WO2021155867A1 - 一种预防和治疗多发性硬化症的方法和药物 - Google Patents

一种预防和治疗多发性硬化症的方法和药物 Download PDF

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WO2021155867A1
WO2021155867A1 PCT/CN2021/075921 CN2021075921W WO2021155867A1 WO 2021155867 A1 WO2021155867 A1 WO 2021155867A1 CN 2021075921 W CN2021075921 W CN 2021075921W WO 2021155867 A1 WO2021155867 A1 WO 2021155867A1
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plasminogen
mice
group
plasmin
sequence
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French (fr)
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李季男
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Talengen International Ltd
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Talengen International Ltd
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Priority to CA3167202A priority Critical patent/CA3167202A1/en
Priority to US17/797,504 priority patent/US20230084586A1/en
Priority to JP2022545965A priority patent/JP7487970B2/ja
Priority to EP21750885.2A priority patent/EP4094775A4/en
Priority to CN202180012709.1A priority patent/CN115427066A/zh
Priority to KR1020227030683A priority patent/KR20220137950A/ko
Publication of WO2021155867A1 publication Critical patent/WO2021155867A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21068—Tissue plasminogen activator (3.4.21.68), i.e. tPA

Definitions

  • the present invention relates to a method for preventing or treating multiple sclerosis, comprising administering to a subject an effective amount of components of the plasminogen activation pathway or related compounds, such as plasminogen, to repair damaged nerves, Improve clinical symptoms and signs.
  • MS Multiple sclerosis
  • MRI Magnetic resonance imaging
  • MRI Magnetic resonance imaging
  • the etiology of the disease is not yet clear, and has a certain relationship with genetic factors, viral infections, and autoimmune reactions.
  • MRI Magnetic resonance imaging
  • In the acute active stage of the disease there are multiple inflammatory demyelinating plaques in the white matter of the central nervous system, while the old lesions form calcified plaques due to the proliferation of glial fibers. They are characterized by multiple foci, remission, and recurrence, and are more likely to occur in the optic nerve, spinal cord and brain. Dry.
  • Current treatment methods include hormone therapy, beta-interferon and immunosuppressive therapy, which are not ideal, and other treatment methods need to be found.
  • plasminogen can obviously promote the regeneration and repair of nerve myelin, and prevent and treat multiple sclerosis.
  • the present invention relates to the following items:
  • this application relates to a method for preventing and treating multiple sclerosis, comprising administering to a subject a therapeutically effective amount of one or more compounds selected from the group consisting of: plasminogen activation pathway Substances, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.
  • plasminogen activation pathway Substances compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin
  • the present application relates to the use of one or more compounds selected from the following in the preparation of drugs for the prevention and treatment of multiple sclerosis, and the one or more compounds selected from the following are: plasminogen Components of the activation pathway, compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating the upstream components of the plasminogen activation pathway, one of which mimics plasminogen or plasmin Active compounds, compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonism of fibrinolytic inhibitors Agent.
  • plasminogen Components of the activation pathway compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating the upstream components of the plasminogen activation pathway, one of which mimics plasminogen or plasmin Active compounds, compounds capable of up-regulating the expression of plasminogen or plasminogen activator,
  • this application relates to drugs for the prevention and treatment of multiple sclerosis, comprising one or more compounds selected from the group consisting of components of the plasminogen activation pathway, capable of directly activating plasminogen or through activation
  • the present application relates to the use of one or more compounds selected from the following for the prevention and treatment of multiple sclerosis, wherein one or more compounds selected from the following are: the group of plasminogen activation pathway Substances, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.
  • the group of plasminogen activation pathway Substances compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasm
  • the component of the plasminogen activation pathway is selected from the group consisting of plasminogen, recombinant human plasmin, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants containing one or more kringle domains and protease domains of plasminogen and plasmin Analogues, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-fibrin Lysinogen, delta-plasmin, plasminogen activator, tPA and uPA.
  • any one of items 1 to 3 wherein the compound has an activity selected from one or more of the following: promoting the regeneration of nerve myelin, promoting the expression of myelin protein (for example, promoting PLP or MBP protein expression), promote the expression of nerve tissue NFP, promote nerve fiber regeneration, promote the expression of nerve tissue NFP, increase the level of nerve tissue MBP, increase the number of nerve tissue microglia, promote nerve tissue inflammation repair, and promote nerve tissue star Glial cell activity, increase the level of BDNF in nervous tissue, promote the expression of GFAP in nervous tissue, improve the subject's social behavior ability, improve the subject's social memory ability, alleviate the subject's depressive behavior, and relieve the subject's anxiety behavior.
  • myelin protein for example, promoting PLP or MBP protein expression
  • promote the expression of nerve tissue NFP promote nerve fiber regeneration
  • promote the expression of nerve tissue NFP increase the level of nerve tissue MBP
  • increase the number of nerve tissue microglia increase the number of nerve tissue microglia
  • the plasminogen comprises the active fragment of plasminogen shown in sequence 14 having at least 75%, 80%, 85%, 90%, Amino acid sequences with 95%, 96%, 97%, 98%, or 99% sequence identity, and have the proteolytic activity of plasminogen.
  • the above-mentioned plasminogen is a conservative substitution variant of the plasminogen described in sequence 2.
  • plasminogen is selected from Glu-plasminogen, Lys-plasminogen, microplasminogen, and microplasminogen , Delta-plasminogen or their variants that retain plasminogen activity.
  • plasminogen is natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • any one of items 1-13 wherein the compound is administered by any one or more methods or routes selected from the group consisting of nasal cavity inhalation, nebulization inhalation, nasal drops, and eye drops Liquid, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial and intramuscular.
  • the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, and the sequence 2, 6, 8, 10 or 12. %, 98%, or 99% sequence identity, and still have plasminogen activity, such as proteolytic activity, lysine binding activity, or proteolytic activity and lysine binding activity.
  • the plasminogen is added, deleted and/or substituted 1-100, 1-90, 1-80, 1-70 on the basis of sequence 2, 6, 8, 10 or 12.
  • plasminogen activity protein such as proteolytic activity, lysine binding activity, or proteolytic activity and lysine binding activity.
  • the plasminogen is a protein that contains plasminogen active fragments and still has plasminogen activity.
  • the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen or their retention Variants of plasminogen activity.
  • the plasminogen is natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • the plasminogen is a human plasminogen ortholog from a primate or rodent or a variant or fragment thereof that still retains plasminogen activity.
  • the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is natural human plasminogen.
  • the subject is a human. In some embodiments, the subject lacks or lacks plasminogen. In some embodiments, the deficiency or deletion is congenital, secondary, and/or local.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and plasminogen used in the aforementioned methods.
  • the kit may be a prophylactic or therapeutic kit comprising: (i) plasminogen for the aforementioned method and (ii) for delivery of the plasminogen to the The means of the subject.
  • the member is a syringe or vial.
  • the kit further includes a label or instructions for use that instructs the plasminogen to be administered to the subject to perform any of the foregoing methods.
  • the article of manufacture comprises: a container containing a label; and a pharmaceutical composition comprising (i) plasminogen used in the foregoing method or a pharmaceutical composition comprising plasminogen, wherein the label indicates that the plasminogen
  • a pharmaceutical composition comprising (i) plasminogen used in the foregoing method or a pharmaceutical composition comprising plasminogen, wherein the label indicates that the plasminogen
  • the prolysozyme or composition is administered to the subject to perform any of the aforementioned methods.
  • the kit or article of manufacture further includes one or more additional components or containers that contain other drugs.
  • the plasminogen is administered systemically or locally, preferably by the following routes: intravenous, intramuscular, or subcutaneous administration of plasminogen for treatment.
  • the plasminogen is administered in combination with an appropriate polypeptide carrier or stabilizer.
  • the plasminogen is at a dose of 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg per day /kg, 10-100mg/kg (calculated per kilogram of body weight) or 0.0001-2000mg/cm2, 0.001-800mg/cm2, 0.01-600mg/cm2, 0.1-400mg/cm2, 1-200mg/cm2, 1-100mg/ cm2, 10-100 mg/cm2 (calculated per square centimeter of body surface area) dose administration, preferably repeated at least once, preferably at least daily administration.
  • the present invention clearly covers all combinations of technical features belonging to the embodiments of the present invention, and the technical solutions after these combinations have been clearly disclosed in this application, just as the above-mentioned technical solutions have been separately and clearly disclosed.
  • the present invention also clearly covers the combinations between the various embodiments and their elements, and the technical solutions after the combination are clearly disclosed herein.
  • FIG. 1A-D LFB staining results of brain corpus callosum in mice of hexanone oxalyl hydrazone-induced demyelination model mice given plasminogen for 14 days.
  • A is the blank control group
  • B is the vehicle PBS control group
  • C is the plasminogen group
  • D is the quantitative analysis result.
  • the results showed that the morphology of the corpus callosum myelin in the blank control group was basically normal.
  • the positive staining of the corpus callosum myelin in the plasminogen group (arrow mark) was significantly more than that in the vehicle PBS control group, and the statistical difference was significant (*P ⁇ 0.05). It shows that plasminogen can reduce the demyelination of the corpus callosum, and promote the repair of myelin damage in the demyelinating model mice induced by hexanooxalyl hydrazone.
  • FIG. 2A-C Representative pictures of immunostaining of brain corpus callosum protein lipoprotein (PLP) in a demyelinating model mouse induced by hexanone oxalyl hydrazone for 3 days after administration of plasminogen.
