the present application includes kringle 1, kringle 2, kringle 3, kringle 4, and / or kringle 5, or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with kringle 1, kringle 2, kringle 3, kringle 4, or kringle 5 and still has lysine-binding activity, and includes proteins having such properties.

[0086] Currently, the methods for measuring plasminogen and its activity in blood include measurement of tissue plasminogen activator activity (t-PAA), measurement of plasma tissue plasminogen activator antigen (t-PAAg), measurement of plasma tissue plasminogen activity (plgA), measurement of plasma tissue plasminogen antigen (plgAg), measurement of inhibitor activity of plasma tissue plasminogen activator, measurement of inhibitor antigen of plasma tissue plasminogen activator, and measurement of plasma plasmin - antiplasmin complex (PAP). The most commonly seen measurement method is the chromogenic substrate method: streptokinase (SK) and a luminescent substrate are added to the plasma of the subject to be measured. PLG in the plasma of the subject to be measured becomes PLM under the action of SK, and the latter acts on the luminescent substrate, and then it is measured with a spectrophotometer. The increase in absorbance is directly proportional to the activity of plasminogen. In addition, immunochemical methods, gel electrophoresis, immunoturbidimetry, radioimmuno-diffusion methods, etc. can be used to measure the activity of plasminogen in blood.

[0087] "Ortholog or ortholog" refers to homologs between different species, including both protein homologs and DNA homologs, and is also called orthologous genes. Specifically, it refers to proteins or genes evolved from the same ancestral gene of different species. The plasminogen of the present invention includes human native plasminogen, and further includes orthologs or orthologs of plasminogen having plasminogen activity derived from different species.

[0088] A "conservative substitution variant" is one in which one of the predetermined amino acid residues has been modified but does not change the overall conformation and function of the protein or enzyme, which includes, but is not limited to, substituting an amino acid in the amino acid sequence in the parent protein with an amino acid of similar characteristics (such as acidic, alkaline, hydrophobic, etc.). Amino acids with similar properties are as 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 proteins or amino acid sequences with similar functions may be different. For example, it has a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. A "conservative substitution variant" further includes polypeptides or enzymes having an amino acid identity of 60% or more based on the BLAST or FASTA algorithm, and it is even better if it reaches 75% or more, most preferably reaches 85% or more, and even more preferably reaches 90% or more, and further has the same or basically similar properties or functions compared to the natural or parent protein or enzyme.

[0089] "Isolated" plasminogen is a plasminogen protein that has been separated and / or recovered from its natural environment. In some embodiments, the plasminogen is purified to a purity of greater than 90%, greater than 95%, or greater than 98% (calculated by weight), for example, as determined by the Lowry method, and is purified to greater than 99% (calculated by weight), (2) purified to such an extent that at least 15 residues of the N-terminal or internal amino acid sequence can be obtained by at least a spinning cup sequencer, or (3) purified to homogeneity. The homogeneity is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions by Coomassie Brilliant Blue or silver staining. Isolated plasminogen is produced from recombinant cells by bioengineering techniques and further includes plasminogen separated in at least one purification step.

[0090] The terms "polypeptide", "peptide" and "protein" can be used interchangeably herein and refer to polymers of amino acids of any length, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusions including leader sequences (with or without an N-terminal methionine residue) from heterologous and homologous sources; and so on.

[0091] The definition of "percentage of amino acid sequence identity" for a reference peptide sequence is the percentage of amino acid residues in the candidate sequence that are the same as the amino acid residues in the reference polypeptide sequence, when no conservative substitutions are considered as part of the sequence identity, after introducing gaps as necessary to achieve the maximum percentage sequence identity. Alignments for the purpose of measuring the percentage of amino acid sequence identity can be achieved by multiple types of methods within the technical scope of this field, for example, by computer software available to the public, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, and the parameters include any algorithms for achieving the maximum comparison requirement for the full size of the sequences to be compared. However, for the purposes of the present invention, the percentage of amino acid sequence identity is that obtained by the sequence comparison computer software ALIGN-2.

[0092] When comparing amino acid sequences by using ALIGN-2, the percentage of amino acid sequence identity of a given amino acid sequence A with respect to a given amino acid sequence B (or also referred to as a given amino acid sequence A having or containing a certain percentage of amino acid sequence identity with respect to or for a given amino acid sequence B) is calculated as follows: Fraction X / Y × 100

[0093] Here, X is the number of amino acid residues that are evaluated as identical and matching in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It should be understood as follows: When the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity of A with respect to B is different from the percentage of amino acid sequence identity of B with respect to A. Unless otherwise specified, all amino acid sequence identity values % used in the present text are as described in the above paragraph and are obtained by the ALIGN-2 computer program.

[0094] The terms "individual", "subject", and "patient" can be used interchangeably herein and refer to a mammal, including but not limited to rodents (e.g., rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats).

[0095] "Therapeutically effective amount" or "effective amount" refers to the amount of plasminogen that can achieve the prevention and / or treatment of a disease when administered to a mammal or other subject for use in the treatment of the disease. The "therapeutically effective amount" varies according to the plasminogen used, the severity of the disease and / or symptoms of the subject to be treated, as well as age, weight, etc.

[0096] The term "treatment" of a disease state includes suppressing or preventing the progression of the disease state or its clinical symptoms, or alleviating the disease state or symptoms, resulting in a temporary or permanent reduction of the disease state or its clinical symptoms.

[0097] Preparation of the plasminogen of the present invention Plasminogen may be isolated and purified from nature or synthesized by standard chemical peptide synthesis techniques for therapeutic use. When synthesizing polypeptides by chemical methods, synthesis can be carried out in the liquid phase or the solid phase. Solid-phase polypeptide synthesis (SPPS) (attaching the C-terminal amino acid of the sequence to an insoluble support and sequentially adding the remaining amino acids in the sequence) is suitable for the chemical synthesis of plasminogen. Various forms of SPPS, such as Fmoc and Boc, can be used for the synthesis of plasminogen. The techniques used in solid-phase synthesis are described below: Barany and Solid-Phase Peptide Synthesis; page 3-284, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, t 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 treated with a functional unit on which the peptide chain is built. After repeated cycles of coupling / deprotection, the free N-terminal amine of the attached solid phase is coupled to a single amino acid unit that is N-protected. Then, the unit is deprotected to expose a new N-terminal amine for ligation with another amino acid. The peptide is left immobilized on the solid phase and then cleaved off.

[0098] 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 a control sequence in the expression vector. Expression control sequences include, but are not limited to, a promoter (e.g., a naturally associated promoter or a promoter of heterologous origin), a signal sequence, an enhancer element, and a transcription termination sequence. Control of expression can be by a eukaryotic promoter system in the vector, and the vector is used to transform or transfect a eukaryotic host cell (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 collection and purification of plasminogen.

