Methods for treating nerve injury and related disorders

Abstract

The present invention relates to methods for treating neuronal injury and related disorders, comprising administering to a subject a therapeutically effective amount of a component of the plasminogen activation pathway. The present invention also relates to medicaments, pharmaceutical compositions, products and kits for treating the above disorders, comprising a component of the plasminogen activation pathway.

Description

[Technical field]

[0001] The present invention relates to a method for treating nerve injury and related disorders, such as paralysis, comprising administering to a subject an effective amount of a component of the plasminogen activation pathway or a related compound, such as plasminogen, to repair damaged nerves and ameliorate clinical symptoms and signs. [Background technology]

[0002] The nervous system includes the central nervous system (brain, spinal cord) and the peripheral nervous system (peripheral nerve tissue). Injuries to the nervous system can be caused by a variety of factors, including (1) physical injuries that directly damage nerve tissue at the site of injury, such as traumatic brain nerve tissue damage or spinal cord injury, (2) temporary or permanent ischemia or hypoxia of parts of the nervous system, such as brain nerve tissue damage caused by stroke or cerebral embolism, (3) exposure to neurotoxins, such as chemicals used to treat cancer, and (4) chronic metabolic diseases, such as peripheral nerve damage caused by diabetes or renal dysfunction.

[0003] Spinal nerve injury is a general term for pathological conditions in which the structure and function of the spinal nerves are destroyed or impaired by trauma or disease. For example, spinal cord trauma, spinal compression fracture, transverse myelitis, spinal tumor, vertebral tuberculosis, and syringomyelia can cause spinal cord injury and paralysis. Paraplegia caused by transverse lesions at or above the cervical vertebrae is severe paraplegia, while paraplegia caused by spinal cord injury below the third thoracic vertebra is paraplegia of both lower limbs. In the acute phase of spinal cord injury, both limbs below the injured level lose sensation, movement, and reflexes, and the bladder and anal sphincter functions are lost, causing spinal shock. Currently, there is no ideal treatment for nerve injuries, including spinal cord injury. Summary of the Invention

[0004] Through research, the present invention has discovered that plasminogen can obviously promote the repair of nerve damage, promote the regeneration of damaged nerve myelin, and improve the associated symptoms.

[0005] The present invention relates to the following.

[0006] 1. A method for treating neuronal injury comprising administering to a subject a therapeutically effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating an upstream component of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

[0007] 2. The method of claim 1, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA, and uPA.

[0008] 3. The method of claim 1, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, alpha 2 antiplasmin or alpha 2 macroglobulin, e.g., an antibody.

[0009] 4. The method according to any one of claims 1 to 3, wherein the nerve injury comprises a spinal nerve injury, in particular a compressive spinal nerve injury.

[0010] In some embodiments, the nerve injury is a central nerve injury (eg, spinal nerve injury) or a peripheral nerve injury.

[0011] 5. The method of claim 4, wherein the spinal nerve injury includes spinal nerve injury due to spinal compression fracture, transverse myelitis, spinal tumor, spinal tuberculosis or syringomyelia.

[0012] 6. The method according to any one of claims 1 to 5, including by treating disorders associated with nerve damage.

[0013] 7. The method of claim 6, wherein the associated damage from the nerve damage includes paralysis.

[0014] 8. The method according to claim 7, wherein the paralysis is severe paraplegia or paraplegia of both lower limbs.

[0015] 9. The method of any one of claims 1 to 8, wherein the compound promotes repair of damaged nerves.

[0016] 10. The method according to any one of claims 1 to 9, wherein the compound promotes regeneration of nerve myelin.

[0017] 11. The method of any one of claims 1 to 10, wherein the compound promotes expression of spinal neurofilament protein.

[0018] 12. The method of any one of claims 1 to 11, wherein the compound promotes recovery of sensory nerve function.

[0019] 13. The method of claim 12, wherein the compound promotes analgesia.

[0020] 14. The method according to any one of claims 1 to 13, wherein the compound is plasminogen.

[0021] In some embodiments, the present application relates to one or more activities or functions of plasminogen selected from the group consisting of promoting regeneration of nerve myelin, promoting expression of spinal neurofilament protein and regeneration of nerve fibers, promoting recovery of sensory nerve function or recovery of motor nerve function, promoting pain relief, promoting expression of synaptophysin, and promoting recovery of damaged neuronal Nissl body levels.

[0022] 15. In some embodiments, the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2 and still has plasminogen activity (e.g., proteolytic activity or lysine-binding activity).

[0023] In some embodiments, the plasminogen is a protein that includes a plasminogen active fragment and still has plasminogen activity (e.g., proteolytic activity or lysine binding activity). In some embodiments, the plasminogen active fragment has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the plasminogen active fragment shown in SEQ ID NO: 14, or has 100% amino acid sequence identity to the plasminogen active fragment shown in SEQ ID NO: 14.

[0024] 17. The method according to any one of claims 1 to 14, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen or a mutant thereof that retains plasminogen activity (e.g., proteolytic activity or lysine-binding activity).

[0025] 18. The method according to any one of claims 1 to 14, wherein the plasminogen is natural or synthetic full-length human plasminogen, or a variant or fragment that still retains plasminogen activity (e.g., proteolytic activity, lysine-binding activity).

[0026] In any one of the above embodiments of the present application, the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12 and still have plasminogen activity (e.g., proteolytic activity or lysine-binding activity). In some embodiments, the plasminogen is a protein in which 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is added, deleted, and / or substituted based on sequence 2, 6, 8, 10 or 12 and still have plasminogen activity (e.g., proteolytic activity or lysine-binding activity), e.g., proteolytic activity. In some embodiments, the plasminogen comprises or has the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12.

[0027] In some embodiments, the plasminogen is a protein that includes plasminogen fragments and still has plasminogen activity, such as proteolytic activity, or lysine-binding activity, or proteolytic activity and lysine-binding activity. In some embodiments, the plasminogen is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, δ-plasminogen, or a variant thereof that retains plasminogen activity (e.g., proteolytic activity or lysine-binding activity). In the above embodiments, the plasminogen is natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity (e.g., proteolytic activity or lysine-binding activity). In some embodiments, the plasminogen is an orthologue of human plasminogen from a primate or rodent, or a variant or fragment thereof that still retains plasminogen activity. In some embodiments, the amino acid of the plasminogen is set forth in sequence 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human native plasminogen.

[0028] In some embodiments, the subject is a human. In some embodiments, the subject is deficient or lacking in plasminogen. In some embodiments, the deficient or lacking is congenital, inherited and / or localized.

[0029] In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier and plasminogen for use in the aforementioned methods. In some embodiments, the kit may be a prophylactic or therapeutic kit comprising (i) plasminogen for use in the aforementioned methods, and (ii) means for delivering the plasminogen to the subject. In some embodiments, the means is a syringe or a vial. In some embodiments, the kit further comprises a label or a protocol for instructing administration of the plasminogen to the subject to perform any of the aforementioned methods.

[0030] In some embodiments, the article of manufacture comprises a container containing a label and (i) plasminogen or a pharmaceutical composition containing plasminogen for use in the aforementioned methods, the label providing instructions for administering the plasminogen or composition to the subject to perform any of the aforementioned methods.

[0031] In some embodiments, the kit or article of manufacture further comprises one or more additional components or containers that contain other agents.

[0032] In some embodiments of the method, the plasminogen is administered systemically or locally, preferably by intravenous, intramuscular, subcutaneous or intrathecal administration of plasminogen. In some embodiments of the method, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In some embodiments of the method, the plasminogen is administered at a daily dose of 0.0001-2000 mg / kg, 0.001-800 mg / kg, 0.01-600 mg / kg, 0.1-400 mg / kg, 1-200 mg / kg, 1-100 mg / kg, 10-100 mg / kg (calculated per kilogram of body weight), or 0.0001-2000 mg / cm 2 , 0.001-800mg / cm 2 , 0.01-600mg / cm 2 , 0.1-400mg / cm 2 , 1-200mg / cm2 , 1-100mg / cm 2 , 10-100mg / cm 2 (calculated per square centimeter of body surface area) and is preferably administered one or more times, preferably at least daily.

[0033] The present invention expressly covers all combinations of the technical features belonging to the embodiments of the present invention, and the technical configurations after the combinations are expressly disclosed in this application as if the above technical configurations were separately expressly disclosed. Moreover, the present invention also expressly covers the combinations between each embodiment and their elements, and the technical configurations after the combinations are expressly disclosed in this specification. [Brief description of the drawings]