  • A is the blank control group
  • B is the vehicle PBS control group
  • C is the plasminogen group.
  • the results showed that the expression of PLP in the corpus callosum in the plasminogen group (marked by the arrow) was significantly higher than that in the vehicle PBS control group, and compared with the latter, the expression was closer to that of the blank control group. It shows that plasminogen promotes the expression of PLP in the corpus callosum and promotes the remyelination of demyelinating model mice induced by hexanooxalyl hydrazone.
  • FIG. 3A-D Observation results of brain neurofilament protein (NFP) immunostaining for 14 days after administration of plasminogen to the demyelinating model mice induced by hexanone oxalyl hydrazone.
  • A is the blank control group
  • B is the vehicle PBS control group
  • C is the plasminogen group
  • D is the quantitative analysis result.
  • FIG. 4 The results of NFP immunohistochemical staining of the corpus callosum of multiple sclerosis model mice 35 days after administration of plasminogen.
  • A is the blank control group
  • B is the vehicle PBS control group
  • C is the plasminogen group
  • D is the average optical density quantitative analysis result.
  • the results showed that the corpus callosum of the blank control group expressed a certain level of NFP (arrow mark), the expression of NFP in the corpus callosum of mice in the vehicle group was significantly reduced, and the expression of NFP in the corpus callosum of the plasminogen group (also referred to as the administration group) was obvious Higher than vehicle mice, and statistically significant (* means P ⁇ 0.05). This result suggests that plasminogen can promote the expression of NFP in the corpus callosum of multiple sclerosis model mice.
  • FIG. 5A-C PLP immunohistochemical staining results of the corpus callosum of multiple sclerosis model mice 35 days after plasminogen administration.
  • A is the blank control group
  • B is the vehicle PBS control group
  • C is the plasminogen group.
  • the results showed that the corpus callosum of the blank control group expressed a certain level of PLP (arrow mark), the expression of PLP in the corpus callosum of mice in the vehicle group was significantly reduced, and the expression level of PLP in the corpus callosum of mice in the administration group was significantly higher than that of the vehicle mice. This result suggests that plasminogen can promote the expression of PLP in the corpus callosum of multiple sclerosis model mice.
  • FIG. 6A-D The results of MBP immunohistochemical staining of the corpus callosum of multiple sclerosis model mice 35 days after plasminogen administration.
  • A is the blank control group
  • B is the vehicle PBS control group
  • C is the plasminogen group
  • D is the average optical density quantitative analysis result.
  • the results showed that the corpus callosum of the blank control group expressed a certain level of MBP (arrow mark).
  • FIG. 7A-D Immunohistochemical staining results of hippocampus Iba-1 in multiple sclerosis model mice 35 days after plasminogen administration.
  • A is the blank control group
  • B is the vehicle PBS control group
  • C is the plasminogen group
  • D is the average optical density quantitative analysis result.
  • the results showed that there were a certain amount of microglia in the hippocampus of the blank control group (arrows), the amount of hippocampal microglia in the vehicle group increased significantly, and the number of hippocampal microglia in the treatment group was significantly more than that of the vehicle. Group, and statistically significant difference (* means P ⁇ 0.05, *** means P ⁇ 0.001). It suggests that plasminogen can promote the repair of hippocampal injury and inflammation in multiple sclerosis model mice.
  • FIG. 8A-D BDNF immunohistochemical staining results of the hippocampus of multiple sclerosis model mice 35 days after plasminogen administration.
  • A is the blank control group
  • B is the vehicle PBS control group
  • C is the plasminogen group
  • D is the average optical density quantitative analysis result.
  • FIG. 9A-D Hippocampal GFAP immunohistochemical staining results of multiple sclerosis model mice 35 days after plasminogen administration.
  • A is the blank control group
  • B is the vehicle PBS control group
  • C is the plasminogen group
  • D is the average optical density quantitative analysis result.
  • mice in the blank control group had a certain percentage of resting time in the border zone, about 57.8%, and the percentage of resting time in the border zone of mice in the vehicle group was significantly reduced, about 49.3%.
  • the percentage of interest time is about 58.4%, which is significantly greater than that of the solvent group, and the statistical difference is significant (* indicates P ⁇ 0.05), and it is similar to the blank control group. It shows that plasminogen can alleviate the depressive behavior of multiple sclerosis model mice to a certain extent.
  • FIG 12 The statistical results of the resting time percentage within the contact range of strange mice 2 in the second stage of the three-box experiment in multiple sclerosis model mice 34 days after plasminogen administration.
  • Percent resting time resting time/total observation time.
  • Figure 13 The statistical results of the percentage of movement distance in the border zone of the open field experiment in multiple sclerosis model rats 6 days after administration of plasminogen.
  • the movement distance of the boundary zone is the length of the movement trajectory of the boundary zone during the open field test time.
  • the results showed that the blank control group had a certain percentage of the boundary zone movement distance, about 91.1%; the vehicle group increased significantly, about 93.6%; the drug group was about 88.1%, which was significantly lower than the vehicle group, and the statistical difference was significant (* Indicates P ⁇ 0.05). It shows that plasminogen can alleviate the depressive behavior of multiple sclerosis model mice.
  • Figure 14 The statistical results of the percentage of exercise distance in the central area of the open field experiment in multiple sclerosis model rats 6 days after the administration of plasminogen.
  • the results show that the blank control group has a certain percentage of the central area distance, about 8.9%; the vehicle group is significantly reduced, about 6.4%; the drug group is about 11.9%, which is significantly higher than the vehicle group, and the statistical difference is significant (* means P ⁇ 0.05). It shows that plasminogen can alleviate the anxiety behavior of multiple sclerosis model rats.
  • Figure 15 The open arm entry percentage statistics of the elevated plus maze test of multiple sclerosis model mice 20 days after the administration of plasminogen.
  • Figure 17 The statistical results of the closed arm movement distance of the elevated plus maze test in multiple sclerosis model mice 27 days after the administration of plasminogen.
  • the total distance of the closed arm movement is the total distance of the closed arm movement of the mice during the test period of the elevated plus maze experiment.
  • the results showed that the mice in the blank control group had a certain total distance of closed arm movement, the total distance of closed arm movement of mice in the vehicle group was significantly increased, and the total distance of closed arm movement of mice in the drug group was significantly less than that of the vehicle group.
  • the statistical difference was significant (* It means P ⁇ 0.05), and it is similar to the blank control group. It shows that plasminogen can alleviate the anxiety and depression behavior of multiple sclerosis model mice to a certain extent.
  • Figure 18 The statistical results of the closed arm resting time percentage of the elevated plus maze test in multiple sclerosis model mice 27 days after administration of plasminogen.
  • mice in the blank control group had a certain percentage of closed arm rest time, about 51.8%; the percentage of closed arm rest time of mice in the vehicle group was significantly reduced, about 36.8%; the percentage of closed arm rest time of mice in the drug group was about 51.8% It is 50.1%, which is significantly larger than that of the vehicle group, and the statistical difference is significant (* means P ⁇ 0.05), and it is similar to the blank control group. It shows that plasminogen can alleviate the anxiety and depression behavior of multiple sclerosis model mice to a certain extent.
  • Fibrinolytic system also known as fibrinolytic system, is a system composed of a series of chemical substances involved in the process of fibrinolysis (fibrinolysis), mainly including fibrinolytic enzyme (plasminogen) and plasmin , Plasminogen activator, fibrinolysis inhibitor.
  • Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA).
  • t-PA tissue-type plasminogen activator
  • u-PA urokinase-type plasminogen activator
  • t-PA activates plasminogen
  • fibrin urokinase-type plasminogen activator
  • u-PA urokinase-type plasminogen activator
  • PLG Plasminogen
  • Plasminase is a serine protease, which has the following functions: degrades fibrin and fibrinogen; hydrolyzes a variety of coagulation factors V, VIII, X, VII, XI, II, etc.; turns plasminogen into fibrinolysis Enzymes; hydrolysis of complement, etc.
  • Fibrinolytic inhibitors including plasminogen activator inhibitor (PAI) and ⁇ 2 antiplasmin ( ⁇ 2-AP).
  • PAI mainly has two forms, PAI-1 and PAI-2, which can specifically bind to t-PA in a ratio of 1:1, thereby inactivating it and activating PLG at the same time.
  • ⁇ 2-AP is synthesized by the liver and combined with PL in a ratio of 1:1 to form a complex, inhibiting PL activity; FXIII makes ⁇ 2-AP covalently bond with fibrin, reducing the sensitivity of fibrin to PL.
  • Substances that inhibit the activity of the fibrinolytic system in the body PAI-1, complement C1 inhibitor; ⁇ 2 anti-plasmin; ⁇ 2 macroglobulin.
  • component of the plasminogen activation pathway covers:
  • Plasminogen activators such as tPA and uPA, and tPA or uPA variants and analogs containing one or more domains of tPA or uPA (such as one or more kringle domains and proteolytic domains) .
  • variants of plasminogen, plasmin, tPA and uPA include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as by addition, deletion and/or substitution such as 1- 100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, Proteins of 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid that still have plasminogen, plasmin, tPA or uPA activity.
  • variants of plasminogen, plasmin, tPA, and uPA include those by, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1- 45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative Mutant variants of these proteins obtained by amino acid substitutions.
  • the "plasminogen variant” of the present invention encompasses sequences 2, 6, 8, 10 or 12 that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99 % Sequence identity, and still have plasminogen activity protein.