[0099] Suitable expression vectors are usually replicated in the host body as episomes or as part of the host chromosomal DNA. Usually, the expression vector contains a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance), which is useful for performing measurements on those cells transformed in vitro with the desired DNA sequence.

[0100] Escherichia coli is an example of a prokaryotic host cell that can be used to clone a polynucleotide encoding plasminogen. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other Enterobacteriaceae, such as the genus Salmonella, the genus Serratia, and various Pseudomonas species. In these prokaryotic hosts, an expression vector can be generated, usually one that contains expression control sequences (such as an origin of replication) compatible with the host cell. There are also many known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the promoter system derived from phage λ. The promoter generally controls expression and may further have a ribosome binding site sequence, etc., when controlling gene sequences as needed to initiate transcription and translation.

[0101] Other microorganisms, such as yeast, can also be used for expression. Yeast (such as Saccharomyces (S. cerevisiae)) and Pichia are examples of suitable yeast host cells, and appropriate carriers among them include expression control sequences (such as a promoter), an origin of replication, a termination sequence, etc., as needed. Typical promoters include those for 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters particularly include those derived from alcohol dehydrogenase, isocytochrome C, and enzymes for the utilization of maltose and galactose.

[0102] In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in in vitro cell cultures) can also be used for the expression and production of the plasminogen of the present invention (e.g., a polynucleotide encoding plasminogen). See, for example, Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (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 used for these cells can include expression control sequences, such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and the required processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from rabbit immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0103] 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 is essentially pure, for example, with a purity of at least about 80% to 85%, at least about 85% - 90%, at least about 90% - 95%, or 98% - 99% or even higher purity, for example, free of contaminants such as cell debris, macromolecules other than plasminogen, etc.

[0104] Drug formulation Mix the desired purity of plasminogen with a pharmaceutical carrier, excipient, or stabilizer as required (Remington’s Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)) to form a lyophilized preparation or an aqueous solution to obtain a therapeutic formulation. Acceptable carriers, excipients, and stabilizers are non-toxic to the subject at the required dosages 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, benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parahydroxybenzoate esters such as methyl or propyl parahydroxybenzoate esters; pyrocatechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (having fewer than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartic acid, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates include glucose, mannose, or dextrin; chelating agents such as EDTA; saccharides such as sucrose, mannitol, fucose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0105] The formulation of the present invention may contain one or more active compounds required for a specific symptom in need of treatment, preferably those with complementary activities and no side effects on each other. Examples include antihypertensive drugs, antiarrhythmic drugs, and antidiabetic drugs.

[0106] The plasminogen of the present invention can be encapsulated in microcapsules produced, for example, by agglutination techniques or interfacial polymerization, and can be incorporated, for example, into colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsion agents, nanoparticles and nanocapsules), or into hydroxymethylcellulose or gel microcapsules and poly-(methyl methacrylate) microcapsules in a coarse emulsion-like liquid. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0107] The plasminogen of the present invention used for administration in the body must necessarily be sterile. This can be easily achieved by freeze-drying and filtering through a sterilizing filter membrane before or after reconstitution.

[0108] The plasminogen of the present invention can be prepared in sustained release formulations. 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 sustained release 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. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid (Sidman, et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al., same source as above), or degradable lactic acid-hydroxyacetic acid copolymers, e.g., Lupron. 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, but some aqueous gels release proteins for a shorter period of time. Rational strategies can be designed to stabilize proteins depending on the mechanism involved. For example, if the aggregation mechanism is the exchange of sulfur disulfide bonds to form intermolecular SS 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.

[0109] 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.

[0110] Products 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, and include saline and buffered media. Parenteral vehicles are sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluids and nutrient supplements, electrolyte supplements, etc. Additionally, preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc., may also be present.

[0111] Medical personnel can determine the dosage regimen based on various clinical factors. For example, as is known in the medical field, the dosage for any patient is determined by a plurality of factors, which include the patient's body size, body surface area, age, specific compound to be administered, gender, frequency and route of administration, overall health, and other drugs administered simultaneously. The dosage range of the pharmaceutical composition containing the plasminogen of the present invention is about 0.0001 - 2000 mg / kg per day, or about 0.001 - 500 mg / kg (such as 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 10 mg / kg, 50 mg / kg, etc.) based on the subject's body weight. For example, the dosage can be 1 mg / kg body weight or 50 mg / kg body weight or in the range of 1 - 50 mg / kg, or at least 1 mg / kg. Dosages higher or lower than this exemplary range are also covered, especially when considering the above-mentioned factors. Intermediate dosages within the said range are also included within the scope of the present invention. The subject can be administered such dosages daily, every other day, weekly, or according to any schedule determined by empirical analysis. An exemplary dosage schedule is to administer 1 - 10 mg / kg for several consecutive days. It is necessary to evaluate the therapeutic effect and safety in real-time during the administration process of the drug of the present invention.

[0112] Product or drug kit One embodiment of the present invention relates to a product or drug kit containing the present application's plasminogen or plasmin that can be used for the treatment of cardiovascular diseases and related disorders caused by diabetes. The product preferably includes a container, a label, or a protocol. Suitable containers include bottles, vials, syringes, etc. The container can be made from various materials such as glass or plastic. The container contains a composition that effectively treats the diseases or symptoms of the present invention and has a sterile inlet (for example, the container is an intravenous infusion pack or vial and includes a stopper penetrated by a subcutaneous injection needle). At least one activator in the composition is plasminogen / plasmin. The label on or attached to the container explains that the composition is used for the treatment of cardiovascular diseases and related disorders caused by diabetes of the present invention. The product further includes a second container containing a pharmaceutical buffer, and the pharmaceutical buffer includes, for example, phosphate-buffered saline, Ringer's solution, and glucose solution. Furthermore, from a commercial and user perspective, it can include other substances required, namely other buffers, diluents, filters, needles, and syringes. Also, the product includes a protocol with instructions for use, which includes, for example, instructing the user of the composition to administer the plasminogen composition and other drugs associated with the treatment of the disease to the patient.

Example

[0113] The human plasminogen used in the following examples was derived from donor plasma, the process was optimized based on the methods described in references [1-3], and it was purified from human donor plasma. Here, human Lys-plasminogen (Lys-plasminogen) and Glu-plasminogen (Glu-plasminogen) exceeded 98%.