[0034] [Figure 1] Figure 1A-C are representative images of H&E staining of the spinal cord after 7 days of plasminogen administration to spinal cord compression injury model mice. A is the sham operation group, B is the solvent PBS administration control group, and C is the plasminogen administration group. The results showed that the neurons and nerve fibers in the sham operation group were all normal in shape and the cytoplasm was stained red. When the junctions of normal and injured nerves were collected in the solvent PBS administration control group and the plasminogen administration group, a large injury cavity (triangle mark) was observed at the junction in the solvent PBS administration control group, whereas the degree of nerve repair at the injury site in the plasminogen administration group was significantly better than that in the solvent PBS administration control group, and no obvious cavity was observed. This indicates that plasminogen can clearly promote the repair of spinal cord injury nerves in spinal cord compression injury model mice. [Diagram 2]Figure 2A-C are representative images of LFB staining of spinal cords after administration of plasminogen for 7 days to spinal cord compression injury model mice. A is the sham operation group, B is the solvent PBS-administered control group, and C is the plasminogen-administered group. The results showed that the structure of nerve myelin in the sham operation group (Figure 2A) was complete and continuous, while the nerve myelin in the solvent PBS-administered control group (Figure 2B) showed obviously larger collapsed areas (marked by arrows), with a paler blue color and broken myelin. Compared with the solvent PBS control group, the myelin structure in the plasminogen-administered group (Figure 2C) was significantly more complete and darker blue. This indicates that plasminogen can promote myelin regeneration in spinal cord compression injury model mice. [Diagram 3] Figure 3A-C are representative images of spinal cord neurofilament protein (NFP) staining after plasminogen administration for 7 days to spinal cord compression injury model mice. A is the sham-operated group, B is the solvent PBS-administered control group, and C is the plasminogen-administered group. As a result, the expression of NFP (marked by arrow) in the plasminogen-administered group was significantly higher than that in the solvent PBS-administered control group, and the expression of NFP was closer to that of the sham-operated group mice. This indicates that plasminogen can promote the expression of NFP and promote the regeneration of spinal nerve fibers in spinal cord compression injury model mice. [Figure 4] Figure 4A-B are representative images of H&E staining of the spinal cord after 7 days of plasminogen administration to the spinal cord compression injury model Plg- / - mice. A is the solvent PBS-administered control group, and B is the plasminogen-administered group. As a result, when the junctions of normal and injured nerves were collected from the solvent PBS-administered control group and the plasminogen-administered group, the normal neuron and nerve fiber structure of both were lost at the injury site and replaced by repaired fibers, the cytoplasm was lightly stained, and inflammatory cells were seen to have infiltrated. In the solvent PBS-administered control group, a large injury cavity (triangle mark) was observed at the junction, whereas the degree of repair of the injury site in the plasminogen-administered group was significantly better than that of the solvent PBS-administered control group, no obvious cavity was observed, and the infiltration of inflammatory cells was also significantly reduced. This indicates that plasminogen can clearly promote the repair of spinal cord injury in the spinal cord compression injury model Plg- / - mice. [Diagram 5] Figure 5A-B are representative images of LFB staining of spinal cords after plasminogen administration for 7 days to spinal cord compression injury model Plg- / - mice. A is the solvent PBS-administered control group, and B is the plasminogen-administered group. As a result, the nerve myelin in the solvent PBS-administered control group clearly showed larger collapsed areas (marked with triangles), lighter blue color, and broken myelin (marked with arrows). In comparison, the myelin structure in the plasminogen-administered group was obviously complete and continuous, and darker blue color. This indicates that plasminogen can promote the repair of myelin damage in spinal cord compression nerve injury model Plg- / - mice. [Figure 6] Figure 6 shows the results of the Von-Frey mechanical pain threshold test after plasminogen was administered to spinal cord compression injury model C57 mice for 6 days. As a result, the mechanical threshold of the mice in the vehicle-administered group was significantly increased compared with that of the mice in the sham-operated group. This indicates that the mice lost their sense of pain after the establishment of the spinal cord compression model. The pain thresholds of the mice in the administration group A (1 mg) and administration group B (0.5 mg) were significantly lower than those of the vehicle group, and the difference was statistically significant (* represents P<0.05, ** represents P<0.01). These results indicate that plasminogen can promote pain recovery in spinal cord compression model mice. [Figure 7] 7A-D are diagrams showing the results of immunohistochemical staining of spinal cord NFP in spinal cord transection model mice 28 days after plasminogen administration. A is the sham operation group, B is the control group administered with solvent PBS (also referred to as the solvent group, the same below), C is the plasminogen administration group (also referred to as the administration group, the same below), and D is the result of quantitative analysis of the average optical density. As a result, the spinal cord of the mice in the sham operation group expressed a certain level of NFP (marked with an arrow), the expression of NFP in the spinal cord at the modeling site of the mice in the solvent group was significantly lower than that of the mice in the sham operation group, and the expression of NFP in the spinal cord at the modeling site of the mice in the administration group was significantly higher than that of the solvent group, and the difference was statistically significant (* represents P<0.05). This indicates that plasminogen can promote the expression of NFP in the spinal cord of the spinal cord transection model mice. [Figure 8]FIG. 8 shows the results of the BMS score of the spinal cord transection model mice. As a result, the BMS score of the mice in the sham operation group was 9 points, and the BMS score of the mice in the vehicle group was 6.75±1.92 points, which was significantly lower than that of the mice in the sham operation group. The BMS score of the mice in the treatment group was 8.29±0.39 points, which was significantly higher than that of the mice in the vehicle group, and the difference was statistically significant (* indicates P<0.05, *** indicates P<0.001). This indicates that plasminogen can promote the recovery of hind limb motor function in paraplegic model mice. [Figure 9] Figure 9 shows the results of plasminogen mRNA RT-PCR detection in the injured spinal cord of spinal cord transection model mice 6 hours after plasminogen administration. The results showed that there was a certain level of plasminogen mRNA in the spinal cord of the blank control group mice, and the level of plasminogen mRNA in the spinal cord of the treatment group mice was significantly higher than that of the vehicle group, and the difference was statistically almost significant (P=0.051). This indicates that plasminogen can promote the transcription of plasminogen gene in the spinal cord of paraplegic model mice. [Figure 10] Figure 10A-D shows the results of immunohistochemical staining of spinal cord synaptophysin in spinal cord transection model mice 28 days after plasminogen administration. A is the sham operation group, B is the vehicle group, C is the treatment group, and D is the result of quantitative analysis of the average optical density. As a result, the spinal cord of the mice in the sham operation group expressed a certain level of synaptophysin (marked with an arrow), and the level of synaptophysin in the spinal cord at the modeling site of the mice in the vehicle group was significantly lower than that of the mice in the sham operation group, and the statistical difference between the two groups was highly significant (*** represents P<0.001). The level of synaptophysin in the spinal cord at the modeling site of the mice in the treatment group was significantly higher than that of the vehicle group, and the difference was statistically significant (* means P<0.05). This indicates that plasminogen can promote the expression of synaptophysin in the injured spinal cord of paraplegic model mice and promote the repair of spinal cord injury. [Figure 11]Figure 11A-D shows the results of Nissl staining of the spinal cord in spinal cord transection model mice 28 days after plasminogen administration. As a result, there was a certain level of Nissl bodies in the spinal cord of mice in the sham operation group (marked with arrows), and the level of Nissl bodies in the spinal cord at the modeling site of mice in the vehicle group was significantly higher than that of mice in the sham operation group, and the statistical difference between the two groups was highly significant (*** represents P<0.001). The level of Nissl bodies in the spinal cord at the modeling site of mice in the treatment group was significantly lower than that of the vehicle group, and the statistical difference was highly significant (** represents P<0.01). This indicates that plasminogen can promote the recovery of Nissl body levels in the damaged spinal cord of paraplegic model mice and promote the repair of spinal cord injury. Detailed Description of the Invention

[0035] The fibrinolytic system, also called the fibrinolytic system, is a system of a series of chemicals involved in the process of fibrinolysis (fibrinolysis), mainly including plasminogen, plasmin, plasminogen activators, and fibrinolysis inhibitors. Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA is a serine protease and is synthesized by vascular endothelial cells. t-PA activates plasminogen, a process that mainly occurs in 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 clots, PLG is adsorbed in large quantities to the fibrin net and activated to plasmin by the action of t-PA or u-PA to promote fibrinolysis. Plasminase (PL) is a serine protease that degrades fibrin and fibrinogen, hydrolyzes various coagulation factors V, VIII, X, VII, XI, II, etc., converts plasminogen to plasmin, hydrolyzes complement, etc. Fibrinolysis inhibitors include plasminogen activator inhibitor (PAI) and α2-antithyplasmin (α2-AP). There are two main forms of PAI, PAI-1 and PAI-2, which can specifically bind to t-PA in a 1:1 ratio, thereby inactivating it and activating PLG at the same time. α2-AP is synthesized in the liver and binds to PL in a 1:1 ratio to form a complex, thereby inhibiting PL activity. FXIII covalently binds α2-AP to fibrin, thereby weakening the susceptibility of fibrin to PL. Substances that inhibit the activity of the fibrinolysis system in vivo include PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin.

[0036] As used herein, the term "a component of the plasminogen activation pathway" refers to a 1. Plasminogen, Lys-plasminogen, Glu-plasminogen, micro-plasminogen, delta-plasminogen, their variants or analogs; 2. Plasmin and its variants or analogues; and 3. Plasminogen activators, including, for example, tPA and uPA, as well as tPA or uPA variants and analogs that contain one or more domains of tPA or uPA, such as one or more of the kringle domain and the proteolytic domain.

[0037] The above "variants" of plasminogen, plasmin, tPA and uPA include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as proteins having, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid added, deleted and / or substituted and still having plasminogen activity, plasmin activity, tPA or uPA activity. For example, "variants" of plasminogen activity, plasmin activity, tPA or uPA include mutants of these proteins resulting from substitution of, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative amino acids.

[0038] "Plasminogen variants" according to the present invention cover proteins having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequences 2, 6, 8, 10 or 12 and still having plasminogen activity (e.g. proteolytic activity, or lysine-binding activity, or proteolytic activity and lysine-binding activity). For example, a "plasminogen variant" of the present invention can be a protein in which 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid has been added, deleted and / or substituted based on sequence 2, 6, 8, 10 or 12, and still has plasminogen activity (e.g., proteolytic activity, or lysine-binding activity, or proteolytic activity and lysine-binding activity). In particular, the plasminogen variants of the present invention include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as mutants of these proteins obtained by conservative substitution of, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids.

[0039] The plasminogen of the present invention can be an orthologue of human plasminogen derived from a primate or rodent, or a variant that still retains plasminogen activity (e.g., proteolytic activity, or lysine-binding activity, or proteolytic activity and lysine-binding activity), such as the plasminogen shown in sequences 2, 6, 8, 10 or 12, e.g., human native plasminogen shown in sequence 2.