  • the "plasminogen variant” of the present invention can be added, deleted and/or substituted 1-100, 1-90, 1-80, 1- on the basis of sequence 2, 6, 8, 10 or 12. 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid, and still have plasminogen activity protein.
  • the plasminogen variants of the present invention include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as through conservative amino acid substitutions such as 1-100, 1-90, 1-80, 1- 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1- 20, 1-15, 1-10, 1-5, 1-4, Mutant variants of these proteins obtained from 1-3, 1-2, 1 amino acids.
  • the plasminogen of the present invention can be a human plasminogen ortholog from a primate or rodent or a variant that still retains plasminogen activity, such as sequence 2, 6, 8, 10 Or the plasminogen shown in 12, for example, the human natural plasminogen shown in sequence 2.
  • plasminogen, plasmin, tPA, and uPA include compounds that provide substantially similar effects to plasminogen, plasmin, tPA, or uPA, respectively.
  • variants and analogs of plasminogen, plasmin, tPA and uPA encompass fibers comprising one or more domains (for example, one or more kringle domains and proteolytic domains)
  • variants and analogs encompass fibers comprising one or more domains (for example, one or more kringle domains and proteolytic domains)
  • variants and analogs encompass plasminogen comprising one or more plasminogen domains (eg, one or more kringle domains and proteolytic domains) Variants and analogs, such as mini-plasminogen.
  • Variants and “analogs” of plasmin encompass plasmin "variants” that include one or more plasmin domains (eg, one or more kringle domains and proteolytic domains) And “analogs” such as mini-plasmin and delta-plasmin.
  • plasminogen, plasmin, tPA or uPA have the activity of plasminogen, plasmin, tPA or uPA respectively, or whether they provide the same
  • the substantially similar effects of plasminogen, plasmin, tPA or uPA can be detected by methods known in the art, for example, by methods based on enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS ( Fluorescence-activated cell sorting method) is measured by the level of activated plasmin activity, for example, it can be measured with reference to a method selected from the following documents: Ny, A., Leonardsson, G., Hagglund, AC, Hagglof, P.
  • the "component of plasminogen activation pathway" of the present invention is plasminogen, selected from Glu-plasminogen, Lys-plasminogen, and microplasmin Pro, microplasminogen, delta-plasminogen or their variants that retain plasminogen activity.
  • the plasminogen is natural or synthetic human plasminogen, or a conservative mutant variant or fragment thereof that still retains plasminogen activity.
  • the plasminogen is a human plasminogen ortholog from a primate or rodent or a conservative mutant variant or a fragment thereof that still retains plasminogen activity.
  • the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is natural human plasminogen.
  • the plasminogen is human natural plasminogen as shown in sequence 2.
  • a compound capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway refers to a compound capable of directly activating plasminogen or by activating plasminogen Any compound that activates upstream components of the pathway and indirectly activates plasminogen, such as tPA, uPA, streptokinase, saruplase,reteplase, reteplase, tenecteplase, aniplase, Monteplase, Lanoteplase, Pamideplase, Staphylokinase.
  • the "antagonist of the fibrinolysis inhibitor" of the present invention is a compound that antagonizes, weakens, blocks, and prevents the action of the fibrinolysis inhibitor.
  • the fibrinolysis inhibitors are, for example, PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, and ⁇ 2 macroglobulin.
  • the antagonist such as PAI-1, complement C1 inhibitor, ⁇ 2 anti-plasmin or ⁇ 2 macroglobulin antibody, or blocking or down-regulating such as PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin Antisense RNA or small RNA expressed by globulin, or occupy the binding site of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin but without PAI-1, complement C1 inhibitor, ⁇ 2 anti-fibrosis Compounds that function as lysozyme or ⁇ 2 macroglobulin", or compounds that block the binding domain and/or active domain of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin.
  • Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease that can hydrolyze several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycan. In addition, plasmin can activate some metalloprotease precursors (pro-MMPs) to form active metalloproteases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis. Plasmin is formed by proteolysis of plasminogen through two physiological PAs: tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA).
  • tPA tissue-type plasminogen activator
  • uPA urokinase-type plasminogen activator
  • PAs Due to the relatively high levels of plasminogen in plasma and other body fluids, it is traditionally believed that the regulation of the PA system is mainly achieved through the synthesis and activity levels of PAs.
  • the synthesis of PA system components is strictly regulated by different factors, such as hormones, growth factors and cytokines.
  • the main inhibitor of plasmin is ⁇ 2-antiplasmin ( ⁇ 2-antiplasmin).
  • the activity of PAs was inhibited by both uPA and tPA's plasminogen activator inhibitor-1 (PAI-1) and mainly inhibited uPA's lysinogen activator inhibitor-2 (PAI-2).
  • PAI-1 uPA and tPA's plasminogen activator inhibitor-1
  • PAI-2 mainly inhibited uPA's lysinogen activator inhibitor-2
  • Certain cell surfaces have uPA-specific cell surface receptors (uPAR) with direct hydrolytic activity.
  • Human plasminogen is a single-chain glycoprotein consisting of 791 amino acids and a molecular weight of approximately 92kDa. Plasminogen is mainly synthesized in the liver and exists in large amounts in the extracellular fluid. Plasminogen content in plasma is about 2 ⁇ M. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids. Plasminogen exists in two molecular forms: Glu-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved form of plasminogen has an amino terminal (N-terminal) glutamate and is therefore called glutamate-plasminogen.
  • glutamate-plasminogen is hydrolyzed at Lys76-Lys77 to lysine-plasminogen.
  • lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate.
  • the Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide bond-linked double-chain protease plasmin.
  • the amino terminal part of plasminogen contains five homologous tricyclic rings, so-called kringles, and the carboxy terminal part contains the protease domain.
  • Some kringles contain lysine binding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor ⁇ 2-AP.
  • Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycan and gelatin, indicating that plasmin also plays an important role in ECM reconstruction.
  • plasmin can also degrade other components of ECM by converting certain protease precursors into active proteases, including MMP-1, MMP-2, MMP-3 and MMP-9. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis.
  • plasmin has the ability to activate certain latent forms of growth factors. In vitro, plasmin can also hydrolyze components of the complement system and release chemotactic complement fragments.
  • Pulminin is a very important enzyme present in the blood, which can hydrolyze fibrin clots into fibrin degradation products and D-dimers.
  • “Plasminogen” is the zymogen form of plasmin. According to the sequence in swiss prot, it is composed of 810 amino acids and the molecular weight is about 90kD, a glycoprotein mainly synthesized in the liver and able to circulate in the blood. The cDNA sequence encoding this amino acid sequence is shown in sequence 3. The full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle1-5).
  • PAp Pan Apple
  • Kringle1 includes residues Cys103-Cys181
  • Kringle2 includes residues Glu184-Cys262
  • Kringle3 includes residues Cys275-Cys352
  • Kringle4 Including residues Cys377-Cys454
  • Kringle5 includes residues Cys481-Cys560.
  • the serine protease domain includes residues Val581-Arg804.
  • Glu-plasminogen is a human natural full-length plasminogen, composed of 791 amino acids (without the signal peptide of 19 amino acids).
  • the cDNA sequence encoding this sequence is shown in sequence 1, and its amino acid sequence is shown in sequence 2 shown.
  • Lys-plasminogen formed by hydrolysis from amino acids 76-77 of Glu-plasminogen.
  • sequence 6 the cDNA sequence encoding this amino acid sequence is as shown in sequence 5.
  • Delta-plasminogen ( ⁇ -plasminogen) is a fragment of the full-length plasminogen with the Kringle2-Kringle5 structure deleted, and only contains Kringle1 and the serine protease domain (also known as the protease domain (PD)).
  • Mini-plasminogen (Mini-plasminogen) is composed of Kringle5 and serine protease domain. It has been reported in the literature that it includes residues Val443-Asn791 (with the Glu residue of the Glu-plasminogen sequence without signal peptide as the starting amino acid). ), its amino acid sequence is shown in sequence 10, and the cDNA sequence encoding the amino acid sequence is shown in sequence 9.
  • Micro-plasminogen (Micro-plasminogen) only contains the serine protease domain.
  • plasminogen includes Kringle 1, 2, 3, 4, and 5 domains and serine protease domains (also called protease domains (protease domain, PD)), among which Kringles is responsible for plasmin Proto binds to low-molecular-weight and high-molecular-weight ligands (ie, lysine binding activity), which causes plasminogen to transform into a more open configuration, which is easier to be activated; the protease domain (PD) is residue Val562 -Asn791, tPA and UPA specifically cleave the Arg561-Val562 activation bond of plasminogen, thereby allowing plasminogen to form plasmin. Therefore, the protease domain (PD) confers plasminogen proteolytic activity area.
  • Plasin can be used interchangeably and have the same meaning;
  • plasminogen is equivalent to “plasminogen” and “plasminogen” “Can be used interchangeably and have the same meaning.
  • the meaning or activity of the "deficiency" of plasminogen means that the content of plasminogen in the subject is lower than that of a normal person, and is low enough to affect the normal physiological function of the subject;
  • the meaning or activity of "deletion" of plasminogen is that the content of plasminogen in the subject is significantly lower than that of normal people, even the activity or expression is minimal, and normal physiological functions can only be maintained through external sources.
  • plasminogen adopts a closed inactive conformation, but when bound to thrombus or cell surface, under the mediation of plasminogen activator (PA), it turns into an open form Active plasmin in conformation.
  • PA plasminogen activator
  • the active plasmin can further hydrolyze the fibrin clot into fibrin degradation products and D-dimers, and then dissolve the thrombus.