[0114] [Example 1] Example 1 relates to the promotion of the formation of mature NGF in the brain tissue of spinal muscular atrophy (SMA) model mice by plasminogen. The FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1MsdTg(SMN2*delta7)4299Ahmb / J gene mutant mice (hereinafter abbreviated as SMNΔ7 SMA mice) have a homozygous mutation in the SMN1 gene and express the human SMN2 gene. The clinical and pathological symptoms of these mice resemble those of human SMA. The breeding mice were purchased from The Jackson Laboratory in the United States (strain number: 005025). Seven 3-day-old SMNΔ7 SMA mice were taken, four of which were used as the solvent group. In the first 9 days, bovine serum albumin solution (5 mg / ml) was administered once daily in the morning and afternoon by intraperitoneal injection at 6 μl / g / time, and then, bovine serum albumin solution (10 mg / ml) was administered once daily by intraperitoneal injection at 6 μl / g / time. Three of the mice in the administration group were used. In the first 9 days, plasminogen was administered once daily in the morning and afternoon by intraperitoneal injection at 30 μg / 6 μl, and then, plasminogen was administered once daily by intraperitoneal injection at 60 μg / 6 μl. Four wild-type mice were used as the blank control group. In the first 9 days, 6 μl of bovine serum albumin solution (5 mg / ml) was administered once daily in the morning and afternoon by intraperitoneal injection, and from the 10th day, 6 μl of bovine serum albumin solution (10 mg / ml) was administered daily by intraperitoneal injection. On the 12th day, the mice were sacrificed and the hindlimb muscle tissue was collected to prepare tissue homogenates, and Western blot detection of NGF protein was performed. According to the instructions for SDS-PAGE gel preparation, a 12% gel was prepared. The samples of each group were uniformly mixed with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100 °C for 5 minutes, cooled and centrifuged for 2 minutes, and then 20 μL was taken for loading. The electrophoresis conditions were carried out at 30 V for 45 minutes and then at 100 V until the bottom of the gel. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 hours.The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution) and blocked overnight in a refrigerator at 4°C. After washing 4 times with TBST (0.01M Tris-NaCl, pH 7.6 buffer), rabbit anti-mouse NGF antibody (Abcam, ab52918) was added and incubated at room temperature for 2 hours. After washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721), a secondary antibody, was added and incubated at room temperature for 1 hour. After washing 4 times with TBST, the PVDF membrane was placed on a clean imaging plate, Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development, photographed with a biomolecular imager, and the optical density values of each band were obtained with Image J for quantitative analysis. Nerve growth factor (NGF) is an important member of the neurotrophic factor family. It is synthesized in vivo in the form of a precursor containing a signal peptide, a leader peptide, and a mature peptide. Studies have reported that the precursor of nerve growth factor NGF (Pro-NGF) plays a role opposite to that of NGF formed by cleavage. Pro-NGF can promote apoptosis of neurons. Mature NGF is involved in the regulation of processes such as neuron growth, development, differentiation, survival, and repair after injury, and also plays an important role in the regulation of functional expression of central and peripheral neurons. [4] . The NGF / ProNGF ratio = NGF optical density (OD) / ProNGF optical density (OD) value. As a result, there was a certain NGF / Pro-NGF ratio in the brain tissues of mice in the blank control group. The NGF / Pro-NGF ratio in the brain tissues of mice in the administration group was significantly higher than that of mice in the solvent group, and the statistical difference was extremely significant (*** represents P < 0.001) (Figure 1). This result suggests that plasminogen can promote the conversion of ProNGF to NGF and the formation of mature NGF in the brain tissues of SMA model mice.

[0115] [Example 2] Example 2 relates to the promotion of Pro-NGF cleavage and mature NGF formation by plasminogen in the brain homogenate of normal mice. Four 18 - 25 g C57BL / 6J male mice aged 11 - 12 weeks were selected, sacrificed, and whole brain tissues were collected. After weighing, 1×PBS (Thermo Fisher, pH 7.4; 10010 - 031) was added at 150 mg tissue / mL PBS respectively, and homogenized at 4°C (1 minute, 3 - 4 times). After homogenization, centrifugation was performed at 4°C (12000 rpm, 20 minutes), and the supernatant, that is, the brain homogenate, was transferred to a new EP tube. An Eppendorf (EP) tube was taken, and five parallel samples were set up in each of the following groups: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. In the blank control group, 21.5 μL of physiological saline, 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added; in the solvent control group, 21.5 μL of Pro-NGF (Shanghai Qiangyao (ChinaPeptides Co., Ltd.,), custom-expressed human Pro-NGF, 1.0 mg / mL), 4.6 μL of solvent solution (citric acid - sodium citrate solution), and 23.9 μL of mouse brain homogenate were added; in the plasminogen group, 21 μL of Pro-NGF (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate were added. After adding samples to each group, they were incubated at 37°C for 6 hours, and then 100 μL of 0.1% trifluoroacetic acid solution was added to each to stop the reaction. According to the instructions of the SDS-PAGE gel preparation kit, a 15% gel was prepared. The samples of each group were mixed with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100 °C for 5 minutes, cooled, centrifuged for 2 minutes, and then 20 μL of the sample was taken for loading. The electrophoresis conditions were run at 30 V for 30 minutes and then at 100 V until the bottom of the gel. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 hours. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed 4 times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), then rabbit anti-human NGF antibody (Abcam, ab52918) was added and incubated at room temperature for 2 hours, washed 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added, incubated at room temperature for 1 hour, washed 4 times with TBST, then the PVDF membrane was placed on a clean imaging plate, Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development, photographed with a biomolecular imager, and the optical density of the band was quantitatively analyzed with Image J. NGF is an important member of the neurotrophic factor family. It is synthesized in vivo in the form of a precursor containing a signal peptide, a leader peptide, and a mature peptide. Studies have reported that the precursor of nerve growth factor NGF (Pro-NGF) plays a role opposite to that of NGF formed by cleavage. Pro-NGF can promote apoptosis of neurons. [5] Mature NGF is involved in the regulation of processes such as the growth, development, differentiation, survival, and repair after injury of neurons, and also plays an important role in the regulation of the functional expression of central and peripheral neurons. [6] 。 As a result, in the brain homogenate of normal mice, the amount of Pro-NGF in the plasminogen-administered group was significantly lower than that in the solvent control group, and the difference was extremely significant (* indicates P < 0.05, *** indicates P < 0.001). The amount of NGF in the plasminogen-administered group was significantly higher than that in the solvent control group, and the difference was significant (Figure 2). This suggests that plasminogen can promote the cleavage of Pro-NGF and the formation of mature NGF in the brain homogenate of normal mice.