[0040] The above "analogs" of plasminogen, plasmin, tPA and uPA include compounds that provide substantially the same effects as plasminogen, plasmin, tPA or uPA, respectively.

[0041] The above "variants" and "analogs" of plasminogen, plasmin, tPA and uPA cover "variants" and "analogs" of plasminogen, plasmin, tPA and uPA that contain one or more domains (e.g., one or more kringle domains and a proteolytic domain). For example, "variants" and "analogs" of plasminogen cover plasminogen variants and analogs, such as mini-plasminogen, that contain one or more plasmin domains (e.g., one or more kringle domains and a proteolytic domain). "Variants" and "analogs" of plasmin cover plasmin "variants" and "analogs" such as mini-plasmin and delta-plasmin that contain one or more plasmin domains (e.g., one or more kringle domains and a proteolytic domain).

[0042] Whether the above-mentioned "variants" or "analogs" of plasminogen, plasmin, tPA or uPA have the activity of plasminogen, plasmin, tPA or uPA, respectively, or whether they provide substantially similar effects as plasminogen, plasmin, tPA or uPA, respectively, can be measured by the level of activated plasmin activity using methods known in the art, such as enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS (fluorescence-activated cell sorting).For example, Ny, A., Leonardsson, G., Hagglund, AC, Hagglof, P., Ploplis, VA, Carmeliet, P. and Ny, T. (1999). 1984). “Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator”. J. Clin. Invest. 74 (5): 1625-33; Gravanis I, Tsirka SE (February 2008). “Tissue-type plasminogen activator as a therapeutic target in stroke”. Expert Opinion on Therapeutic Targets. 12 (2): 159-70;Geiger M, Huber The measurement can be performed by referring to the methods described in the literature selected from 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.

[0043] In some embodiments of the present invention, the "component of the plasminogen activation pathway" of the present invention is plasminogen, which is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, δ-plasminogen or a variant thereof that retains plasminogen activity (e.g., proteolytic activity, or lysine-binding activity, or proteolytic activity and lysine-binding activity). In some embodiments, the plasminogen is natural or synthetic human plasminogen, or a conservative mutant or fragment thereof that still retains plasminogen activity. In some embodiments, the plasminogen is an orthologue of human plasminogen from a primate or rodent, or a conservative mutant or fragment thereof that still retains plasminogen activity. In some embodiments, the amino acid sequence of the plasminogen is shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is human native plasminogen. In some embodiments, the plasminogen is human native plasminogen shown in sequence 2.

[0044] "A compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway" refers to any compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway, including, for example, tPA, uPA, streptokinase, saruplase, alteplase, reteplase, tenecteplase, anistreplase, monteplase, lanoteplase, pamiteplase, and staphylokinase.

[0045] The "antagonist of fibrinolysis inhibitors" of the present invention is a compound that antagonizes, weakens, blocks, or prevents the action of fibrinolysis inhibitors, such as PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin. Said antagonists are antibodies to PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or for example antisense RNA or miniRNA that blocks or downregulates the expression of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that occupy the binding site of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin but do not have the function of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that block the binding and / or activity domains of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0046] 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 proteoglycans. Plasmin can also activate some pro-matrix metalloproteinases (pro-MMPs) to become active matrix metalloproteinases (MMPs). Thus, plasmin is an important upstream regulator of extracellular proteolysis. Plasmin is formed by proteolysis of two physiological PAs: tissue-type plasminogen activator (tPA) or urokinase plasminogen activator (uPA). Plasminogen is present at relatively high relative levels in plasma and other body fluids, and traditionally, it has been believed that the regulation of the PA system is mainly achieved by the synthesis and activity levels of PA. The synthesis of the components of the PA system is tightly 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), which mainly inhibits uPA. Some cells have a uPA-specific cell surface receptor (uPAR) with direct hydrolytic activity.

[0047] Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. It is synthesized mainly in the liver and is present in large amounts in extracellular fluids. The plasminogen content in plasma is approximately 2 μM. Thus, plasminogen is a large potential source of proteolytic activity in tissues and body fluids. There are two molecular forms of plasminogen: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). The naturally secreted and undegraded form of plasminogen has one amino-terminal (N-terminal) glutamic acid and is therefore called glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys77. Compared to glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PA at a higher rate. The Arg560-Val561 peptide bond of the two forms of plasminogen is cleaved by uPA or tPA, leading to the formation of the double-chain protease plasmin connected by disulfide bonds. The amino-terminal part of plasminogen contains five homologous tricycles, the so-called kringles, and the carboxyl-terminal part contains the protease domain. Some kringles contain a lysine-binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor α2-AP. The most recently discovered fibrin-plasminogen fragment of 38 kDa, kringlel-4, is a potent inhibitor of angiogenesis. This fragment, named angiostatin, is generated from several protease-hydrolyzed plasminogens.

[0048] The main substrate of plasmin is fibrin, and fibrin lysis is a key point to prevent the formation of pathological thrombi. Plasmin also has substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycan, and gelatin, indicating that plasmin plays an important role in ECM reconstruction. Indirectly, plasmin also degrades other components of the ECM by converting some protease precursors, including MMP-1, MMP-2, MMP-3, and MMP-9, into active proteases. Therefore, some have proposed that: Plasmin is an important upstream regulator of extracellular protein hydrolysis; Plasmin also has the ability to activate some latent forms of growth factors; In vitro, plasmin can also hydrolyze components of the complement system to release chemotactic complement fragments.

[0049] "Plasmin" is a very important enzyme present in blood, which hydrolyzes fibrin clots into fibrin degradation products and D-dimers.

[0050] "Plasminogen" is the proenzyme form of plasmin, which, based on the sequence in swiss prot, is calculated as the amino acid sequence of natural human plasminogen including the signal peptide (Sequence 4), consisting of 810 amino acids, has a molecular weight of about 90 kD, is synthesized mainly in the liver, and is a glycoprotein that can circulate in the blood, and the cDNA sequence encoding said amino acid sequence is as shown in Sequence 3. Full-sized 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). With reference to the sequences in swiss prot, the signal peptide comprises residues Met1-Gly19, Pap comprises residues Glu20-Val98, Kringle1 comprises residues Cys103-Cys181, Kringle2 comprises residues Glu184-Cys262, Kringle3 comprises residues Cys275-Cys352, Kringle4 comprises residues Cys377-Cys454, and Kringle5 comprises residues Cys481-Cys560. According to NCBI data, the serine protease domain comprises residues Val581-Arg804.

[0051] Glu-plasminogen is a natural full-sized human plasminogen, and consists of 791 amino acids (not including a signal peptide consisting of 19 amino acids). The cDNA sequence encoding this sequence is as shown in sequence 1, and its amino acid sequence is as shown in sequence 2. In the body, Lys-plasminogen is further formed by hydrolysis at the 76th-77th amino acid positions of Glu-plasminogen, and is, for example, as shown in sequence 6, and the cDNA sequence encoding this amino acid sequence is as shown in sequence 5. Delta-plasminogen (δ-plasminogen) is a fragment of full-sized plasminogen lacking the Kringle2-Kringle5 structure, and contains only Kringle1 and a serine protease domain (also called the protease domain (PD)). There is a literature that reports the amino acid sequence of δ-plasminogen (sequence 8), and the cDNA sequence encoding this amino acid sequence is, for example, sequence 7. Mini-plasminogen consists of Kringle5 and serine protease domains, and the literature reports residues Val443-Asn791 (starting with the Glu residue of the Glu-plasminogen sequence without the signal peptide), and its amino acid sequence is as shown in sequence 10, and the cDNA sequence encoding this amino acid sequence is as shown in sequence 9. However, the literature reports that microplasminogen only contains a serine protease domain, and its amino acid sequence is residues Ala543-Asn791 (the Glu residue in the Glu-plasminogen sequence without the signal peptide is the starting amino acid), and patent document CN102154253A discloses that its sequence contains residues Lys531-Asn791 (the Glu residue in the Glu-plasminogen sequence without the signal peptide is the starting amino acid). In this patent application, the sequence of microplasminogen can be referred to patent document CN102154253A, and its amino acid sequence is as shown in sequence 12, and the cDNA sequence encoding the amino acid sequence is as shown in sequence 11.

[0052] The structure of full-length plasminogen is also described in a paper by Aisina et al. (Aisina RB, Mukhametova L I. Structure and function of plasminogen / plasmin system[J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6):590-605). According to the above-mentioned article by Aisina et al., plasminogen contains Kringle 1, 2, 3, 4, 5 domains and a serine protease domain (also called protease domain (PD)), and Kringles are responsible for binding plasminogen to low and high molecular weight ligands, thereby converting plasminogen into a more open configuration and making it more accessible to activation. The protease domain (PD) is located at residues Val562-Asn791, and tPA and UPA specifically cleave the Arg561-Val562 activating bond of plasminogen, thereby allowing plasminogen to form plasmin.

[0053] In the present invention, "plasmin", "fibrin plasmin", and "fibrous protein plasmin" can be used interchangeably and have the same meaning. "Plasminogen", "fibrin plasminogen", and "fibrous protein plasminogen" can be used interchangeably and have the same meaning.

[0054] In this application, the "deficiency" of plasminogen refers to the content or activity of plasminogen in the subject's body being lower than that of a normal person and being sufficiently low to affect the normal physiological function of the subject. The "deficiency" of plasminogen means that the content or activity of plasminogen in the subject's body is obviously lower than that of a normal person, the activity or expression is extremely low, and the normal physiological function can only be maintained by external supply.