  • the PAp domain of plasminogen contains important determinants that maintain plasminogen in an inactive closed conformation, while the KR domain can bind to lysine residues present on the receptor and substrate.
  • a variety of enzymes that can act as plasminogen activators are known, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hager Man factor) and so on.
  • “Plasminogen active fragment” refers to the activity of binding to lysine in the target sequence of the substrate (lysine binding activity), or the activity of exerting a proteolytic function (proteolytic activity), or proteolytic activity and lysine Amino acid-binding active fragments.
  • the technical scheme of the present invention related to plasminogen covers the technical scheme of replacing plasminogen with active fragments of plasminogen.
  • the active fragment of plasminogen according to the present invention comprises or consists of the serine protease domain of plasminogen, preferably, the plasminogen according to the present invention
  • the original active fragment contains sequence 14, or an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identical to sequence 14, or is composed of sequence 14, or has at least 80% identity with sequence 14. %, 90%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the active plasminogen fragment of the present invention comprises a region selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, Kringle 5, or is selected from Kringle 1, Kringle 2.
  • Kringle 3, Kringle 4, Kringle 5 are composed of one or more areas.
  • the plasminogen of the present invention includes a protein containing the above-mentioned active fragment of plasminogen.
  • the methods for measuring plasminogen and its activity in blood include: the detection of tissue plasminogen activator activity (t-PAA), the detection of plasma tissue plasminogen activator antigen (t-PAAg), Detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator inhibition Detection of substance antigens, plasma plasmin-antiplasmin complex detection (PAP).
  • t-PAA tissue plasminogen activator activity
  • t-PAAg the detection of plasma tissue plasminogen activator antigen
  • plgA Detection of plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma tissue plasminogen activator inhibition
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the tested plasma, the PLG in the tested plasma is transformed into PLM under the action of SK, and the latter acts on The chromogenic substrate is subsequently measured with a spectrophotometer, and the increase in absorbance is proportional to the activity of plasminogen.
  • SK streptokinase
  • immunochemical methods, gel electrophoresis, immunoturbidimetry, radioimmuno-diffusion methods, etc. can also be used to determine the plasminogen activity in the blood.
  • orthologs or orthologs refer to homologs between different species, including both protein homologs and DNA homologs, and are also called orthologs and vertical homologs. It specifically refers to proteins or genes in different species that evolved from the same ancestor gene.
  • the plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs derived from different species that have plasminogen activity.
  • Constant substitution variant refers to a given amino acid residue that changes but does not change the overall conformation and function of the protein or enzyme. This includes, but is not limited to, those with similar characteristics (such as acidic, basic, hydrophobic, etc.) Amino acids replace amino acids in the amino acid sequence of the parent protein. Amino acids with similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and can be interchanged. Similarly, isoleucine is a hydrophobic amino acid and can be replaced by leucine, methionine or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different.
  • Constant substitution variants also include polypeptides or enzymes that are determined by BLAST or FASTA algorithms to have more than 60% amino acid identity. If it can reach more than 75%, it is better, preferably more than 85%, or even more than 90%. It is the best, and has the same or substantially similar properties or functions compared with the natural or parent protein or enzyme.
  • isolated plasminogen refers to plasminogen protein separated and/or recovered from its natural environment.
  • the plasminogen will be purified (1) to a purity (by weight) greater than 90%, greater than 95%, or greater than 98%, as determined by the Lowry method, for example, greater than 99% (By weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, which is achieved by using Coomassie blue or silver staining is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions.
  • the isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering technology and separated by at least one purification step.
  • polypeptide refers to polymerized forms of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived Modified amino acids, and polypeptides with modified peptide backbones.
  • the term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without N-terminal methionine residues); and so on.
  • the “percent amino acid sequence identity (%)" with respect to the reference polypeptide sequence is defined as when gaps are introduced when necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity, the candidate sequence is Refers to the percentage of amino acid residues that are identical to amino acid residues in the polypeptide sequence.
  • the comparison for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithm that achieves the maximum comparison requirements over the entire length of the sequence being compared. However, for the purposes of the present invention, the percent amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2.
  • the% amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or can be expressed as having or containing relative to, with, or against a given amino acid sequence)
  • a given amino acid sequence A) of a certain% amino acid sequence identity of B is calculated as follows:
  • X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's A and B alignment
  • Y is the total number of amino acid residues in B. It should be appreciated that in the case where the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% amino acid sequence identity of A relative to B may not be equal to the% amino acid sequence identity of B relative to A. Unless expressly stated otherwise, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.
  • the term "treatment” refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be to completely or partially prevent the occurrence and onset of the disease or its symptoms, partially or completely reduce the disease and/or its symptoms, and/or partially or completely cure the disease and/or its symptoms, including: (a) prevention of disease Occurs or attacks in a subject, the subject may have the cause of the disease, but has not yet been diagnosed as having the disease; (b) inhibit the disease, that is, block its formation; and (c) reduce the disease and/or its symptoms , That is, causing the disease and/or its symptoms to subside or disappear.
  • mice rats, mice
  • non-human primates humans
  • dogs and cats
  • Hoofed animals such as horses, cows, sheep, pigs, goats
  • “Therapeutically effective amount” or “effective amount” refers to a component or component of the plasminogen activation pathway that is sufficient to achieve the prevention and/or treatment of the disease when administered to a mammal or other subject to treat the disease
  • the amount of related compounds e.g. plasminogen.
  • the “therapeutically effective amount” will depend on the components of the plasminogen activation pathway used or related compounds (such as plasminogen), the severity of the disease and/or symptoms of the subject to be treated, and the age. , Weight, etc.
  • Plasminogen can be isolated from nature and purified for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When a polypeptide is synthesized chemically, it can be synthesized via a liquid phase or a solid phase.
  • Solid phase peptide synthesis (SPPS) (where the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence) is a suitable method for the chemical synthesis of plasminogen.
  • SPPS Solid phase peptide synthesis
  • Various forms of SPPS, such as Fmoc and Boc can be used to synthesize plasminogen.
  • the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. Then, the unit is deprotected, exposing a new N-terminal amine that can be attached to other amino acids.
  • the peptide remains immobilized on the solid phase, after which it is cut off.
  • Standard recombinant methods can be used to produce the plasminogen of the present invention.
  • a nucleic acid encoding plasminogen is inserted into an expression vector so that it is operably linked to the regulatory sequence in the expression vector.
  • Expression control sequences include, but are not limited to, promoters (such as naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression control can be a eukaryotic promoter system in a vector that can transform or transfect eukaryotic host cells (such as COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of plasminogen.
  • Suitable expression vectors are usually replicated in the host organism as an episome or as an integrated part of the host chromosomal DNA.
  • the expression vector contains a selection marker (for example, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate the transformation of the desired DNA sequence for exogenous use Those cells are tested.
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone a polynucleotide encoding a subject antibody.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various pseudomonas. Genus (Pseudomonas) species.
  • expression vectors can also be produced, which usually contain expression control sequences compatible with the host cell (for example, an origin of replication).
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from bacteriophage lambda. Promoters usually control expression, optionally in the case of manipulating gene sequences, and have ribosome binding site sequences, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast such as S. cerevisiae
  • Pichia Pichia
  • suitable yeast host cells in which suitable vectors have expression control sequences (such as promoters), origins of replication, termination sequences, etc., as required.
  • suitable promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeasts are initiated by specifically including promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose.
  • mammalian cells e.g., mammalian cells cultured in in vitro cell culture
  • the anti-Tau antibodies of the present invention e.g., polynucleotides encoding the subject anti-Tau antibodies.
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas.
  • Expression vectors used in these cells may contain expression control sequences such as an origin of replication, promoters and enhancers (Queen et al., Immunol. Rev.
  • ribosome binding Site RNA splice site
  • polyadenylation site RNA splice site
  • transcription terminator sequence RNA splice site
  • suitable expression control sequences are promoters derived from white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149 (1992).
  • the plasminogen is substantially pure, for example at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure Or purer, for example, free of contaminants, such as cell debris, macromolecules other than the target product, and so on.
  • plasminogen activation pathway with the required purity or related compounds (such as plasminogen) with optional pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16 Edition, Osol, A. ed. (1980)) mixed to form a lyophilized preparation or an aqueous solution to prepare a therapeutic formulation.
  • pharmaceutical carriers such as plasminogen
  • excipients such as plasminogen
  • stabilizers Remington's Pharmaceutical Sciences, 16 Edition, Osol, A. ed. (1980)
  • Acceptable carriers, excipients, and stabilizers are non-toxic to recipients at the dose and concentration used, and include buffers such as phosphate, citrate and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (such as Octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoic acid Esters such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues) ; Proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • formulations of the present invention may also contain more than one active compound required for the specific condition to be treated, preferably those with complementary activities and no side effects between each other.
  • the plasminogen of the present invention can be encapsulated in microcapsules prepared by techniques such as coacervation or interfacial polymerization, for example, can be placed in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules) or placed in hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a coarse emulsion.
  • colloidal drug delivery system e.g., liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules
  • hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a coarse emulsion.
  • the components of the plasminogen activation pathway of the present invention or related compounds (such as plasminogen) for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after freeze-drying and reformulation.
  • sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers having a certain shape and containing glycoproteins, such as films or microcapsules.
  • sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.
  • Polymers such as ethylene- Vinyl acetate and lactic-glycolic acid can continue to release molecules for more than 100 days, but some hydrogels release proteins for a short time.