[0116] [Example 3] Example 3 relates to the promotion of the cleavage of Pro-NGF and the formation of mature NGF by plasminogen in the brain homogenate of Alzheimer's disease model mice. Four 11-week-old B6SJLTg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas / Mmjax(FAD)(stock number: 034840)(abbreviated as FAD) mice were selected, sacrificed, and the whole brain tissue was collected. After weighing, it was placed in an Eppendorf (EP) tube, and 1×PBS (Thermo Fisher, pH 7.4; 10010-031) was added to each at 150 mg tissue / mL PBS, and homogenized at 4°C (1 minute, 3 - 4 times). After homogenization, centrifugation was performed at 4°C (12,000 rpm, 15 minutes), and the supernatant, that is, the brain homogenate, was transferred to a new EP tube. Take Eppendorf (EP) tubes and set up 5 parallel samples for each of the following groups: (1) blank group, (2) blank control group, (3) solvent control group, and (4) plasminogen group. To the blank control group, add 21.5 μL of physiological saline, 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate; to the solvent control group, add 21.5 μL of Pro-NGF (Shanghai Qiangyao (ChinaPeptides Co., Ltd.,), custom-expressed human Pro-NGF, 1.0 mg / mL), 4.6 μL of solvent solution (citric acid-sodium citrate solution), and 23.9 μL of mouse brain homogenate; to the plasminogen group, add 21. μL of Pro-NGF (1.0 mg / mL), 4.6 μL of plasminogen solution (2 mg / mL), and 23.9 μL of mouse brain homogenate. After adding the samples to each group, incubate at 37 °C for 6 hours, and then add 100 μL of 0.1% trifluoroacetic acid solution to each to stop the reaction. According to the instructions for SDS-PAGE gel preparation, a 15% gel was prepared. The samples of each group were mixed with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100 °C for 5 minutes, cooled, centrifuged for 2 minutes, and then 20 μL of the sample was taken for loading. The electrophoresis conditions were run at 30 V for 30 minutes and then at 100 V until the bottom of the gel. After electrophoresis, the gel was peeled off and transferred to an activated PVDF membrane (GE, A29433753), and the electrophoresis conditions were 15 V for 2.5 hours. The transferred PVDF membrane was immersed in a blocking solution (5% skim milk solution), blocked overnight in a refrigerator at 4 °C, washed 4 times with TBST (0.01 M Tris-NaCl, pH 7.6 buffer), then rabbit anti-human NGF antibody (Abcam, ab6721) was added and incubated at room temperature for 2 hours, washed 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was added, incubated at room temperature for 1 hour, washed 4 times with TBST, then the PVDF membrane was placed on a clean imaging plate, Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) was added for color development, photographed with a biomolecular imager, and the band optical density was quantitatively analyzed with Image J. As a result, in the brain homogenate of Alzheimer's disease model mice, the amount of Pro-NGF in the plasminogen administration group was significantly lower than that in the solvent control group, and the difference was extremely significant (***P < 0.001); the amount of NGF in the plasminogen administration group was significantly higher than that in the solvent control group, and the difference was significant (Figure 3). This suggests that plasminogen can promote the cleavage of Pro-NGF and the formation of mature NGF in the brain homogenate of Alzheimer's disease model mice.

[0117] [Example 4] Example 4 relates to the promotion of NGF expression by plasminogen in the spinal cord tissue of SMA mice. Eight 3-day-old or 7-day-old SMNΔ7 SMA mutant mice and four wild-type mice of the same age were taken. The SMNΔ7 SMA mutant mice were randomly divided into a solvent group and an administration group, with four mice in each group, and the four wild-type mice were used as a blank control group. The mice in the administration group were intraperitoneally injected with TP01HN106 at a concentration of 10 mg / ml at 60 mg / kg / day, and the mice in the solvent group and the blank control group were intraperitoneally injected with the solvent at 6 ml / kg / day. All were administered until the 11th day after birth. Two hours after administration on the 11th day after birth, all the mice were sacrificed and dissected, a part of the spinal cord tissue was collected, and fixed in 10% formaldehyde solution for 24 to 48 hours. The fixed spinal cord tissue was dehydrated with an alcohol gradient, cleared with xylene, and then embedded in paraffin. The thickness of the tissue section was 3 μm. The section was deparaffinized, rehydrated, and washed once with water. The tissue was circled with a PAP marker, incubated with 3% hydrogen peroxide for 15 minutes, washed twice with 0.01M PBS for 5 minutes each time. Blocked with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 minutes, then discarded the goat serum when the time was up, dropped rabbit anti-mouse NGF antibody (Abcam, ab52918), and incubated overnight at 4°C, washed twice with 0.01M PBS for 5 minutes each time. Incubated with goat anti-rabbit IgG (HRP) antibody (Vector, MP-7451-50) secondary antibody at room temperature for 1 hour, washed twice with 0.01M PBS for 5 minutes each time. Stained with DAB kit (Vector laboratories, Inc., USA), washed three times with water, then counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. Dehydrated with an alcohol gradient, cleared with xylene, and sealed with neutral gum, and the section was observed under an optical microscope at 400 times magnification. As a result, it is shown that the expression level of NGF in the administration group (Figure 4C) was significantly higher than that in the solvent group (Figure 4B), and the statistical difference was significant (P < 0.05) (Figure 4D). This indicates that plasminogen can promote the expression of NGF in the spinal cord tissue of SMNΔ7 SMA mice.

[0118] [Example 5] Example 5 relates to the administration of plasminogen increasing the level of plasminogen in the brain tissue of SMA mice. Eight 3-day-old or 7-day-old FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1MsdTg(SMN2*delta7)4299Ahmb / J gene mutant mice (abbreviated as SMNΔ7 SMA mice, purchased from Jackson Laboratory, strain number: 005025) and four wild-type SMNΔ7 SMA mice of the same age were taken. The SMNΔ7 SMA mice were randomly divided into a solvent group and an administration group, with 4 mice in each group, and the four wild-type mice were used as a blank control group. The mice in the administration group were administered plasminogen by intraperitoneal injection at a dose of 60 mg / kg / day and a concentration of 10 mg / ml, and the mice in the solvent group and the blank control group were administered plasminogen by intraperitoneal injection at a dose of 6 ml / kg / day. All were administered until postnatal day 11. Two hours after administration on postnatal day 11 of the mice, all the mice were sacrificed and dissected, a part of the brain tissue was collected and homogenized, and then detected according to the instructions of the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). Using the human plasminogen working standard as an internal standard, the concentration of each sample was calibrated, and the calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per unit total protein mass of each sample, and statistical analysis was performed. SMNΔ7 SMA mice have a homozygous mutation in the SMN1 gene and express the human SMN2 gene. The clinical and pathological symptoms of these mice are similar to those of human spinal muscular atrophy (SMA). As a result, the level of plasminogen in the brain tissue of wild-type mice in the blank control group was 1.18 ± 1.54 ng / mg, and the level of plasminogen in the brain tissue of mice in the solvent group was 1.49 ± 1.59 ng / mg. Compared with the blank control group, the level of plasminogen in the brain tissue of mice in the solvent group did not change significantly; the level of plasminogen in SMA transgenic mice in the administration group was 12.09 ± 5.32 ng / mg, which was about 8 times that of the solvent group (Figure 5). This suggests that when plasminogen is supplemented, the permeability of the blood-brain barrier to plasminogen increases under SMA disease conditions, and plasminogen can reach the brain by passing through the blood-brain barrier.