[0055] Those skilled in the art can understand as follows. All technical configurations of plasminogen in the present invention can be applied to plasmin, so the technical configurations described in the present invention cover plasminogen and plasmin. In the circulation process, plasminogen is in a closed inactive conformation, but when it binds to a thrombus or cell surface, it becomes active plasmin with an open conformation under the mediation of a plasminogen activator (PA). The active plasmin further hydrolyzes fibrin clots into fibrin degradation products and D-dimers, thereby dissolving the thrombus. Among them, the PAp domain of plasminogen is an important epitope that makes plasminogen in an inactive closed conformation, but the KR domain can bind to lysine residues on receptors and substrates. There are already several types of enzymes known as plasminogen activators, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hageman factor), etc.

[0056] "Plasminogen active fragment" refers to an active fragment that binds to lysine in the target sequence of a substrate (lysine binding activity), or an active fragment that exerts a proteolytic function (proteolytic activity), or a fragment that has both proteolytic activity and lysine binding activity. The plasminogen technical configuration of the present invention encompasses technical configurations that replace plasminogen with an active fragment of plasminogen. In some embodiments, the plasminogen active fragment of the present invention comprises or consists of the serine protease domain of plasminogen, and preferably, the plasminogen active fragment of the present invention comprises sequence 14, or comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with sequence 14, or consists of an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with sequence 14. In some embodiments, the plasminogen active fragments of the present invention comprise one or more domains selected from Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5, or consist of one or more domains selected from Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5. In some embodiments, the plasminogen of the present invention comprises a protein comprising an active fragment of plasminogen as described above.

[0057] At present, the methods for measuring plasminogen and its activity in blood include tissue plasminogen activator activity (t-PAA), plasma tissue plasminogen activator antigen (t-PAAg), plasma tissue plasminogen activity (plgA), plasma tissue plasminogen antigen (plgAg), plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator inhibitor antigen, and plasma plasmin-antiplasmin complex (PAP). The most common method is the chromogenic substrate method: streptokinase (SK) and luminescent substrate are added to the plasma of the subject, and the PLG in the subject's plasma becomes PLM under the action of SK, which acts on the luminescent substrate, and then measured by a spectrophotometer, and the increase in absorbance is directly proportional to the activity of plasminogen. In addition, immunochemical methods, gel electrophoresis, immunoturbidimetry, radial immunodiffusion, etc. can also be used to measure plasminogen activity in blood.

[0058] "Ortholog" refers to a homologue between different species, including both protein and DNA homologues. It specifically refers to a protein or gene that is evolved from the same ancestral gene between different species. The plasminogen of the present invention includes human native plasminogen, and further includes plasminogen orthologs having plasminogen activity derived from different species.

[0059] A "conservative substitution variant" is one in which a specified amino acid residue is modified but does not change the overall conformation and function of a protein or enzyme, including but not limited to those that substitute an amino acid in the amino acid sequence of a parent protein with an amino acid of similar properties (e.g., acidic, alkaline, hydrophobic, etc.). Amino acids with similar properties are known. For example, arginine, histidine, and lysine are hydrophilic alkaline amino acids and can be substituted for each other. Similarly, isoleucine is a hydrophobic amino acid and can be substituted by leucine, methionine, or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may vary. For example, they have a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variant" further includes polypeptides or enzymes with an amino acid identity of 60% or more based on the BLAST or FASTA algorithm, preferably 75% or more, most preferably 85% or more, and most preferably 90% or more, and have the same or essentially similar properties or functions compared to the natural or parent protein or enzyme.

[0060] "Isolated" plasminogen is plasminogen protein that has been separated and / or recovered from its natural environment. In some embodiments, the plasminogen is (1) purified to greater than 90%, greater than 95%, or greater than 98% purity (by weight), e.g., greater than 99% (by weight), as determined by the Lowry method; (2) purified to at least 15 residues of N-terminal or internal amino acid sequence as determined by a spinning cup sequencer; or (3) purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions with Coomassie brilliant blue or silver staining. Isolated plasminogen includes plasminogen produced from recombinant cells by bioengineering techniques and separated by at least one further purification step.

[0061] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having modified peptide backbones. The terms also include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions containing leader sequences of heterologous and homologous origin (with or without an N-terminal methionine residue); and the like.

[0062] The definition of "percentage (%) amino acid sequence identity" of a reference peptide sequence is the percentage of amino acid residues in a candidate sequence that are the same as those in the reference polypeptide sequence, after introducing gaps as necessary to achieve the maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Comparisons aimed at determining percentage amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example by publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm that achieves maximum comparison requirements for the full size of the sequences to be compared. However, for the purposes of the present invention, the percentage amino acid sequence identity is obtained by the sequence comparison computer software ALIGN-2.

[0063] When comparing amino acid sequences by using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (also referred to as a given amino acid sequence A that has or contains identity to, with, or with an amino acid sequence for a given amino acid sequence B) is calculated as follows: Fraction X / Y×100

[0064] where X is the number of amino acid residues that the sequence alignment program ALIGN-2 evaluates as identical and matching in its alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood as follows: if the length of amino acid sequence A and the length of amino acid sequence B are not equal, then the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise stated, all % amino acid sequence identity values ​​used in this text are as described in the previous paragraph and are obtained by the ALIGN-2 computer program.

[0065] As used herein, the terms "treatment" and "treating" refer to obtaining a desired pharmacological and / or physiological effect. The effect may be to completely or partially prevent a disease or its symptoms, and / or to partially or completely cure a disease and / or its symptoms. It further includes: (a) preventing a disease from occurring in a subject, where the subject has the causative agent for the disease but has not been diagnosed with the disease; (b) inhibiting the disease, inhibiting its formation; and (c) mitigating a disease and / or its symptoms, i.e., reducing the disease and / or its symptoms.

[0066] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0067] A "therapeutically effective amount" or "effective amount" is an amount of plasminogen that, when administered to a mammal or other subject for use in treating a disease, is capable of achieving said prevention and / or treatment of the disease. The "therapeutically effective amount" will vary according to the plasminogen used, the severity of the disease and / or symptoms, and the age, weight, etc., of the subject to be treated.

[0068] Preparation of the Plasminogen of the Invention For therapeutic use, plasminogen may be isolated and purified from nature or may be synthesized by standard chemical peptide synthesis techniques. When polypeptides are synthesized by chemical methods, the synthesis may be performed in liquid phase or solid phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support and the remaining amino acids in the sequence are added in order, is suitable for the chemical synthesis of plasminogen. Various forms of SPPS, e.g., Fmoc and Boc, can be used to synthesize plasminogen. The techniques used for solid-phase synthesis are described in: Barany and Solid-Phase Peptide Synthesis; pages 3-284, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6: 3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12: 723-8. Briefly, insoluble porous beads are treated with functional units on which the peptide chain is built. After repeated coupling / deprotection cycles, the free N-terminal amine of the attached solid phase is coupled with a single N-protected amino acid unit. The unit is then deprotected, revealing a new N-terminal amine that is attached to another amino acid. The peptide remains immobilized on the solid phase and is then excised.

[0069] The plasminogen of the present invention is produced by standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector and operably linked to control sequences in the expression vector. Expression control sequences include, but are not limited to, a promoter (e.g., a naturally associated promoter or a heterologous promoter), a signal sequence, an enhancer element, and a transcription termination sequence. Control of expression can be a eukaryotic promoter system in a vector, which is transformed or transfected into 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 for collection and purification of the plasminogen.

[0070] Suitable expression vectors are usually replicated within the host as an adduct or integral part of the host chromosomal DNA. Usually, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) which is useful for detecting those cells which have been transformed in vitro with the desired DNA sequence.

[0071] Escherichia coli is an example of a prokaryotic host cell in which a polynucleotide encoding a target antibody may be cloned. Other suitable microbial hosts include bacilli, such as Bacillus subtilis and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can be generated, which will usually contain expression control sequences (e.g., an origin of replication) that are 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 a promoter system derived from phage lambda. Promoters generally control expression and may further contain ribosome binding site sequences to initiate transcription and translation when controlling gene sequences, if necessary.

[0072] Other microorganisms, such as yeast, can also be used for expression. Yeast (e.g., Saccharomyces (S. cerevisiae) and Pichia are examples of suitable yeast host cells, in which suitable carriers include expression control sequences (e.g., promoters), origins of replication, termination sequences, etc., as necessary. Exemplary promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, promoters for alcohol dehydrogenase, isocytochrome C, and enzymes for maltose and galactose utilization.

[0073] In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in in vitro cell culture) can also be used to express and produce the anti-Tau antibodies of the present invention (e.g., polynucleotides encoding such anti-Tau antibodies). See, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for use in these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and required processing information sites, such as ribosome binding sites, RNA cleavage sites, polyadenosine oxidation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from rabbit immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0074] Once synthesized (chemically or recombinantly), the plasminogen according to the 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, e.g., at least about 80% to 85% pure, at least about 85%-90% pure, at least about 90%-95% pure, or 98%-99% pure or even more pure, e.g., free of contaminants, such as cellular debris, large molecules other than the antibody of interest, etc.

[0075] Drug combinations Plasminogen of the desired purity is mixed with pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)) as required to form a lyophilized formulation or aqueous solution to obtain the therapeutic combination. Acceptable carriers, excipients, and stabilizers are non-toxic to subjects 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., octadecyl dimethyl benzyl ammonium chloride; hexamethylenediamine chloride; benzalkonium chloride). chloride), benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parahydroxybenzoic acid esters, such as methyl or propyl parahydroxybenzoic acid ester; pyrocatechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptides (having at least 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates include glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, fucose or sorbitol; salt forming counterions, such as sodium; metal complexes (such as zinc-protein complexes); and / or non-ionic surfactants, such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). A preferred lyophilized anti-VEGF antibody formulation is described in WO97 / 04801, incorporated herein by reference.