  • a reasonable strategy for stabilizing the protein can be designed according to the relevant mechanism. For example, if the mechanism of aggregation is found to be The formation of intermolecular SS bonds through the exchange of thiodisulfide bonds can be stabilized by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions .
  • nasal inhalation nebulization
  • nasal drops or eye drops intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (for example, via the carotid artery), intramuscular, and rectal Drugs to achieve the administration of the pharmaceutical composition of the present invention.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, and so on. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, among others.
  • the dosage range of the pharmaceutical composition containing plasminogen of the present invention can be, for example, about 0.0001 to 2000 mg/kg, or about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg per day). mg/kg, 10 mg/kg, 50 mg/kg, etc.) subject weight.
  • 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 above or below this exemplified range are also covered, especially taking into account the factors mentioned above.
  • the intermediate dose in the above range is also included in the scope of the present invention.
  • the subject can administer such doses every day, every other day, every week, or according to any other schedule determined through empirical analysis.
  • An exemplary dosage schedule includes 0.01-100 mg/kg for consecutive days. During the administration of the drug of the present invention, it is necessary to evaluate the therapeutic effect and safety in real time.
  • One embodiment of the present invention relates to a product or a kit comprising a component of the plasminogen activation pathway or a related compound (for example, plasminogen).
  • the article preferably includes a container, label or package insert. Suitable containers are bottles, vials, syringes, etc.
  • the container can be made of various materials such as glass or plastic.
  • the container contains a composition that can effectively treat the disease or condition of the present invention and has a sterile access (for example, the container may be an intravenous solution pack or a vial, which contains a stopper that can be penetrated by a hypodermic injection needle of).
  • At least one active agent in the composition is a component of the plasminogen activation pathway or a related compound (for example, plasminogen).
  • the label on or attached to the container indicates that the composition is used to treat the condition of the present invention.
  • the preparation may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further contain other substances required from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.
  • the product includes a package insert with instructions for use, including, for example, a composition that instructs the user of the composition to include components of the plasminogen activation pathway or related compounds (such as plasminogen) As well as other medications for the treatment of concomitant diseases.
  • a composition that instructs the user of the composition to include components of the plasminogen activation pathway or related compounds (such as plasminogen) As well as other medications for the treatment of concomitant diseases.
  • the human plasminogen used in the following examples is derived from donor plasma, based on the method described in the following literature: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry,1965,240(1):541-550; Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu-and Lys-plasminogens and unplasmins. J Biol Chem. 1976 25; 251(12): 3693-9; HAGAN JJ, ABLONDI FB, DE RENZO EC. Purification and biochemical properties of human plasmainogen. J Biol Chem. 1960 Apr; 235: 1005-10, and process optimization from human plasma The purified product is obtained from human donor plasma, and the human plasminogen monomer is >98%.
  • Example 1 Plasminogen promotes the regeneration of the myelin sheath of the corpus callosum in a mouse model of demyelination induced by hexanooxalyl hydrazone
  • mice Twenty 8-week-old C57 male mice were randomly divided into 2 groups, 6 in the blank control group and 14 in the model group.
  • the mice in the blank control group were fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the mice in the model group were fed with 0.2% dicyclohexanone oxalyl hydrazone (cuprizone) model feed (Nantong Trofe Feed Technology Co., Ltd.) ), fed for 6 weeks, induced myelin sheath in mice [1] .
  • the mice in the model group were again randomly divided into two groups according to their body weight, the plasminogen group and the vehicle PBS control group, with 7 mice in each group.
  • mice in the plasminogen group were given plasminogen by tail vein injection of 1mg/0.1ml/mouse/day, the vehicle PBS control group was given the same volume of PBS in the same way, and the mice in the blank control group were not injected.
  • the administration was continued for 14 days. During the administration period, all mice were fed with normal maintenance feed. The first day of administration was set as the first day. On the 15th day, the mice were dissected and their brains were fixed in 4% paraformaldehyde and embedded in dehydration. The fixed tissue samples were dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin. The thickness of the coronal section of brain tissue is 3 ⁇ m. After deparaffinization to water, LFB staining is performed with myelin staining solution. Gradient alcohol dehydration, transparent xylene, and sealing with neutral gum. Under the optical microscope, observe and take pictures.
  • Example 2 Plasminogen promotes the expression of PLP in the corpus callosum of a mouse model of demyelination induced by hexanooxalyl hydrazone
  • mice Twenty 8-week-old C57 male mice were randomly divided into 2 groups, 6 in the blank control group and 14 in the model group.
  • the mice in the blank control group were fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the mice in the model group were fed with 0.2% hexanone oxalyl dihydrazone model feed (Nantong Trofe Feed Technology Co., Ltd.).
  • the mouse model of myelin sheath shedding was induced [1] .
  • the mice in the model group were again randomly divided into two groups according to their body weight, the plasminogen group and the vehicle PBS control group, with 7 mice in each group.
  • mice in the plasminogen group were given plasminogen by tail vein injection of 1mg/0.1ml/mouse/day, the vehicle PBS control group was given the same volume of PBS in the same way, and the mice in the blank control group were not injected.
  • the administration was continued for 3 days. During the administration period, all mice were fed with normal maintenance feed. The first day of administration was set as the first day. On the fourth day, the mice were dissected and their brains were fixed in 4% paraformaldehyde and embedded in dehydration. The fixed tissue samples were dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the coronal section of brain tissue was 3 ⁇ m, and the section was washed once with water after deparaffinization and rehydration.
  • the citric acid repairs for 30 minutes, and after cooling at room temperature for 10 minutes, rinse gently with water.
  • 10% goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour; when the time was up, the goat serum was discarded.
  • Rabbit-derived anti-proteolipid protein (PLP) antibody (Abcam) was incubated overnight at 4°C and washed twice with PBS for 5 minutes each time.
  • the goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, and washed twice with PBS for 5 minutes each.
  • the color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, returned to blue in running water for 5 minutes, and then washed once with PBS. Gradient dehydration is transparent and mounted, and the slices are observed under a 200x optical microscope.
  • PGP Proteolipid protein
  • Example 3 Plasminogen promotes the expression of neurofilament protein (NFP) in the corpus callosum of a mouse model of demyelination induced by hexanooxalyl hydrazone
  • mice Twenty 8-week-old C57 male mice were randomly divided into 2 groups, 6 in the blank control group and 14 in the model group.
  • the mice in the blank control group were fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the mice in the model group were fed with 0.2% hexanone oxalyl dihydrazone model feed (Nantong Trofe Feed Technology Co., Ltd.).
  • the mouse model of myelin sheath shedding was induced [1] .
  • the mice in the model group were again randomly divided into two groups according to their body weight, the plasminogen group and the vehicle PBS control group, with 7 mice in each group.
  • mice in the plasminogen group were given plasminogen by tail vein injection of 1mg/0.1ml/mouse/day, the vehicle PBS control group was given the same volume of PBS in the same way, and the mice in the blank control group were not injected.
  • the administration was continued for 14 days. During the administration period, all mice were fed with normal maintenance feed. The first day of administration was set as the first day. On the 15th day, the mice were dissected and their brains were fixed in 4% paraformaldehyde and embedded in dehydration. The fixed tissue samples were dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the coronal section of brain tissue was 3 ⁇ m, and the section was washed once with water after deparaffinization and rehydration.
  • the citric acid repairs for 30 minutes, and after cooling at room temperature for 10 minutes, rinse gently with water.
  • 10% goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour; when the time was up, the goat serum was discarded.
  • Rabbit-derived anti-NF antibody (Abcam, ab207176) was incubated overnight at 4°C and washed twice with PBS for 5 minutes each time.
  • the goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, and washed twice with PBS for 5 minutes each.
  • the color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, returned to blue in running water for 5 minutes, and then washed once with PBS. Gradient dehydration is transparent and mounted, and the slices are observed under a 200x optical microscope.
  • Neurofilament protein (Neurofilament protein, NFP) is a protein that composes the intermediate filaments of nerve cell axons. Its function is to provide elasticity to make nerve fibers easy to stretch and prevent breakage. It is of great significance in maintaining the cytoskeleton, stabilizing cell morphology and axon transport [3] .
  • Example 4 Plasminogen promotes increased expression of neurofilament protein in the corpus callosum of multiple sclerosis model mice
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item No.: S30349), continuously fed for 42 days to induce multiple sclerosis [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 35 consecutive days. During the administration period, all mice were fed with normal maintenance feed. The mice were sacrificed on the 36th day, and the brain tissues were taken and fixed in 10% formaldehyde solution, dehydrated and embedded. The fixed tissue samples were dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the coronal section of brain tissue was 3 ⁇ m, and the section was washed once with water after deparaffinization and rehydration.
  • the citric acid repairs for 30 minutes, and after cooling at room temperature for 10 minutes, rinse gently with water.
  • 10% goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour; when the time was up, the goat serum was discarded.
  • Rabbit-derived anti-NFP antibody (Abcam, ab207176) was incubated overnight at 4°C and washed twice with PBS for 5 minutes each time.
  • the goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, and washed twice with PBS for 5 minutes each.
  • the color was developed according to DAB kit (Vector laboratories, Inc., USA), after washing 3 times, it was counterstained with hematoxylin for 30 seconds, returning to blue in running water for 5 minutes, and then washed once with PBS. Gradient dehydration was transparent and mounted, and the slices were observed under a 100x optical microscope.