[0119] [Example 6] Example 6 relates to the administration of plasminogen increasing the level of plasminogen in the spinal cord tissue of SMA mice. Eight 3-day-old or 7-day-old SMNΔ7 SMA mice, four wild-type mice of the same age, and three SMA heterozygous mice of the same age were taken. The SMNΔ7 SMA mice were randomly divided into a solvent group and an administration group, with four mice in each group. Four wild-type mice were used as the blank control group, and three SMA heterozygous mice were used as the normal administration control group. Mice in the administration group and the normal administration control group were administered plasminogen by intraperitoneal injection at a concentration of 10 mg / ml at 60 mg / kg / day, and mice in the solvent group and the blank control group were administered the solvent by intraperitoneal injection at 6 ml / kg / day. All were administered until the 11th day after birth. Two hours after administration on the 11th day after birth, all the mice were sacrificed and dissected, a part of the spinal cord tissue was collected, homogenized, and detected for plasminogen by ELISA. As a result, the level of spinal cord plasminogen in wild-type mice of the blank control group was 8.51 ± 9.51 ng / mg. The level of plasminogen in the spinal cord of solvent group SMA transgenic mice was 19.95 ± 4.06 ng / mg, slightly increased compared with the blank control group. The level of plasminogen in the normal administration control group was 13.03 ± 7.51 ng / mg. The level of spinal cord plasminogen in the administration group of SMA transgenic mice was 62.33 ± 17.37 ng / mg, about 3 times that of the solvent group mice. The P value of the comparative statistical analysis between the administration group and the solvent group was 0.029, about 4.8 times that of the normal administration control group. The P value of the comparative statistical analysis between the administration group and the normal administration control group was 0.026 (Figure 6). This indicates that the administration of plasminogen promotes the increase in the permeability of the blood-spinal cord barrier to plasminogen under the condition of SMA, and plasminogen can reach the spinal cord by passing through the blood-spinal cord barrier.

[0120] [Example 7] Example 7 relates to the fact that the administration of plasminogen promotes an increase in the level of plasminogen in the spinal cord tissue of LPS-induced pneumonia SMA heterozygous mice. Nine SMA heterozygous mice (purchased from Jackson Laboratory, strain number: 005025) were randomly divided into two groups according to body weight, with 3 mice in the blank control group and 6 mice in the model group. After all the mice were anesthetized with Zoletil 50, the mice in the model group were modeled by injecting a 2.5 mg / ml solution of bacterial lipopolysaccharide (LPS) into the trachea at a dosage of 5 mg / kg, and the mice in the blank control group were injected with 2 ml / kg of physiological saline. After 2 hours of modeling, all the mice in the model group were randomly divided into two groups according to body weight, with 3 mice in the solvent group and 3 mice in the administration group. Administration began after grouping. The mice in the administration group were administered plasminogen by tail vein injection at 50 mg / kg per mouse, and the mice in the solvent group and the blank control group were administered the solvent by tail vein injection at 5 ml / kg per mouse. Two hours after administration, all the mice were sacrificed and dissected, spinal cord tissue was collected, homogenized, and detected according to the instructions of the Human Plasminogen ELISA Kit (manufacturer: AssayMax, catalog number: EP1200-1). Using a human plasminogen working standard as an internal standard, the concentration of each sample was calibrated, and the calibrated concentration was divided by the total protein concentration to calculate the amount of plasminogen per unit total protein mass of each sample, and statistical analysis was performed. As a result, the level of plasminogen in the spinal cord tissue homogenate of the mice in the blank control group was 2.70 ± 0.74 ng / mg. After tracheal injection of LPS, the level of plasminogen in the spinal cord tissue homogenate of the mice in the solvent group was 3.17 ± 1.51 ng / mg, showing no significant change compared with the blank control group. After supplementing the mice in the administration group with 2.5 times the physiological dose of plasminogen, the level of plasminogen was 121.16 ± 44.68 ng / mg, approximately 38.2 times that of the mice in the solvent group (Figure 7). This suggests that supplementing plasminogen promotes an increase in the permeability of the blood-spinal cord barrier to plasminogen in pneumonia SMA heterozygous mice induced by LPS, and under this condition, plasminogen can pass through the blood-spinal cord barrier and enter the central nervous system.

[0121] [Example 8] Example 8 relates to the fact that administration of plasminogen promotes an increase in plasminogen levels in the spinal cord tissue of LPS-induced pneumonia SMA heterozygous mice. Nine SMA heterozygous mice (purchased from Jackson Laboratory, strain number: 005025) were randomly divided into two groups according to body weight, with 3 mice in the blank control group and 6 mice in the model group. After all mice were anesthetized with Zoletil 50, mice in the model group were modeled by injecting a 2.5 mg / ml solution of bacterial lipopolysaccharide (LPS) into the trachea at a dosage of 5 mg / kg, and mice in the blank control group were injected with 2 ml / kg of physiological saline. After 2 hours of modeling, all mice in the model group were randomly divided into two groups according to body weight, with 3 mice in the solvent group and 3 mice in the administration group. Administration began after grouping. Mice in the administration group were administered plasminogen by tail vein injection at 50 mg / kg per mouse, and mice in the solvent group and the blank control group were administered the solvent by tail vein injection at 5 ml / kg per mouse. Two hours after administration, all mice were sacrificed and dissected, spinal cord tissue was collected, homogenized, and then detected by the enzymatic substrate kinetic method for plasminogen. To a microplate (manufacturer: NUNC, catalog number: 446469), 85 μL / well of seven different concentrations of standard solution, blank, and sample were added, and then a mixture of 15 μL of 20 mM S-2251 solution (manufacturer: Chromogenix, catalog number: 82033239) and 100 ng / μL uPA solution (mixed at a volume ratio of 2:1 immediately before use) was added to each well and incubated at 37°C. From 0 minutes to 90 minutes of the reaction, the A405 absorbance value was read every 5 minutes with a multifunctional microplate reader. All reactions were fitted linearly based on time and absorbance value, and the slope of the line was the reaction rate of the standard product / sample (ΔA405 / min). Finally, the potency of the test sample was calculated using the potency value of the standard product and ΔA405 / min as the standard curve. The activity of plasminogen per unit total protein mass of each sample was calculated. As a result, the level of plasminogen in the spinal cord tissue of mice in the blank control group was 0.00011 ± 4.51x10 -5 U / mg; after intratracheal injection of LPS, the level of plasminogen in the spinal cord of mice in the solvent group was 0.00010 ± 9.72×10 -6 U / mg, and there was no significant change compared with the blank control group; after supplementing the mice in the administration group with 2.5 times the physiological dose of plasminogen, the level of plasminogen increased to 0.00034 ± 1.04×10 -4 U / mg, and the P value of statistical analysis was 0.058 compared with the solvent group (Figure 8). This indicates that supplementing plasminogen promotes an increase in the permeability of the blood-spinal cord barrier to plasminogen in LPS-induced pneumonia SMA heterozygous mice, suggesting that plasminogen can pass through the blood-spinal cord barrier and accumulate in the spinal cord.