[0076] The combination preparation of the present invention may contain one or more active compounds as required for the specific condition requiring treatment, preferably with complementary activities and no side effects to each other, such as antihypertensives, antiarrhythmics, antidiabetics, etc.

[0077] The plasminogen of the present invention can be encapsulated in microcapsules made, for example, by aggregation techniques or interfacial polymerization, can be incorporated, for example, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or can be incorporated into hydroxymethylcellulose or gel microcapsules and poly(methyl methacrylate) microcapsules in crude emulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0078] The plasminogen of the present invention to be administered internally must be sterile, which is readily accomplished by filtration through sterile filtration membranes, prior to or after lyophilization and recombination.

[0079] The plasminogen of the present invention can be prepared in a buffered formulation. Suitable examples of buffered formulations include semipermeable matrices of solid hydrophobic polymers having a defined shape and containing the glycoprotein, such as membranes or microcapsules. Examples of 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 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.

[0080] Dosage and dosage The administration of the pharmaceutical composition of the present invention can be achieved by different ways, for example, intravenously, intraperitoneally, subcutaneously, intracranially, intrathecally, intraarterially (eg, carotid artery), intramuscularly.

[0081] Preparations for use in parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolytic replenishers, and the like. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, inert gases, and the like.

[0082] A medical practitioner may determine a dosage regimen based on various clinical factors. For example, as is well known in the medical arts, the dosage regimen for any given patient is determined by several factors, including the patient's body type, body surface area, age, the specific compound being administered, sex, frequency and route of administration, overall health, and other drugs being administered at the same time. The dosage range of the plasminogen pharmaceutical composition of the present invention may be, for example, about 0.0001-2000 mg / kg of subject body weight daily, or about 0.001-500 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 10 mg / kg, 50 mg / kg, etc.). For example, the dosage may be 1 mg / kg body weight or 50 mg / kg body weight or in the range of 1-50 mg / kg, or at least 1 mg / kg. Doses higher or lower than this illustrative range are also covered, particularly when considering the above factors. Intermediate doses within the above range are also within the scope of the present invention. Subjects may be administered such doses daily, every other day, weekly, or according to any schedule determined by empirical analysis. An exemplary dose schedule is 1-10 mg / kg on consecutive days. During the administration of the drug of the present invention, it is necessary to evaluate the therapeutic effect and safety in real time.

[0083] Product or drug kit One embodiment of the present invention is directed to an article of manufacture or pharmaceutical kit, which contains the plasminogen or plasmin of the present invention for treating cardiovascular and related diseases caused by diabetes. The article of manufacture preferably includes a container, a label, or a protocol. Suitable containers are bottles, vials, syringes, and the like. The containers can be made of a variety of materials, such as glass or plastic. The container contains a composition, which is effective for treating a disease or condition of the present invention, and has a sterile inlet (e.g., the container is an intravenous infusion pack or vial, which includes a stopper that can be pierced by a hypodermic needle). At least one activator in the composition is plasminogen / plasmin. A label on or attached to the container describes the composition as being used for treating cardiovascular and related diseases caused by diabetes of the present invention. The article of manufacture may further include a second container containing a pharmaceutical buffer, such as phosphate buffered saline, Ringer's solution, and glucose solution. It may further include other materials as may be required from a commercial and user perspective, i.e., other buffers, diluents, filters, needles, and syringes. The article of manufacture also includes a protocol with instructions, for example, instructing a user of the composition to administer the plasminogen composition and other medications associated with treating a disease to a patient. EXAMPLES

[0084] The human plasminogen used in the following examples was derived from the plasma of a human donor and was prepared according to the following publications: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry, 1965, 240(1):541-550; Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J Biol Chem. 1976 Jun 25; 251(12):3693-9; HAGAN JJ, ABLONDI FB, DE RENZO EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 Based on the method described in Apr;235:1005-10, the process was optimized and purified from human donor plasma, where the human plasminogen alone was greater than 98%. Example 1

[0085] Example 1 relates to the promotion of repair of nerve damage in a mouse model of spinal cord compression injury by plasminogen. Fifteen 8-week-old C57 female mice were taken and weighed. The mice were randomly divided into three groups: sham-operated group, vehicle PBS-treated group, and plasminogen-treated group, with five mice in each group. All mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution (50 mg / kg), the midline skin of the back was incised, the dorsal T7-T9 muscles were separated, and the T7-T9 lamina was removed. For the vehicle PBS-treated control group and the plasminogen-treated group, a microvascular clip (3.5 × 1.0 mm, RWD) was clamped on the T8 spinal cord for 1 min. For the sham-operated group, only the lamina was removed to avoid dural damage. All mice were sutured immediately after surgery, injected intramuscularly with Tolfedine (0.1 mg / kg) and penicillin potassium (40,000 units / kg), and placed on a heating pad until revived. To prevent infection during surgery, mice were manually assisted to urinate after surgery until they urinated naturally. [1] The administration was started 30 minutes after the operation, and plasminogen was administered to the mice in the plasminogen-treated group at 1 mg / 0.1 ml / mouse / day via tail vein injection. The mice in the PBS control group were similarly administered the same amount of PBS, and the mice in the sham-operated group were not administered plasminogen. The day administration began was counted as day 1, and the administration cycle was 7 days. On the 8th day of administration, the mice were dissected, and the spinal cord T7-T9 segments were fixed with 4% paraformaldehyde. The fixed tissue samples were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The spinal cord tissue was cut into 4 μm-thick transverse sections, which were deparaffinized, rehydrated, stained with hematoxylin and eosin (HE staining), fractionated with 1% hydrochloric acid ethanol, blued with ammonia water, dehydrated in an alcohol gradient, mounted, and observed under an optical microscope at 200x magnification. The results showed that the neurons and nerve fibers in the sham-operated group (Figure 1A) had normal shapes and the cytoplasm was stained red. When the junctions of normal and injured nerves were collected from the PBS-treated control group (Figure 1B) and the plasminogen-treated group, a large injury cavity (triangle mark) was observed at the junction in the PBS-treated control group, whereas the degree of nerve repair at the injury site in the plasminogen-treated group (Figure 1C) was significantly better than that in the PBS-treated control group, with no obvious cavity observed. This indicates that plasminogen can obviously promote the repair of spinal cord injury nerves in spinal cord compression injury model mice. Example 2

[0086] Example 2 relates to the promotion of nerve myelin regeneration by plasminogen. Fifteen 8-week-old C57 female mice were taken and weighed. The mice were randomly divided into three groups: sham-operated group, vehicle PBS-treated group, and plasminogen-treated group, with five mice in each group. All mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution (50 mg / kg), the midline skin of the back was incised, the dorsal T7-T9 muscles were separated, and the T7-T9 lamina was removed. For the vehicle PBS-treated control group and the plasminogen-treated group, a microvascular clip (3.5 × 1.0 mm, RWD) was clamped on the T8 spinal cord for 1 min. For the sham-operated group, only the lamina was removed to avoid dural damage. All mice were sutured immediately after surgery, injected intramuscularly with Tolfedine (0.1 mg / kg) and penicillin potassium (40,000 units / kg), and placed on a heating pad until revived. To prevent infection during surgery, mice were manually assisted to urinate after surgery until they urinated naturally. [1]Administration was started 30 minutes after surgery, and plasminogen was administered to the mice in the plasminogen-treated group at 1 mg / 0.1 ml / mouse / day via tail vein injection. The mice in the vehicle PBS control group were similarly administered the same amount of PBS, and the mice in the sham-operated group were not administered plasminogen. The day administration began was counted as day 1, and the administration cycle was 7 days. On the 8th day of administration, the mice were dissected, and the spinal cord T7-T9 segments were fixed in 4% paraformaldehyde. The fixed tissue samples were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The transverse sections of the spinal cord tissue were 4 μm thick, and the sections were deparaffinized, immersed in water, and stained with LFB using a myelin staining solution. The sections were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in neutral rubber. The sections were observed under an optical microscope and photographed. LFB (loxol fast blue) staining stains myelin using the fast blue staining method. The degree of blue indicates the amount of myelin. LFB staining is an effective method for studying the localization of the corticospinal tract, and the morphological observation of myelin lesions, injury, and regeneration repair. [2,3] . The results showed that the structure of nerve myelin in the sham-operated group (Figure 2A) was intact and continuous, while the nerve myelin in the solvent PBS-treated control group (Figure 2B) showed obviously larger collapsed areas (marked with arrows), with a paler blue color and broken myelin. Compared with the solvent PBS control group, the myelin structure in the plasminogen-treated group (Figure 2C) was significantly intact and darker blue. This indicates that plasminogen can promote the regeneration of myelin in spinal cord compression injury model mice. Example 3