  • Example 5 Plasminogen promotes the increase in the level of corpus callosum proteoprotein in multiple sclerosis model mice
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with maintenance feed, and the model group was fed with a model-making feed containing 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., item number: S30349). Feeding for 42 days, induced multiple sclerosis model [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 35 consecutive days.
  • all mice were fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.). The mice were sacrificed on the 36th day, and the brain tissues were taken and fixed in 10% formaldehyde solution, dehydrated and embedded.
  • the fixed tissue samples were dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the coronal section of brain tissue was 3 ⁇ m, and the section was washed once with water after deparaffinization and rehydration.
  • the citric acid repairs for 30 minutes, and after cooling at room temperature for 10 minutes, rinse gently with water.
  • 10% goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour; when the time was up, the goat serum was discarded.
  • Rabbit-derived anti-PLP antibody (Abcam) was incubated overnight at 4°C and washed twice with PBS for 5 minutes each time.
  • the goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, and washed twice with PBS for 5 minutes each.
  • the color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, returned to blue in running water for 5 minutes, and then washed once with PBS. Gradient dehydration was transparent and mounted, and the slices were observed under a 100x optical microscope.
  • Example 6 Plasminogen promotes the increase of MBP level in the corpus callosum of multiple sclerosis model mice
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item No.: S30349), continuously fed for 42 days to induce multiple sclerosis model [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group are injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group are given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 35 consecutive days. During the administration period, all mice were fed with normal maintenance feed. The mice were sacrificed on the 36th day, and the brain tissues were taken and fixed in 10% formaldehyde solution, dehydrated and embedded. The fixed tissue samples were dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the coronal section of brain tissue was 3 ⁇ m, and the section was washed once with water after deparaffinization and rehydration.
  • the citric acid repairs for 30 minutes, and after cooling at room temperature for 10 minutes, rinse gently with water.
  • 10% goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour; when the time was up, the goat serum was discarded.
  • Rabbit-derived anti-myelin basic protein (MBP) antibody (Abcam) was incubated overnight at 4°C and washed twice with PBS for 5 minutes each time.
  • MBP myelin basic protein
  • the goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, and washed twice with PBS for 5 minutes each.
  • the color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, returned to blue in running water for 5 minutes, and then washed once with PBS. Gradient dehydration was transparent and mounted, and the slices were observed under a 100x optical microscope.
  • Myelin basic protein is a strong basic membrane protein synthesized by oligodendrocytes of the vertebrate central nervous system and Schwann cells of the peripheral nervous system. It contains a variety of basic amino acids.
  • Example 7 Plasminogen promotes the repair of hippocampal injury and inflammation in multiple sclerosis model mice
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item No.: S30349), continuously fed for 42 days to induce multiple sclerosis model [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 35 consecutive days. During the administration period, all mice were fed with normal maintenance feed. The mice were sacrificed on the 36th day, and the brain tissues were taken and fixed in 10% formaldehyde solution, dehydrated and embedded. The fixed tissue samples were dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the coronal section of brain tissue was 3 ⁇ m, and the section was washed once with water after deparaffinization and rehydration.
  • the citric acid repairs for 30 minutes, and after cooling at room temperature for 10 minutes, rinse gently with water.
  • 10% goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour; when the time was up, the goat serum was discarded.
  • Rabbit-derived anti-Ionized calcium binding adaptor molecule-1 (Iba-1) antibody (Abcam, ab178847) was incubated overnight at 4°C and washed twice with PBS for 5 minutes each time.
  • the goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, and washed twice with PBS for 5 minutes each. The color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, returned to blue in running water for 5 minutes, and then washed once with PBS. Gradient dehydration is transparent and mounted, the slices are observed and photographed under a 200x optical microscope, and the photographed pictures are analyzed for optical density of positive staining with Imaging-Pro software.
  • Ionized calcium binding adaptor molecule-1 (Iba-1) is a surface marker of microglia in the central nervous system. As the immune cells in the central nervous system, microglia can quickly sense neurological disorders and be activated when they are pathologically or injured. Activated microglia change significantly in number and morphology and migrate to the injured site to perform a variety of functions, such as phagocytosis of dead cells and increased production of pro-inflammatory cytokines [4] .
  • Example 8 Plasminogen promotes the increase of BDNF levels in the hippocampus of multiple sclerosis model mice
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item No.: S30349), continuously fed for 42 days to induce multiple sclerosis model [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 35 consecutive days. During the administration period, all mice were fed with normal maintenance feed. The mice were sacrificed on the 36th day, and the brain tissues were taken and fixed in 10% formaldehyde solution, dehydrated and embedded. The fixed tissue samples were dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the coronal section of brain tissue was 3 ⁇ m, and the section was washed once with water after deparaffinization and rehydration.
  • the citric acid repairs for 30 minutes, and after cooling at room temperature for 10 minutes, rinse gently with water.
  • 10% goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour; when the time was up, the goat serum was discarded.
  • Rabbit-derived anti-brain-derived neurotrophic factor (BDNF) antibody (BosterBio, PB9075) was incubated overnight at 4°C and washed twice with PBS for 5 minutes each time.
  • BDNF brain-derived neurotrophic factor
  • the goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, and washed twice with PBS for 5 minutes each. The color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, returned to blue in running water for 5 minutes, and then washed once with PBS. Gradient dehydration, transparency and mounting, slices were observed and photographed under a 400x optical microscope, and the photographed pictures were analyzed for optical density of positive staining with Imaging-Pro software.
  • BDNF Mature brain-derived neurotrophic factor
  • BDNF Mature brain-derived neurotrophic factor
  • Example 9 Plasminogen promotes increased activity of hippocampal astrocytes in multiple sclerosis model mice
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item No.: S30349), continuously fed for 42 days to induce multiple sclerosis model [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 35 consecutive days. During the administration period, all mice were fed with normal maintenance feed. The mice were sacrificed on the 36th day, and the brain tissues were taken and fixed in 10% formaldehyde solution, dehydrated and embedded. The fixed tissue samples were dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the coronal section of brain tissue was 3 ⁇ m, and the section was washed once with water after deparaffinization and rehydration.
  • the citric acid repairs for 30 minutes, and after cooling at room temperature for 10 minutes, rinse gently with water.
  • 10% goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour; when the time was up, the goat serum was discarded.
  • Rabbit-derived anti-glial fibrillary acidic protein (GFAP) antibody (Abcam, ab4648) was incubated overnight at 4°C, washed twice with PBS, 5 minutes each time.
  • the goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, and washed twice with PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), after washing 3 times, it was counterstained with hematoxylin for 30 seconds, returning to blue in running water for 5 minutes, and then washed once with PBS. Gradient dehydration is transparent and mounted, the slices are observed and photographed under a 200x optical microscope, and the photographed pictures are analyzed for optical density of positive staining with Imaging-Pro software.
  • Glial fibrillary acidic protein is a hallmark intermediate filament protein in astrocytes, which participates in the formation of the cytoskeleton and maintains its tensile strength [6] .
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item: S30349), continuously fed for 42 days to induce multiple citation syndrome [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 28 consecutive days. During the administration period, all mice were fed with normal maintenance feed. The open field experiment was conducted on the 29th day.
  • Psychiatric symptoms often appear in patients with multiple sclerosis, most of which are depression, irritability, and temper tantrums. Some patients experience euphoria, excitement, or apathy, lethargy, strong crying and strong laughter, unresponsiveness, low intelligence, and repetition. Language, suspicion and delusion of doctrine, etc. There may also be cognitive dysfunctions such as memory loss and attention impairment [7] .
  • the Smart system is a complete and easy-to-use video tracking system for evaluating the behavior of experimental animals. It allows to record trajectories, activities, specific behaviors (such as rotation, stretching and feeding) and events, and perform calculations of various analysis parameters.
  • This experiment uses the Smart3.0 system to record and analyze the movement of the mice. The parameters include the total moving distance, the border resting time rate, the average movement speed of the central area and the average movement speed of the border. In each experiment, 70% alcohol was used to wipe the box to prevent the preference of smell generation [8] .
  • the design principle of the open field experiment is based on the avoidance of mice, which means that the mice are afraid to open
  • the avoidance is evaluated by the activities of the mice in the surrounding areas (four corners and four sides) of the wilderness. Looking at the activity time in the surrounding area, which reflects the avoidance, the time is reduced, indicating that the mice are more "adventurous". The significant increase in activity time in the central area indicates that avoidance and anxiety (depression) levels are low.
  • the total movement distance is the total movement distance of the mice in each area during the open field experiment.
  • the results showed that the mice in the blank control group had a certain total distance of movement, and the total distance of movement of mice in the vehicle group was significantly increased.
  • the total distance of movement of mice in the drug group was significantly smaller than that in the vehicle group, and the statistical difference was significant (* indicates P ⁇ 0.05) ( Figure 10), and is similar to the blank control group. It shows that plasminogen can alleviate the depressive behavior of multiple sclerosis model mice to a certain extent.
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item No.: S30349), continuously fed for 42 days to induce multiple sclerosis [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 28 consecutive days. During the administration period, all mice were fed with normal maintenance feed. The open field experiment was conducted on the 29th day.
  • Percentage of resting time in border zone resting time in border zone/total observation time.
  • the results showed that the blank control group had a certain percentage of resting time in the border zone, about 57.8%; the percentage of resting time in the border zone of the vehicle group was significantly reduced, about 49.3%, and the mice in the administration group were resting in the border zone.