[0122] [Example 9] Example 9 relates to the fact that administration of plasminogen promotes an increase in the level of plasminogen in the spinal cord tissue of LPS-induced pneumonia mice. Fifteen male C57 mice were weighed and then randomly divided into two groups, with 3 mice in the blank control group and 12 mice in the model group. After all the mice in the model group were anesthetized with Zoletil 50, a 2.5 mg / ml LPS solution was injected intratracheally for modeling, and the modeling dose was 5 mg / kg. Two hours after modeling, all the mice in the model group were randomly divided into four groups according to body weight, with 3 mice in the solvent group, 3 mice in administration group A, 3 mice in administration group B, and 3 mice in administration group C. After grouping, administration was started. The mice in administration group A were administered plasminogen by tail vein injection at 50 mg / kg per mouse, the mice in administration group B were administered plasminogen by tail vein injection at 17 mg / kg per mouse, and the mice in administration group C were administered plasminogen by tail vein injection at 6 mg / kg per mouse. The mice in the solvent group and the blank control group were administered the solvent by tail vein injection at 5 ml / kg per mouse. Two hours after administration, the spinal cord tissue was dissected and detected by ELISA for specific human plasminogen. The results of detecting the plasminogen level in the spinal cord tissue homogenate by ELISA showed that the Plg content in the spinal cord tissue of the blank control group was 7.71 ± 0.51 ng / mg, and the Plg content in the spinal cord tissue of the solvent group mice was 14.04 ± 3.25 ng / mg. After intratracheal injection of LPS, the level of plasminogen in the spinal cord of mice increased; after administering 50 mg / kg (about 1 mg per mouse) of plasminogen to the mice in administration group A, the level of plasminogen in the spinal cord tissue was 85.10 ± 11.59 ng / mg, which was about 6.1 times that of the mice in the solvent group. After administering 17 mg / kg (about 0.34 mg per mouse) of plasminogen to the mice in administration group B, the level of plasminogen in the spinal cord tissue was 77.90 ± 21.39 ng / mg, which was about 5.5 times that of the mice in the solvent group; after administering 6 mg / kg (about 0.1 mg per mouse) of plasminogen to the mice in administration group C, the level of plasminogen in the spinal cord tissue was 64.00 ± 19.63 ng / mg, which was about 4.6 times that of the mice in the solvent group (Figure 9). This indicates that 1) supplementing plasminogen can promote the increase in the permeability of the blood-brain barrier to plasminogen in LPS-induced pneumonia mice, enabling plasminogen to pass through the blood-brain barrier and accumulate in the spinal cord tissue; 2) the enrichment of plasminogen in the spinal cord has a dose-dependent effect, suggesting that within the dose range of 6 mg / kg to 50 mg / kg, the enrichment level of plasminogen in the spinal cord increases with the increase in the dose of plasminogen administered.

[0123] [Example 10] Example 10 relates to the promotion of an increase in the plasminogen level in the spinal cord tissue of LPS-induced pneumonia mice by the administration of plasminogen. Fifty-four 6- to 9-week-old male C57 mice were weighed and randomly divided into two groups: a blank control group of 15 mice and a model group of 39 mice. After all the mice were anesthetized with Zoletil 50, the model group mice were modeled by injecting a 2.5 mg / ml LPS solution into the trachea at a dosage of 5 mg / kg, and the blank group mice were injected with 2 ml / kg of physiological saline. Two hours after LPS modeling in the model group mice, they were randomly divided into three groups according to body weight: an LPS + solvent group of 15 mice, an LPS + 50 mg / kg plasminogen group of 12 mice, and an LPS + 6 mg / kg plasminogen group of 12 mice. The blank group mice were randomly divided into two groups according to body weight: a blank + solvent group of 3 mice and a blank + 50 mg / kg plasminogen group of 12 mice, and administration was started. The LPS + 50 mg / kg plasminogen group and the blank + 50 mg / kg plasminogen group were administered plasminogen by tail vein injection at a dose of 50 mg / kg body weight, the LPS + solvent group and the blank + solvent group were administered the solvent by tail vein injection at 5 ml / kg per mouse, and the LPS + 6 mg / kg plasminogen group was administered plasminogen by tail vein injection at a dose of 6 mg / kg body weight. Mice in the three blank + solvent groups and the three LPS + solvent groups were sacrificed at 0 h after administration, and three mice from each group were randomly sacrificed at each time point of 2, 6, 12, and 24 h. The spinal cord was collected, homogenized, and then detected by ELISA for specific human plasminogen. As a result, the mice in the sham operation + solvent group and the LPS + solvent group did not receive plasminogen injection, and human plasminogen was not detected in the spinal cord tissue homogenates at 0, 2, 6, 12, and 24 hours. When mice in the sham operation + 50 mg / kg plasminogen group and the LPS + 50 mg / kg plasminogen group were injected with 50 mg / kg body weight of plasminogen, the human plasminogen in the spinal cord tissue homogenates significantly increased 2 hours after injection, the level of plasminogen significantly decreased 6 - 12 hours after injection, and human plasminogen could basically not be detected at 24 hours. When mice in the LPS + 6 mg / kg plasminogen group were injected with 6 mg / kg body weight of plasminogen, the level of plasminogen detected in the spinal cord tissue homogenates 2 hours after injection was significantly higher than that in the mice of the LPS + solvent group and significantly lower than that in the mice of the LPS + 50 mg / kg plasminogen group (Figure 10). This result indicates that: 1) After administering plasminogen under physiological and pathological conditions, plasminogen can pass through the blood-brain barrier and promote its accumulation in spinal cord tissue; 2) Intravenous injection of plasminogen into normal mice significantly increases the level of plasminogen in spinal cord tissue; 3) The enrichment of plasminogen in the spinal cord has a time-dependent effect, first increasing, gradually decreasing from 2 to 12 hours, and being almost completely metabolized from 12 to 24 hours; 4) The enrichment of plasminogen in the spinal cord has a dose-dependent effect, suggesting that the higher the dose, the higher the level of plasminogen in spinal cord tissue.