[0087] Example 3 relates to the fact that plasminogen promotes the expression of neurofilament proteins. Fifteen 8-week-old C57 female mice were taken and weighed. The mice were randomly divided into three groups: sham-operated group, vehicle PBS-treated group, and plasminogen-treated group, with five mice in each group. All mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution (50 mg / kg), the midline skin of the back was incised, the dorsal T7-T9 muscles were separated, and the T7-T9 lamina was removed. For the vehicle PBS-treated control group and the plasminogen-treated group, a microvascular clip (3.5 × 1.0 mm, RWD) was clamped on the T8 spinal cord for 1 min. For the sham-operated group, only the lamina was removed to avoid dural damage. All mice were sutured immediately after surgery, injected intramuscularly with Tolfedine (0.1 mg / kg) and penicillin potassium (40,000 units / kg), and placed on a heating pad until revived. To prevent infection during surgery, mice were manually assisted in urinating after surgery until spontaneous urination. Administration began 30 minutes after surgery, and plasminogen was administered to mice in the plasminogen-treated group at 1 mg / 0.1 ml / mouse / day via tail vein injection. The same amount of PBS was administered to mice in the PBS vehicle control group, and no administration was given to mice in the sham-operated group. The day administration began was counted as day 1, and the administration cycle was 7 days. On the 8th day of administration, mice were dissected, and the spinal cord T7-T9 was fixed in 4% paraformaldehyde. The fixed tissue samples were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The spinal cord tissue was cut into transverse sections with a thickness of 3 μm, and the sections were deparaffinized, rehydrated, and washed once with water. The sections were fixed in citric acid for 30 minutes, cooled at room temperature for 10 minutes, and gently rinsed with water. The tissue was incubated with 3% hydrogen peroxide for 15 minutes and circled with a PAP marker. It was blocked with 10% sheep serum solution (Vector Laboratories, Inc., USA) for 1 hour. After the time was up, the sheep serum solution was discarded. Rabbit anti-neurofilament protein (NFP) antibody (Abeam, ab207176) was added and incubated overnight at 4°C, then washed twice with PBS, 5 minutes each time. The secondary antibody of goat anti-rabbit IgG (HRP) antibody (Abcam) was incubated at room temperature for 1 hour and washed twice with PBS, 5 minutes each time.The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, stained with hematoxylin for 30 seconds, blued with running water for 5 minutes, and washed once with PBS. They were dehydrated in a gradient, cleared, and mounted, and the sections were observed under an optical microscope at 200x magnification. Neurofilament proteins (NFPs) are proteins that make up the intermediate filaments of nerve cell axons. Their function is to provide the elasticity that allows nerve fibers to easily stretch and prevent breakage, which is crucial for maintaining the cytoskeleton, stabilizing cell morphology, and axonal transport. [4] . As a result, the expression of NFP (marked by arrow) in the plasminogen-treated group (Figure 3C) was significantly higher than that in the solvent PBS-treated control group (Figure 3B), and the expression of NFP was closer to that in the sham-operated group mice (Figure 3A). This indicates that plasminogen can promote the expression of NFP and promote the regeneration of spinal nerve fibers in spinal cord compression injury model mice. Example 4

[0088] Example 4 relates to plasminogen promoting repair of nerve damage. Ten 8-week-old Plg- / - female mice were taken and weighed, then the mice were randomly divided into two groups, a PBS-treated group and a plasminogen-treated group, with 5 mice in each group. All mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution (50 mg / kg), the midline skin of the back was incised, the T7-T9 dorsal muscles were separated, and the T7-T9 lamina was removed. For the PBS-treated control group and the plasminogen-treated group, a microvascular clip (3.5 × 1.0 mm, RWD) was clamped on the T8 spinal cord for 1 min. All mice were sutured immediately after surgery, injected intramuscularly with Tolfedine (0.1 mg / kg) and penicillin potassium (40,000 units / kg), and placed on a heating pad until revived. To prevent infection during surgery, mice were manually assisted to urinate after surgery until spontaneous urination. [1]Plasminogen was administered to mice in the plasminogen-treated group at 1 mg / 0.1 ml / mouse / day via tail vein injection, starting 30 minutes after surgery, and the same amount of PBS was administered to mice in the vehicle PBS control group. The day administration began was counted as day 1, and the administration cycle was 7 days. On the 8th day of administration, mice were dissected and the spinal cord T7-T9 segments were fixed in 4% paraformaldehyde. The fixed tissue samples were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The spinal cord tissue was cut into 4-μm-thick transverse sections, which were deparaffinized, rehydrated, stained with hematoxylin and eosin (H&E staining), fractionated in 1% hydrochloric acid ethanol, bluing with ammonia water, dehydrated in an alcohol gradient, mounted, and observed under an optical microscope at 200x magnification. The results showed that the neurons and nerve fibers in the sham-operated group (Figure 4A) had normal shapes and the cytoplasm was stained red. When the junctions of normal and injured nerves were collected from the PBS-treated control group (Figure 4B) and the plasminogen-treated group (Figure 4C), the normal neuron and nerve fiber structures in both groups were lost at the injury site and replaced by repaired fibers, the cytoplasm was lightly stained, and inflammatory cells were infiltrated. In the PBS-treated control group, a large injury cavity (triangle mark) was observed at the junction, whereas the degree of repair of the injury site in the plasminogen-treated group was significantly better than that in the PBS-treated control group, no obvious cavity was observed, and the infiltration of inflammatory cells was also significantly reduced. This indicates that plasminogen can obviously promote the repair of spinal cord injury in the spinal cord compression injury model Plg- / - mice. Example 5

[0089] Example 5 relates to the promotion of regeneration of nerve myelin in Plg − / − spinal cord compression injury model mice. Ten 8-week-old Plg- / - female mice were taken and weighed, then the mice were randomly divided into two groups, a PBS-treated group and a plasminogen-treated group, with 5 mice in each group. All mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution (50 mg / kg), the midline skin of the back was incised, the T7-T9 dorsal muscles were separated, and the T7-T9 lamina was removed. For the PBS-treated control group and the plasminogen-treated group, a microvascular clip (3.5 × 1.0 mm, RWD) was clamped on the T8 spinal cord for 1 min. All mice were sutured immediately after surgery, injected intramuscularly with Tolfedine (0.1 mg / kg) and penicillin potassium (40,000 units / kg), and placed on a heating pad until revived. To prevent infection during surgery, mice were manually assisted to urinate after surgery until spontaneous urination. [1] The administration was started 30 minutes after the operation, and plasminogen was administered to the mice in the plasminogen-treated group at 1 mg / 0.1 ml / mouse / day via tail vein injection, while the mice in the vehicle PBS control group were similarly administered the same amount of PBS. The day administration began was counted as day 1, and the administration cycle was 7 days. On the 8th day of administration, the mice were dissected, and the spinal cord T7-T9 segments were fixed in 4% paraformaldehyde. The fixed tissue samples were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The transverse sections of the spinal cord tissue were 4 μm thick, and the sections were deparaffinized, rehydrated, and stained with LFB using a myelin staining solution. The sections were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in neutral rubber. The sections were observed under an optical microscope and photographed. As a result, the nerve myelin in the solvent PBS-treated control group (Figure 5A) clearly showed larger collapsed areas (marked with triangles), lighter blue color, and broken myelin (marked with arrows). In comparison, the myelin structure in the plasminogen-treated group (Figure 5B) was clearly intact and continuous, and darker blue color. This indicates that plasminogen can significantly promote myelin regeneration in the spinal cord compression nerve injury model Plg- / - mice. Example 6

[0090] Example 6 relates to the promotion of pain recovery in spinal cord compression injury model mice by plasminogen. Thirty-eight 7-week-old C57BL / 6J female mice were taken and weighed one day before modeling, and the mice were randomly divided into two groups, sham operation group and model group, according to the weight results, with 7 mice in the sham operation group and 31 mice in the modeling group. All mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution (50 mg / kg), the midline skin of the back was incised, the T5-T8 muscles of the back were separated, and the T6-T7 lamina was removed. For the model group, a microvascular clip (30 g, Kent Scientific Corporation) was clamped on the T6-T7 spinal cord for 1 min. The forceps should be inserted into the vertebral channel so that its tip is located at the top of the lamina to form a 90° angle. [5] For the sham-operated group, only the lamina was removed to avoid dural damage. All mice were sutured immediately after surgery, injected intramuscularly with Tolfedine (0.1 mg / kg) and penicillin potassium (40,000 units / kg), and placed on a heating pad until revived. To prevent infection during surgery, mice were manually assisted to urinate after surgery until spontaneous urination and were kept under observation for 7 days. Seven days after surgery, all mice were weighed and scored (scored by two people independently in a blinded manner). [5] The mice were randomly divided into three groups, treatment group A, treatment group B, and solvent control group, according to their body weight and BMS score, and the day when treatment started was designated as day 1. The mice in treatment group A were administered plasminogen solution at 1 mg / 100 μl / mouse via tail vein injection, the mice in treatment group B were administered plasminogen solution at 0.5 mg / 100 μl / mouse via tail vein injection, and the mice in the solvent control group were administered solvent solution at 100 μl / mouse via tail vein injection, and the administration was continued for 6 days. On the 7th day, the Von-Frey mechanical pressure pain test was performed on the left paw of the mice. As a result, the mechanical threshold of the mice in the vehicle-treated group was significantly increased compared with that of the mice in the sham-operated group. This indicates that the mice lost their sense of pain after the establishment of the spinal cord compression injury model. The pain thresholds of the mice in the treated group A (1 mg) and treated group B (0.5 mg) were significantly lower than those of the vehicle group, and the difference was statistically significant (* indicates P<0.05, ** indicates P<0.01) (Figure 6). These results indicate that plasminogen can promote pain recovery in the spinal cord compression injury model mice. Example 7