  • the time percentage is about 58.4%, which is significantly greater than that of the vehicle group, and the statistical difference is significant (* indicates P ⁇ 0.05) ( Figure 11), and it is similar to the blank control group. It shows that plasminogen can alleviate the depressive behavior of multiple sclerosis model mice to a certain extent.
  • Example 12 Plasminogen improves the social memory ability of multiple sclerosis model mice
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item No.: S30349), continuously fed for 42 days to induce multiple sclerosis [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 34 consecutive days. During the administration period, all mice were fed with normal maintenance feed. A three-box social test experiment was conducted on the 35th day.
  • mice Before the start of the three-box experiment, put the mice in the behavioral testing room for half an hour; separate the three boxes with a transparent glass resin plate, and put the test mice in the middle box to adapt for 5 minutes; Mice 1 is randomly placed in the metal cage in the left or right box, and the metal cage in the other box is empty; remove the glass resin plate separating the boxes, so that the test mice can move freely in the three boxes. Minutes; immediately start shooting and record the relevant parameters: the first stage: 1) The number and duration of direct contact between the experimental mouse and the unfamiliar mouse 1 or an empty metal cage, 3-5cm around the metal cage is defined as contact Scope; 2) The number of times the experimental mouse enters each box and the duration.
  • mice When the head and four paws of the mouse enter a box, it is considered to be in that box.
  • the second stage In the second stage of the experiment, put a second strange mouse (Strange Mouse 2) into the empty metal cage, and then record for ten minutes, observe the experimental mice and strange mice 1 and strange mice 2 The time and number of contacts between each other. Normal mice will show social behaviors. In a one-stage experiment, the time and frequency of communication between normal mice and unfamiliar mice 1 is usually significantly longer than that of empty metal cages. At the same time, mice also have the characteristics of memory and "love the new and dislike the old". Therefore, in the second stage, the mice will prefer to communicate with unfamiliar mice 1 that have not been wiped before, rather than the strange mice that have been communicated for ten minutes. .
  • Percent resting time resting time/total observation time.
  • rats in the vehicle group and the administration group start to take the drug, which is recorded as the first day of administration.
  • the rats in the administration group are injected with 35mg/kg plasminogen through the tail vein, and the rats in the vehicle group are injected with 3.5ml through the tail vein. /kg vehicle, continuous administration for 6 days, the blank control group rats are not treated.
  • the open field experiment was performed on the 7th day of administration.
  • rats in the vehicle group and the administration group start to take the drug, which is recorded as the first day of administration.
  • the rats in the administration group are injected with 35mg/kg plasminogen through the tail vein, and the rats in the vehicle group are injected with 3.5ml through the tail vein. /kg vehicle, continuous administration for 6 days, the blank control group rats are not treated.
  • the open field experiment was performed on the 7th day of administration.
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item No.: S30349), continuously fed for 42 days to induce multiple sclerosis model [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 20 consecutive days. During the administration period, all mice were fed with normal maintenance feed. The elevated plus maze experiment was performed on the 21st day.
  • the elevated plus maze uses the animal's exploring characteristics of new environments and the fear of hanging open arms to form conflicting behaviors to investigate the animal's anxiety state.
  • the elevated plus maze has a pair of open arms and a pair of closed arms. Rodents tend to move in the closed arms due to their dark addiction, but they will move in the open arms out of curiosity and exploration, facing novelty.
  • the anti-anxiety drugs can significantly increase the number and time of entering the open arm.
  • the distance between the plus maze is higher than the ground, which is equivalent to standing on a cliff, causing fear and anxiety in the experimental subjects.
  • the elevated plus maze is widely used in the fields of science-research and computer-assisted teaching in multiple disciplines such as new drug development/screening/evaluation, pharmacology, toxicology, preventive medicine, neurobiology, animal psychology, and behavioral biology. Medical colleges and scientific research institutions conduct classic experiments on behavioral research, especially anxiety and depression research.
  • mice were put into the maze from the central grid to the closed arm, and their activities within 5 minutes were recorded. Observation indicators include: open arm entry times (two forepaws must enter the arm), open arm stay time, closed arm entry times, and closed arm stay time. Calculate the proportion of open arm residence time, the proportion of open arm entry times, and the total number of entries in the elevated plus maze. After the experiment was completed, the mice were taken out, the arms were cleaned, and alcohol was sprayed to remove the odor. Finally, use animal behavior software for data analysis.
  • Percentage of open arm entry total number of open arm entry/sum of total number of open arm and closed arm entry.
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with maintenance feed, and the model group was fed with a model-making feed containing 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., item number: S30349). Feeding for 42 days, induced multiple sclerosis model [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 20 consecutive days. During the administration period, all mice were fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.). The elevated plus maze experiment was performed on the 21st day.
  • Percentage of closed arm entry total number of closed arm entry/total number of open arm and closed arm entry.
  • the results showed that the blank control group mice had a certain closed arm entry percentage, about 34.4%; the vehicle group mice’s closed arm entry percentage significantly decreased, about 28.1%; the drug group mice’s closed arm entry percentage was about 37.1% , Significantly larger than the vehicle group, statistically significant (P 0.007) (Figure 16), and similar to the blank control group. It shows that plasminogen can alleviate the anxiety and depression behavior of multiple sclerosis model mice to a certain extent.
  • Example 17 Plasminogen relieves anxiety and depression behavior in multiple sclerosis model mice
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item No.: S30349), continuously fed for 42 days to induce multiple sclerosis model [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group are injected with the solvent at 0.1ml/mouse/day through the tail vein, and the mice in the administration group are taken at 1mg/mouse. Plasminogen was injected into the tail vein every day for 27 consecutive days. During the administration period, all mice were fed with normal maintenance feed. The elevated plus maze experiment was performed on the 28th day.
  • the total distance of the closed arm movement is the total distance of the closed arm movement of the mice during the test period of the elevated plus maze experiment.
  • the results showed that the mice in the blank control group had a certain total distance of closed arm movement, the total distance of closed arm movement of mice in the vehicle group was significantly increased, and the total distance of closed arm movement of mice in the drug group was significantly less than that of the vehicle group.
  • the statistical difference was significant (* It means P ⁇ 0.05) ( Figure 17), and it is similar to the blank control group. It shows that plasminogen can alleviate the anxiety and depression behavior of multiple sclerosis model mice to a certain extent.
  • Example 18 Plasminogen relieves anxiety and depression behavior in multiple sclerosis model mice
  • mice Thirty female C57 mice were selected and weighed before modeling. All mice were randomly divided into 2 groups after the abnormal mice were excluded according to their body weights, with 8 mice in the blank control group and 22 mice in the model group. After the grouping was completed, the blank control group was fed with normal maintenance feed (purchased from Beijing Keyao Xieli Co., Ltd.), and the model group was fed with 0.6% dicyclohexanone oxalyl dihydrazone (CPZ) (manufacturer: Shanghai Yuanye Biological Technology Co., Ltd.) , Item No.: S30349), continuously fed for 42 days to induce multiple sclerosis model [1] . After the completion of the model building, all mice were tested in an open field experiment.
  • CPZ dicyclohexanone oxalyl dihydrazone
  • the model group mice were divided into groups, 11 mice in the vehicle group and 11 mice in the drug group. After the grouping is completed, all mice start the administration, which is recorded as the first day of administration.
  • the mice in the blank control group and the vehicle group will be injected with 0.1ml/mouse/day through the tail vein, and the mice in the administration group will be given 1mg/mouse.
  • Plasminogen was injected into the tail vein every day for 27 consecutive days. During the administration period, all mice were fed with normal maintenance feed. The elevated plus maze experiment was performed on the 28th day.
  • the percentage of closed arm rest time closed arm rest time / the sum of closed arm exercise time and closed arm rest time.
  • the results showed that the mice in the blank control group had a certain percentage of closed arm rest time, about 51.8%; the percentage of closed arm rest time of mice in the vehicle group was significantly reduced, about 36.8%; the percentage of closed arm rest time of mice in the drug group was about 51.8% It was 50.1%, which was significantly larger than the vehicle group, and the statistical difference was significant (* means P ⁇ 0.05) ( Figure 18), and it was similar to the blank control group. It shows that plasminogen can alleviate the anxiety and depression behavior of multiple sclerosis model mice to a certain extent.