[0124] [Example 11] Example 11 relates to the fact that administration of plasminogen promotes an increase in the level of plasminogen in the brain tissue of LPS-induced pneumonia mice. Fifty-four 6- to 9-week-old male C57 mice were weighed and randomly divided into two groups: a blank control group of 15 mice and a model group of 39 mice. After all the mice were anesthetized with Zoletil 50, the model group mice were modeled by injecting a 2.5 mg / ml LPS solution into the trachea at a dosage of 5 mg / kg, and the blank group mice were injected with 2 ml / kg of physiological saline. Two hours after LPS modeling in all model group mice, they were randomly divided into three groups according to body weight: a LPS + solvent group of 15 mice, a LPS + 50 mg / kg plasminogen group of 12 mice, and a LPS + 6 mg / kg plasminogen group of 12 mice. The blank group mice were randomly divided into two groups according to body weight: a blank + solvent group of 3 mice and a blank + 50 mg / kg plasminogen group of 12 mice, and administration was started. The LPS + 50 mg / kg plasminogen group and the blank + 50 mg / kg plasminogen group were administered plasminogen by tail vein injection at 50 mg / kg body weight, the LPS + solvent group and the blank + solvent group were administered the solvent by tail vein injection at 5 ml / kg per mouse, and the LPS + 6 mg / kg plasminogen group was administered plasminogen by tail vein injection at 6 mg / kg body weight. Three mice in the blank + solvent group and three mice in the LPS + solvent group were sacrificed 0 hours after administration, and three mice were randomly sacrificed from each group of mice at each time point of 2, 6, 12, and 24 hours. The brains were collected, homogenized, and then detected by ELISA for specific human plasminogen. As a result, the mice in the blank + solvent group and the LPS + solvent group did not receive plasminogen injection, and human plasminogen was not detected in the brain tissue homogenate at 0, 2, 6, 12, and 24 hours. When mice in the blank + 50 mg / kg plasminogen group and the LPS + 50 mg / kg plasminogen group were injected with 50 mg / kg body weight of plasminogen, human plasminogen significantly increased in the brain tissue homogenate 2 hours after injection. The level of plasminogen significantly decreased 6 - 12 hours after injection, and human plasminogen was basically undetectable at 24 hours. When mice in the LPS + 6 mg / kg plasminogen group were injected with 6 mg / kg body weight of plasminogen, the level of plasminogen detected in the brain tissue homogenate 2 hours after injection was significantly higher than that in the mice of the LPS + solvent group and significantly lower than that in the mice of the LPS + 50 mg / kg plasminogen group (Figure 11). This result indicates that 1) after administering plasminogen under physiological and pathological conditions, plasminogen can pass through the blood-brain barrier and promote its accumulation in brain tissue; 2) when plasminogen is intravenously injected into normal mice, the level of plasminogen in brain tissue significantly increases; 3) the enrichment of plasminogen in brain tissue has a time-dependent effect, first increasing, gradually decreasing from 2 to 12 hours, and being almost completely metabolized from 12 to 24 hours; 4) the enrichment of plasminogen in brain tissue has a dose-dependent effect, suggesting that the higher the dose, the higher the level of plasminogen in brain tissue.

[0125] [Example 12] Example 12 relates to the promotion of an increase in the plasminogen level in the spinal cord tissue of SOD1-G93A mice by the administration of plasminogen. Twenty-seven 15-week-old B6.Cg-Tg(SOD1-G93A)1Gur / J transgenic mice (hereinafter abbreviated as SOD1-G93A mice, purchased from Jackson Laboratory, strain number: 004435) were randomly divided into three groups: 3 mice in the solvent group, 12 mice in the 50 mg / kg administration group, and 12 mice in the 6 mg / kg administration group. The mice in the solvent group were administered the solvent by tail vein injection, the mice in the 50 mg / kg plasminogen administration group were administered plasminogen at a dose of 50 mg / kg body weight by tail vein injection, and the mice in the 6 mg / kg plasminogen administration group were administered plasminogen at a dose of 6 mg / kg body weight by tail vein injection. Two hours after administration to the mice in the solvent group, the mice were sacrificed. For the other mice, three mice from each group were sacrificed at 2, 6, 12, and 24 hours after administration, the spinal cords were collected, homogenized, and then detected by ELISA for specific human plasminogen. SOD1-G93A mice overexpress the human mutant SOD1 gene, and the clinical and pathological symptoms of the mice are similar to those of human amyotrophic lateral sclerosis. As a result of ELISA level detection of the spinal cords of SOD1-G93A mice, the level of plasminogen in the spinal cords of SOD1-G93A mice increased significantly after tail vein injection of 50 mg / kg and 6 mg / kg of plasminogen, and the level of plasminogen in the 50 mg / kg administration group was significantly higher than that in the 6 mg / kg administration group. Plasminogen gradually decreased from 2 hours after administration and was almost completely metabolized at 12-24 hours (Figure 12). This result suggests that 1) after administration of plasminogen at physiological dose levels, plasminogen can pass through the blood-brain barrier of SOD1-G93A mice and accumulate in spinal cord tissue; 2) the concentration of plasminogen in the spinal cord has a dose-dependent effect, and the higher the dose of plasminogen administered, the more concentrated it is; 3) the concentration of plasminogen in the spinal cord has a time-dependent effect, first increasing, gradually decreasing from 2 to 12 hours, and being almost completely metabolized from 12 to 24 hours.

[0126] [Example 13] Example 13 relates to the administration of plasminogen promoting an increase in plasminogen levels in the brain tissue of SOD1-G93A mice. Twenty-seven 15-week-old B6.Cg-Tg(SOD1-G93A)1Gur / J transgenic mice (hereinafter abbreviated as SOD1-G93A mice, purchased from Jackson Laboratory, strain number: 004435) were randomly divided into three groups: 3 mice in the solvent group, 12 mice in the 50 mg / kg administration group, and 12 mice in the 6 mg / kg administration group. The mice in the solvent group were administered the solvent by tail vein injection, the mice in the 50 mg / kg plasminogen administration group were administered plasminogen at 50 mg / kg body weight by tail vein injection, and the mice in the 6 mg / kg plasminogen administration group were administered plasminogen at 6 mg / kg body weight by tail vein injection. Two hours after administration to the mice in the solvent group, the mice were sacrificed. For the other mice, three mice from each group were sacrificed at 2, 6, 12, and 24 hours after administration. The brains were collected, homogenized, and then detected for specific human plasminogen by ELISA. As a result of ELISA level detection in the brains of SOD1-G93A mice, the level of plasminogen in the brain tissue of SOD1-G93A mice increased significantly after tail vein injection of 50 mg / kg and 6 mg / kg plasminogen, and the level of plasminogen in the 50 mg / kg administration group was significantly higher than that in the 6 mg / kg administration group. Plasminogen gradually decreased from 2 hours after administration and was almost completely metabolized at 12 - 24 hours (Figure 13). This result indicates that: 1) after administering plasminogen at physiological dosage levels, plasminogen can pass through the blood-brain barrier of SOD1-G93A mice and accumulate in the brain tissue; 2) the concentration of plasminogen in the brain tissue has a dose-dependent effect, and the higher the dose of plasminogen administered, the more concentrated it is; 3) the concentration of plasminogen in the brain tissue has a time-dependent effect, first increasing, gradually decreasing from 2 to 12 hours, and being almost completely metabolized from 12 to 24 hours.