[0091] Example 7 relates to the promotion of expression of neurofilament protein in the spinal cord of a paraplegic model mouse. Thirty-two C57 female mice aged 8 to 10 weeks were taken and weighed before modeling, and all mice were randomly divided into two groups according to the weight results, 8 in the sham operation group and 24 in the model group. Before surgery, the mice in the model group were under light anesthesia with Zoletil 50, and the hair on the back of the mice was removed. After that, the mice were fully anesthetized with respiratory anesthesia and placed in a prone position in a respiratory anesthesia machine with 2.5% isoflurane turned on. The skin and muscles were surgically incised to expose the T9 and T10 segments, the lamina was removed, and the spinal cord was exposed. After that, the right half of the spinal cord between T9 and T10 was cut along the coronal plane to create a hemisection injury, and the wound was sutured after completion. The mice in the sham operation group were anesthetized in the same way, and then a laminectomy was performed and directly sutured. All mice were placed on a heating pad until revived. Postoperative care included daily injections of the analgesic Tolfedine and penicillin potassium (40,000 units / kg) for a total of 4 days. Mice were given access to food and water, and urination was assisted until voluntary bladder function was restored. [6]One week after the surgical modeling was completed, the postoperative mice were subjected to an open-field behavioral test and scored. According to the test results, the mice in the model group were randomly divided into two groups, a solvent group and a treatment group, with 12 mice in each group, and the mice in the sham-operated group were not divided into groups. After the grouping was completed, the mice in the solvent group and the sham-operated group were administered the solvent at 0.1 ml / mouse / day via tail vein injection, and the mice in the treatment group were administered plasminogen at 1 mg / mouse / day via tail vein injection. The day of administration was the first day, and administration was continued for 28 days. On the 29th day, the mice were sacrificed, and the spinal cords at the modeling site were removed and fixed in 4% paraformaldehyde. The fixed tissue samples were dehydrated in an alcohol gradient, permeabilized with xylene, and then embedded in paraffin. The thickness of the tissue sections was 3 μm, and the sections were deparaffinized, rehydrated, and washed once with water. The sections were fixed with citric acid for 30 minutes, cooled at room temperature for 10 minutes, and then gently rinsed with water. The sections were incubated with 3% hydrogen peroxide for 15 minutes, and the tissue was circled with a PAP marker. The sections were blocked with 10% sheep serum solution (Vector Laboratories, Inc., USA) for 1 hour. After the time was up, the sheep serum solution was discarded. Rabbit anti-neurofilament protein (NFP) antibody (Abeam, ab207176) was added and incubated overnight at 4°C, then washed twice with PBS, for 5 minutes each time. The secondary antibody of goat anti-rabbit IgG (HRP) antibody (Abcam) was incubated at room temperature for 1 hour, washed twice with PBS, for 5 minutes each time. The sections were stained with DAB kit (Vector Laboratories, Inc., USA), washed three times with water, then stained with hematoxylin for 30 seconds, blued with running water for 5 minutes, and washed once with PBS. After gradient dehydration, clearing and mounting, sections were examined under a light microscope at 400x magnification. As a result, the spinal cord of the sham-operated mice (Figure 7A) expressed a certain level of NFP (marked with an arrow), the expression of NFP in the spinal cord of the modeling site of the vehicle-operated mice (Figure 7B) was significantly lower than that of the vehicle-operated mice, and the expression of NFP in the spinal cord of the modeling site of the administration-operated mice (Figure 7C) was significantly higher than that of the vehicle group, and the difference was statistically significant (* represents P<0.05) (Figure 7D). This indicates that plasminogen can promote the expression of NFP in the spinal cord of the spinal cord transection model mice. Example 8

[0092] Example 8 relates to the effect of plasminogen on improving hind limb motor function in paraplegic model mice. Forty-two C57 female mice aged 8 to 10 weeks were taken and weighed before modeling, and all mice were randomly divided into two groups according to the weight results, 14 in the sham operation group and 28 in the model group. The experimental mice were taken, and after removing the hair on their backs after pre-anesthesia, the mice were fully anesthetized with respiratory anesthesia and placed in a prone position in a respiratory anesthesia machine with 2.5% isoflurane turned on. The skin and muscles were surgically incised to expose the T9 and T10 segments, the lamina was removed, and the spinal cord was exposed, after which the right half of the spinal cord between T9 and T10 was cut along the coronal plane to create a hemisection injury, and the wound was sutured after completion. The mice in the sham operation group were anesthetized in the same way, and then the skin and muscles were surgically incised to expose the T9 and T10 segments, which were directly sutured. The analgesics Tolfedine and penicillin potassium (200,000 units / kg, 80,000 units / ml) were administered before surgery, 24 hours, and 72 hours after surgery. After surgery, all mice should be placed on a heating pad, with their necks kept horizontal and not bent until the mouse is revived. For postoperative care, a normal animal needs approximately 40-80 ml / kg of fluid per day, and if the mouse is unable to eat or drink after waking up from anesthesia, saline should be injected intraperitoneally to replenish the body fluids. In addition, urination was assisted until the voluntary voiding function of the bladder of the mouse was restored. [6]On the day of surgery, clean wipes should be placed on the litter in the PC box to prevent the wound from being contaminated by feces, urine, and litter. Three days after the completion of surgical modeling, the postoperative mice were subjected to an open-field behavioral test and scored, and according to the test results, the 28 mice in the model group were randomly divided into two groups, a solvent group and a treatment group, with 14 mice in each group, and the sham-operated group mice were not grouped. After the grouping was completed, the solvent was administered to the solvent group and the sham-operated group mice by tail vein injection at 0.1 ml / mouse / day, and the treatment group mice were administered plasminogen by tail vein injection at 1 mg / mouse / day. The day when the administration began was regarded as day 1, and the administration was continued for 7 days. On the 8th day, the Basso Mouse Scale (BMS) test was performed according to Table 1. The BMS score is specifically used to evaluate the changes in hind limb function in spinal cord-injured mice, and includes a main and sub-scoring system. The main score is scored by observing the mobility, coordination, foot posture, trunk stability, and tail posture of the mouse's hind limb ankle joints, and the sub-score is a supplementary correction of the main score based on coordination, stability, etc. [7] . JPEG0007641025000001.jpg174136As a result, the BMS score of the mice in the sham operation group was 9 points, and the BMS score of the mice in the vehicle group was 6.75 ± 1.92 points, which was significantly lower than that of the mice in the sham operation group. The BMS score of the mice in the treatment group was 8.29 ± 0.39 points, which was significantly higher than that of the mice in the vehicle group, and the difference was statistically significant (* indicates P < 0.05, *** indicates P < 0.001) (Figure 8). This indicates that plasminogen can promote the recovery of hind limb motor function in paraplegic model mice. Example 9

[0093] Example 9 relates to the promotion of transcription of the spinal cord plasminogen gene in a paraplegic model mouse. Fifteen 8-week-old C57 female mice were taken and weighed before modeling, and all mice were randomly divided into two groups according to the weight results, 5 in the blank control group and 10 in the model group. The experimental mice were taken, and after removing the hair on their backs after pre-anesthesia, the mice were fully anesthetized with respiratory anesthesia and placed in a prone position in a respiratory anesthesia machine with 2.5% isoflurane turned on. The skin and muscle were surgically incised to expose the T9 and T10 segments, the lamina was removed, and the spinal cord was exposed, and then the right half of the spinal cord between T9 and T10 was cut along the coronal plane to create a hemisection injury, and the wound was sutured after completion. The mice in the blank control group were similarly anesthetized, and then the skin and muscle were surgically incised to expose the T9 and T10 segments and directly sutured. [6] Painkillers Tolfedine and penicillin potassium (200,000 units / kg, 80,000 units / ml) were administered before surgery, 24 hours, and 72 hours after surgery. After surgery, all mice should be placed on a heating pad, with their necks kept horizontal and not bent until the mouse is revived. For postoperative care, a normal animal needs approximately 40-80 ml / kg of fluid per day, and if the mouse is unable to eat or drink after waking up from anesthesia, saline should be injected intraperitoneally to replenish the fluid. In addition, urination was assisted until the voluntary voiding function of the mouse's bladder was restored. To prevent the wound from being contaminated with feces, urine, or litter on the day of surgery, clean wipes should be placed on top of the litter in the PC box. Three days after the surgical modeling was completed, the postoperative mice were subjected to an open field behavioral test and scored. According to the test results, the 10 mice in the model group were randomly divided into two groups, a solvent group and an administration group, with 5 mice in each group, and the mice in the sham-operated group were not grouped. After the grouping was completed, all experimental mice were administered plasminogen solution (10 mg / ml) by tail vein injection at 50 mg / kg / day to the administration group, and the blank control group and the solvent group were administered by tail vein injection. Six hours after administration, the mice were sacrificed, and spinal cord homogenates were collected from the modeling site, after which RT-PCR detection of Plg gene transcription mRNA was performed and the CT value was recorded. The primer information is shown in Table 1 below. The CT value of Plg gene transcription mRNA in 200 ng of total RNA was calculated. JPEG0007641025000002.jpg49118CT value: indicates the number of cycles each PCR reaction tube has passed when the fluorescent signal reaches a set threshold. Studies have shown that there is a linear relationship between the CT value of each template and the logarithm of the initial copy number of the template, and the higher the initial copy number, the smaller the CT value, and vice versa. The results showed that there was a certain level of Plg mRNA in the spinal cord of the mice in the blank control group, and the level of Plg mRNA in the spinal cord of the mice in the treatment group was significantly higher than that in the vehicle group, and the difference was almost statistically significant (P=0.051) (Figure 9). This indicates that plasminogen can promote the transcription of plasminogen gene in the spinal cord of paraplegic model mice. Example 10