[0127] [Example 14] Example 14 relates to the promotion of an increase in the plasminogen level in the spinal cord tissue of FAD mice by the administration of plasminogen. Twenty-seven 15-week-old B6-Tg(APPSwFlLon, PSEN1*M146L*L286V)6799Vas / Mmjax (hereinafter abbreviated as FAD mice) (purchased from Jackson Laboratory, strain number: 034840) were randomly divided into three groups: 3 mice in the solvent group, 12 mice in the 50 mg / kg administration group, and 12 mice in the 6 mg / kg administration group. The solvent was administered to the mice in the solvent group by tail vein injection, plasminogen was administered to the mice in the 50 mg / kg plasminogen administration group at 50 mg / kg body weight by tail vein injection, and plasminogen was administered to the mice in the 6 mg / kg plasminogen administration group at 6 mg / kg body weight by tail vein injection. The mice in the solvent group were sacrificed 2 hours after administration, and for the other mice, 3 mice from each group were sacrificed at 2, 6, 12, and 24 hours after administration, the spinal cord was collected, homogenized, and then detected by ELISA for specific human plasminogen. FAD mice are an animal model commonly used in Alzheimer's disease research. As a result of ELISA level detection of spinal cord tissue homogenates, the level of plasminogen in the spinal cord tissue of FAD mice increased significantly after tail vein injection of 50 mg / kg and 6 mg / kg of plasminogen, and the level of plasminogen in the 50 mg / kg administration group was significantly higher than that in the 6 mg / kg administration group. Plasminogen gradually decreased from 2 hours after administration and was almost completely metabolized at 12 - 24 hours (Figure 14). This result suggests that: 1) after administration of plasminogen at physiological dosage levels, plasminogen can pass through the blood-brain barrier of FAD mice and accumulate in the spinal cord tissue; 2) the enrichment of plasminogen in the spinal cord tissue has a dose-dependent effect, and the higher the dose of plasminogen administered, the more concentrated it is; 3) the enrichment of plasminogen in the spinal cord tissue has a time-dependent effect, first increasing, gradually decreasing from 2 to 12 hours, and being almost completely metabolized from 12 to 24 hours.

[0128] [Example 15] Example 15 relates to the fact that administration of plasminogen promotes an increase in plasminogen levels in the brain tissue of FAD mice. Twenty-seven 15-week-old B6-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas / Mmjax (hereinafter abbreviated as FAD mice) (purchased from Jackson Laboratory, strain number: 034840) were randomly divided into three groups: 3 mice in the solvent group, 12 mice in the 50 mg / kg administration group, and 12 mice in the 6 mg / kg administration group. The solvent was administered to the mice in the solvent group by tail vein injection, plasminogen was administered to the 50 mg / kg plasminogen administration group by tail vein injection at 50 mg / kg body weight, and plasminogen was administered to the 6 mg / kg plasminogen administration group by tail vein injection at 6 mg / kg body weight. The mice in the solvent group were sacrificed 2 hours after administration, and for the other mice, 3 mice from each group were sacrificed at 2, 6, 12, and 24 hours after administration. The brains were collected, homogenized, and then detected for specific human plasminogen by ELISA. As a result of ELISA level detection of brain tissue homogenates, the level of plasminogen in the brain tissue of FAD mice increased significantly after tail vein injection of 50 mg / kg and 6 mg / kg of plasminogen, and the level of plasminogen in the 50 mg / kg administration group was significantly higher than that in the 6 mg / kg administration group. Plasminogen gradually decreased from 2 hours after administration and was almost completely metabolized at 12 - 24 hours (Figure 15). This result suggests that: 1) after administration of plasminogen at physiological dose levels, plasminogen can pass through the blood-brain barrier of FAD mice and accumulate in the brain tissue; 2) the enrichment of plasminogen in the brain tissue has a dose-dependent effect, and the higher the dose of plasminogen administered, the more concentrated it is; 3) the enrichment of plasminogen in the brain tissue has a time-dependent effect, first increasing, gradually decreasing from 2 to 12 hours, and being almost completely metabolized from 12 to 24 hours.

[0129] References: [1]KENNETH C. ROBBINS, LOUIS SUMMARIA, DAVID ELWYN et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry , 1965 , 240 (1) :541-550. [2]Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J Biol Chem. 1976 Jun 25;251(12):3693-9. [3]HAGAN JJ, ABLONDI FB, DE RENZO EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 Apr;235:1005-10. [4]Aloe L, Rocco M L , Bianchi P , et al. Nerve growth factor: from the early discoveries to the potential clinical use[J]. Journal of Translational Medicine, 2012, 10(1). [5] Aloe L , Rocco M L , Bianchi P , et al. Nerve growth factor: from the early discoveries to the potential clinical use[J]. Journal of Translational Medicine, 2012, 10(1). [6] Lewin G R , Lechner S G , Smith E S J . Nerve Growth Factor and Nociception: From Experimental Embryology to New Analgesic Therapy[J]. 2014.

Claims

1. A pharmaceutical composition comprising plasminogen for use in promoting the formation of mature NGF and / or increasing the level of NGF, wherein the plasminogen comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2 and has proteolytic activity of plasminogen.

2. The pharmaceutical composition according to claim 1, wherein the plasminogen promotes the formation of mature NGF and / or the expression of NGF by cleavage of pro-NGF in the nerve tissue of a subject.

3. The pharmaceutical composition according to claim 1 or 2, wherein the plasminogen has one or more uses or activities selected from nourishing nerves, protecting nerves, promoting nerve regeneration, inhibiting nerve damage, promoting repair of wound tissue, and promoting wound healing.

4. A pharmaceutical composition comprising plasminogen for use in the prevention or treatment of NGF-related diseases or conditions, wherein the plasminogen comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2 and has proteolytic activity of plasminogen, wherein the NGF-related disease or condition is 1) stroke, transient ischemic attack (TIA), subarachnoid hemorrhage, subdural hemorrhage and hematoma, and epidural hemorrhage; 2) a nerve structure damage disease selected from any one of brain or spinal cord injury, Bell palsy, cervical spondylosis, carpal tunnel syndrome, brain or spinal cord tumor, peripheral neuropathy, and Guillain-Barré syndrome; 3) a neurodegenerative disease selected from any one of Alzheimer's disease, Parkinson's disease, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), spinocerebellar ataxia, and Pick's disease; 4) a motor neuron disease selected from any one of spinal muscular atrophy (SMA), progressive bulbar palsy, progressive muscular atrophy, and primary lateral sclerosis; 5) peripheral neuropathy, nerve injury due to trauma, optic nerve disorder, polyneuritis, herpes zoster, facial nerve paralysis, burn, bedsores, corneal ulcer, side effects of radiotherapy or chemotherapy; The pharmaceutical composition comprising any one selected from the group consisting of.

5. The pharmaceutical composition according to claim 1 or 2, wherein the plasminogen increases the level of plasminogen in the target nerve tissue.

6. The pharmaceutical composition according to claim 1 or 2, wherein the plasminogen is used in combination with one or more other agents or treatment methods.

7. The pharmaceutical composition according to claim 1 or 2, wherein the plasminogen is administered by intravenous, intramuscular, intrathecal, nasal inhalation, aerosol inhalation, nasal drops, or eye drops.

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