[0094] Example 10 relates to the promotion of synaptophysin expression in the injured spinal cord of a paraplegic model mouse. Forty-two C57 female mice aged 8 to 10 weeks were taken and weighed before modeling, and all mice were randomly divided into two groups according to the weight results, 14 in the sham operation group and 28 in the model group. The experimental mice were taken, and after removing the hair on their backs after pre-anesthesia, the mice were fully anesthetized with respiratory anesthesia and placed in a prone position in a respiratory anesthesia machine with 2.5% isoflurane turned on. The skin and muscles were surgically incised to expose the T9 and T10 segments, the lamina was removed, and the spinal cord was exposed, after which the right half of the spinal cord between T9 and T10 was cut along the coronal plane to create a hemisection injury, and the wound was sutured after completion. The mice in the sham operation group were anesthetized in the same way, and then the skin and muscles were surgically incised to expose the T9 and T10 segments, which were directly sutured. The analgesics Tolfedine and penicillin potassium (200,000 units / kg, 80,000 units / ml) were administered before surgery, 24 hours, and 72 hours after surgery. After surgery, all mice should be placed on a heating pad, with their necks kept horizontal and not bent until the mouse is revived. For postoperative care, a normal animal needs approximately 40-80 ml / kg of fluid per day, and if the mouse is unable to eat or drink after waking up from anesthesia, saline should be injected intraperitoneally to replenish the body fluids. In addition, urination was assisted until the voluntary voiding function of the bladder of the mouse was restored. [6]On the day of surgery, clean wipes should be placed on the litter in the PC box to prevent the wound from being contaminated by feces, urine, and litter. Three days after the completion of surgical modeling, the postoperative mice were subjected to an open-field behavioral test and scored. According to the test results, the 28 mice in the model group were randomly divided into two groups, a vehicle group and a treatment group, with 14 mice in each group, and the sham-operated group mice were not divided into groups. After the grouping was completed, the vehicle group and the sham-operated group mice were administered the vehicle at 0.1 ml / mouse / day by tail vein injection, and the treatment group mice were administered plasminogen at 1 mg / 0.1 ml / mouse / day by tail vein injection. The day when the administration began was counted as day 1, and the administration continued for 28 days. On the 30th day, the mice were sacrificed, and the injured spinal cords were fixed in 10% formaldehyde solution. The fixed tissue samples were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The tissue sections were 3 μm thick, and the sections were deparaffinized and rehydrated, then washed once with water. They were fixed with citric acid for 30 min, cooled at room temperature for 10 min, and gently rinsed with water. They were incubated with 3% hydrogen peroxide for 15 min, and the tissue was circled with a PAP marker. They were blocked with 10% sheep serum solution (Vector laboratories, Inc., USA) for 1 h. After the time was up, the sheep serum solution was discarded. Rabbit anti-synaptophysin antibody (Wuhan Mitaka, 10842-1-AP) was added and incubated overnight at 4 °C, then washed twice with PBS, 5 min each time. The secondary antibody of goat anti-rabbit IgG (HRP) antibody (Abcam) was incubated at room temperature for 1 h, and washed twice with PBS, 5 min each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, stained with hematoxylin for 30 seconds, blued with running water for 5 minutes, and washed once with PBS. They were dehydrated in a gradient, cleared, and mounted, and the sections were observed under an optical microscope at 200x magnification. Synaptophysin, also known as SYP (major synaptic vesicle protein p38), is a calcium-binding protein with a molecular weight of 38KDa located on the membrane of synaptic vesicles and is a membrane protein closely related to the structure and function of synapses. It is widely present in all nerve terminals in the body, is specifically distributed in the membrane of presynaptic vesicles, and is involved in calcium ion-dependent neurotransmitter release and synaptic vesicle circulation, and is considered an important marker for synapse formation and synaptic remodeling. [8] . The results showed that the spinal cord of the sham-operated mice (Figure 10A) expressed a certain level of synaptophysin (marked with an arrow), and the level of synaptophysin in the spinal cord of the modeling site of the vehicle-operated mice (Figure 10B) was significantly lower than that of the vehicle-operated mice, and the statistical difference between the two groups was highly significant (*** means P<0.001). The level of synaptophysin in the spinal cord of the modeling site of the treatment-operated mice (Figure 10C) was significantly higher than that of the vehicle group, and the difference was statistically significant (* means P<0.05) (Figure 10D). This indicates that plasminogen can promote the expression of synaptophysin in the injured spinal cord of paraplegic model mice and promote the repair of spinal cord injury. Example 11

[0095] Example 11 relates to the promotion of functional recovery of injured spinal cord neurons in a paraplegic mouse model by plasminogen. Forty-two C57 female mice aged 8 to 10 weeks were taken and weighed before modeling, and all mice were randomly divided into two groups according to the weight results, 14 in the sham operation group and 28 in the model group. The experimental mice were taken, and after removing the hair on their backs after pre-anesthesia, the mice were fully anesthetized with respiratory anesthesia and placed in a prone position in a respiratory anesthesia machine with 2.5% isoflurane turned on. The skin and muscles were surgically incised to expose the T9 and T10 segments, the lamina was removed, and the spinal cord was exposed, after which the right half of the spinal cord between T9 and T10 was cut along the coronal plane to create a hemisection injury, and the wound was sutured after completion. The mice in the sham operation group were anesthetized in the same way, and then the skin and muscles were surgically incised to expose the T9 and T10 segments, which were directly sutured. The analgesics Tolfedine and penicillin potassium (200,000 units / kg, 80,000 units / ml) were administered before surgery, 24 hours, and 72 hours after surgery. After surgery, all mice should be placed on a heating pad, with their necks kept horizontal and not bent until the mouse is revived. For postoperative care, a normal animal needs approximately 40-80 ml / kg of fluid per day, and if the mouse is unable to eat or drink after waking up from anesthesia, saline should be injected intraperitoneally to replenish the body fluids. In addition, urination was assisted until the voluntary voiding function of the bladder of the mouse was restored. [6]On the day of surgery, clean wipes should be placed on the litter in the PC box to prevent the wound from being contaminated by feces, urine, and litter. Three days after the completion of surgical modeling, the postoperative mice were subjected to an open-field behavioral test and scored. According to the test results, the 28 mice in the model group were randomly divided into two groups, a vehicle group and a treatment group, with 14 mice in each group, and the sham-operated group mice were not divided into groups. After the grouping was completed, the vehicle group and the sham-operated group mice were administered the vehicle by tail vein injection at 0.1 ml / mouse / day, and the treatment group mice were administered plasminogen by tail vein injection at 1 mg / mouse / day. The day when the administration began was counted as day 1, and the administration was continued for 28 days. The fixed striatal tissues were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. Tissue sections were 3 μm thick, deparaffinized, rehydrated, and stained with 0.4% tar violet solution (pH = 3), dehydrated in an alcohol gradient, permeabilized with xylene, and mounted in neutral rubber. Sections were observed under a light microscope at 400x magnification and photographed. Nissl bodies, also known as chromatin, are structures unique to neurons that consist of rough endoplasmic reticulum and free ribosomes distributed in parallel rows and function to synthesize proteins. Their number and distribution are closely related to the functional state of neurons and are considered to be markers of neural activity. [9] . The results showed that the spinal cord of the mice in the sham-operated group (Figure 11A) had a certain level of Nissl bodies (marked by arrows), and the level of Nissl bodies in the spinal cord of the modeling site of the mice in the vehicle group (Figure 11B) was significantly higher than that of the mice in the sham-operated group, and the statistical difference between the two groups was highly significant (*** represents P<0.001). The level of Nissl bodies in the spinal cord of the modeling site of the mice in the treatment group (Figure 11C) was significantly lower than that of the vehicle group, and the statistical difference was highly significant (** represents P<0.01) (Figure 11D). This indicates that plasminogen can promote the recovery of Nissl body levels in the injured spinal cord of paraplegic model mice and promote the repair of spinal cord injury. References: [1] Suelen Adriani Marques .A simple, inexpensive and easily reproducible model of spinal cord injury in mice: Morphological and functional assessment. Journal of Neuroscience Methods 177 (2009) 183-193. [2] Sun SW, Liang HF, Trinkaus K et al. Noninvasive detection of cuprizone induced axonal damage and demyelination in the mouse corpus callosum. Magn Reson Med. 2006 Feb;55(2):302-8. [3]Vallieres N1, Berard JL, David S et al. Systemic injections of lipopolysaccharide accelerates myelin phagocytosis during Wallerian degeneration in the injured mouse spinal cord. Glia. 2006 Jan 1;53(1):103-13. [4]Gotow T.Neurofilaments in health and disease. Med Electron Microsc. 2000; 33 (4):173-99. [5]Joshi M. Development and characterization of a graded, in vivo, compressive, murine model of spinal cord injury [J]. American Journal of Mathematics, 2000, 97(2):308-311. [6]Wen-Ge Ding,Spinal Cord Injury Causes More Damage to Fracture Healing of Later Phase than Ovariectomy in Young Mice, Connective Tissue Research, 53(2): 142-148, (2012) [7]RV Ung, NP Lapointe,Spontaneous recovery of hindlimb movement in completely spinal cord transected mice: a comparison of assessment methods and conditions,Spinal Cord (2007) 45, 367-379. [8] Li GL, Farooque M, Isaksson J, Olsson Y. Changes in synapses and axons demonstrated by synaptophysin immunohistochemistry following spinal cord compression trauma in the rat and mouse. Biomed Environ Sci. 2004 Sep;17(3):281-90. [9] HEBERT AE, DASH PK. Nonredundant roles for hippocampal and entorhinal cortical plasticity in spatial memory storage [J]. Pharmacology Biochemistry and Behavior, 2004, 79(1) :143-153.

Claims

1. A pharmaceutical composition for treating nerve damage comprising plasminogen, wherein the plasminogen has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 2 and has plasminogen proteolytic activity, and the nerve damage is compressive spinal nerve injury or paralysis.

2. The pharmaceutical composition of claim 1 , wherein the plasminogen promotes the expression of spinal neurofilament protein and the regeneration of nerve fibers.

3. The pharmaceutical composition of claim 1 , wherein the plasminogen further promotes recovery of sensory nerve function or recovery of motor nerve function.

4. The pharmaceutical composition of claim 1 , wherein the plasminogen further promotes analgesia.

5. The pharmaceutical composition of claim 1 , wherein the plasminogen further promotes expression of synaptophysin.

6. The pharmaceutical composition of claim 1 , wherein the plasminogen further promotes the restoration of damaged neuronal Nissl body levels.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the plasminogen is a conservatively substituted variant of the plasminogen set forth in SEQ ID NO:

2.

8. 7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the plasminogen comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a plasminogen active fragment shown in SEQ ID NO: 14 and has the proteolytic activity of plasminogen.

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