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Abstract

一种预防和治疗多发性硬化症的方法，包括给药受试者治疗有效量的纤维蛋白溶酶原激活途径组分，还涉及用于治疗上述病症的包含纤维蛋白溶酶原激活途径组分的药物、药物组合物、制品、试剂盒。

Description

一种预防和治疗多发性硬化症的方法和药物 技术领域
本发明涉及一种预防或治疗多发性硬化症的方法，包括给药受试者有效量的纤维蛋白溶酶原激活途径的组分或其相关化合物，例如纤溶酶原，以修复损伤神经，改善临床症状和体征。
背景技术
多发性硬化症(multiple sclerosis，MS)是最常见的一种中枢神经脱髓鞘疾病，磁共振成像检查(MRI)可以看见大脑以及脊髓内多发的斑块样的病灶。该病的病因尚不明确，与遗传因素、病毒感染、自身免疫反应等有一定关系。本病急性活动期中枢神经白质有多发性炎性脱髓鞘斑，陈旧病变则由于胶质纤维增生而形成钙化斑，以多发病灶、缓解、复发病程为特点，好发于视神经、脊髓和脑干。目前的治疗方法包括激素治疗、β-干扰素和免疫抑制剂治疗，不甚理想，需要寻找其他的治疗方法。
发明概述
本发明研究发现纤溶酶原可以明显促进神经髓鞘的再生和修复，预防和治疗多发性硬化症。
具体地，本发明涉及如下各项：
1.一方面，本申请涉及一种预防和治疗多发性硬化症的方法，包括给药受试者治疗有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请涉及选自如下的一种或多种化合物在制备预防和治疗 多发性硬化症的药物中的用途，所述选自如下的一种或多种化合物为：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请涉及预防和治疗多发性硬化症的药物，包含选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请涉及选自如下的一种或多种化合物用于预防和治疗多发性硬化症的用途，其中选自如下的一种或多种化合物为：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
2.项1所述的方法、用途、或药物，其中所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro-plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。
3.项1的方法、用途、或药物，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抑制剂，例如抗体。
4.项1-3任一项的方法、用途、或药物，其中所述化合物具有选自如下一项或多项的活性：促进神经髓鞘的再生、促进髓鞘蛋白表达(例如促进PLP或MBP蛋白的表达)、促进神经组织NFP的表达、促进神经纤维再生、促进神经组织NFP的表达、提高神经组织MBP水平、增加神经组织小胶质细胞数量、促进神经组织炎症修复、促进神经组织星形胶质细胞活性、提高神经组织BDNF水平、促进神经组织GFAP表达、改善受试者的社交行为能力、改善受试者的社交记忆能力、缓解受试者的抑郁行为、缓解受试者的焦虑行为。
5.项1-4任一项的方法、用途、或药物，其中所述纤溶酶原缓解受试者的精神症状，例如抑郁症状或焦虑症状。
6.项1-5任一项的方法、用途、或药物，其中所述化合物为纤溶酶原(也称纤维蛋白原)。
7.项1-6任一项的方法、用途、或药物，其中所述纤溶酶原与序列2具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性，例如蛋白水解活性、赖氨酸结合活性、或蛋白水解活性和赖氨酸结合活性。
8.项1-6任一项的方法、用途、或药物，所述纤溶酶原包含与序列14所示的纤溶酶原活性片段具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列、并且具有纤溶酶原的蛋白水解活性。
在一些实施方案中，上述纤溶酶原为序列2所述纤溶酶原的保守取代变体。
9.项1-6任一项的方法、用途、或药物，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。
10.项1-6任一项的方法、用途、或药物，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。
11.项1-10任一项的方法、用途、或药物，其中所述化合物与一种或多种其他治疗方法或药物联合使用。
12.项11的方法、用途、或药物，其中所述其他治疗方法包括手术治 疗、细胞治疗(包括干细胞治疗)和物理治疗(例如物理的支持治疗，例如医疗器械辅助治疗，比如人机接口技术)。
13.项11的方法、用途、或药物，其中所述其他药物包括激素、免疫抑制剂、神经营养药物、抗菌素和抗病毒药物。
14.项1-13任一项的方法、用途、或药物，其中所述化合物通过选自如下的任一或多种方式或途径给药：鼻腔吸入、雾化吸入、滴鼻液、滴眼液、滴耳液、静脉内、腹膜内、皮下、颅内、鞘内、动脉内和肌肉内。
在本发明的上述任一实施方案中，所述纤溶酶原可与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性，例如蛋白水解活性、赖氨酸结合活性、或蛋白水解活性和赖氨酸结合活性。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质，例如蛋白水解活性、赖氨酸结合活性、或蛋白水解活性和赖氨酸结合活性。
在一些实施方案中，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。在一些实施方案中，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。在一些实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。
在一些实施方案中，所述受试者是人。在一些实施方案中，所述受试者缺乏或缺失纤溶酶原。在一些实施方案中，所述缺乏或缺失是先天的、继发的和/或局部的。
在一些实施方案中，所述药物组合物包含药学上可接受的载剂和用于前述方法的纤溶酶原。在一些实施方案中，所述试剂盒可以是预防性或治 疗性试剂盒，其包含：(i)用于前述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。在一些实施方案中，所述构件为注射器或小瓶。在一些实施方案中，所述试剂盒还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施前述任一方法。
在一些实施方案中，所述制品包含：含有标签的容器；和包含(i)用于前述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施前述任一方法。
在一些实施方案中，所述试剂盒或制品还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。
在前述方法的一些实施方案中，所述纤溶酶原通过全身或局部给药，优选通过以下途径施用：静脉内、肌内、皮下给予纤溶酶原来进行治疗。在前述方法的一些实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在前述方法的一些实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。
本发明明确涵盖了属于本发明实施方案之间的技术特征的所有组合，并且这些组合后的技术方案在本申请中已经明确公开，就像上述技术方案已经单独且明确公开一样。另外，本发明还明确涵盖各个实施方案及其要素的之间的组合，该组合后的技术方案在本文中明确公开。
附图简述
图1A-D己酮草酰双腙诱导脱髓鞘模型小鼠给予纤溶酶原14天脑胼胝体LFB染色结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，空白对照组胼胝体髓鞘形态基本正常，给纤溶酶原组胼胝体髓鞘阳性着色(箭头标识)明显多于给溶媒PBS对照组，且统计差异显著(*为P<0.05)。说明纤溶酶原能够减少胼胝体脱髓鞘，促进己酮草酰双腙诱导脱髓鞘模型小鼠髓鞘损伤的修复。
图2A-C己酮草酰双腙诱导脱髓鞘模型小鼠给予纤溶酶原3天脑胼胝体蛋白脂蛋白(PLP)免疫染色代表性图片。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。结果显示，给纤溶酶原组胼胝体PLP的表达(箭头标识)明显多于给溶媒PBS对照组，与后者相比表达更加接近空白对照组小鼠。说明纤溶酶原促进胼胝体PLP的表达，促进己酮草酰双腙诱导脱髓鞘模型小鼠髓鞘再生。
图3A-D己酮草酰双腙诱导脱髓鞘模型小鼠给予纤溶酶原14天脑神经丝蛋白(NFP)免疫染色观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，给纤溶酶原组小鼠胼胝体NFP的表达(箭头标识)明显多于给溶媒PBS对照组，统计差异显著(*为P<0.05)，且与给溶媒PBS对照组(全文也简称为溶媒组)相比给纤溶酶原组胼胝体NFP的表达更加接近空白对照组。说明纤溶酶原能够促进NFP的表达，从而促进神经纤维再生。
图4给予纤溶酶原35天后多发性硬化症模型小鼠胼胝体NFP免疫组织化学染色结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为平均光密度定量分析结果。结果显示，空白对照组胼胝体表达一定水平的NFP(箭头标识)，溶媒组小鼠胼胝体NFP的表达明显减少，给纤溶酶原组(全文也简称为给药组)小鼠胼胝体NFP表达水平明显高于溶媒小鼠，且统计差异显著(*表示P<0.05)。该结果提示纤溶酶原能够促进多发性硬化症模型小鼠胼胝体NFP的表达。
图5A-C给予纤溶酶原35天后多发性硬化症模型小鼠胼胝体PLP免疫组织化学染色结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。结果显示，空白对照组胼胝体表达一定水平的PLP(箭头标识)，溶媒组小鼠胼胝体PLP的表达明显减少，给药组小鼠胼胝体PLP的表达水平明显高于溶媒小鼠。该结果提示纤溶酶原能够促进多发性硬化症模型小鼠胼胝体PLP的表达。
图6A-D给予纤溶酶原35天后多发性硬化症模型小鼠胼胝体MBP免疫组织化学染色结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为平均光密度定量分析结果。结果显示，空白对照组胼胝体表达一定水平的MBP(箭头标识)，给药组小鼠胼胝体MBP明显高于溶 媒组小鼠，且统计差异接近显著(P＝0.063)。该结果提示纤溶酶原能够促进多发性硬化症模型小鼠胼胝体MBP的水平增加。
图7A-D给予纤溶酶原35天后多发性硬化症模型小鼠海马Iba-1免疫组织化学染色结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为平均光密度定量分析结果。结果显示，空白对照组小鼠海马存在一定量的小胶质细胞(箭头标识)，溶媒组小鼠海马小胶质细胞量明显增加，给药组小鼠海马小胶质细胞数量明显多于溶媒组，且统计差异显著(*表示P<0.05，***表示P<0.001)。提示纤溶酶原能够促进多发性硬化症模型小鼠海马损伤炎症修复。
图8A-D给予纤溶酶原35天后多发性硬化症模型小鼠海马BDNF免疫组织化学染色结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为平均光密度定量分析结果。结果显示，空白对照组小鼠海马具有一定水平的BDNF(箭头标识)，溶媒组小鼠海马BDNF水平增加，给药组小鼠海马BDNF水平明显高于溶媒组,且统计差异接近显著(P＝0.095)。提示纤溶酶原能够促进多发性硬化症模型小鼠海马BDNF水平增加。
图9A-D给予纤溶酶原35天后多发性硬化症模型小鼠海马GFAP免疫组织化学染色结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为平均光密度定量分析结果。结果显示，空白对照组小鼠海马表达一定水平的GFAP(箭头标识)，溶媒组小鼠海马GFAP表达减少，给药组小鼠海马GFAP表达明显高于溶媒组，且统计差异接近显著(P＝0.051)。提示纤溶酶原能够促进多发性硬化症模型小鼠海马GFAP表达增加，促进星形胶质细胞活性增加。
图10给予纤溶酶原28天后多发性硬化症模型小鼠旷场实验总运动路程统计结果。结果显示，空白对照组小鼠具有一定的总运动路程，溶媒组小鼠总运动路程明显增加，给药组小鼠总运动路程明显小于溶媒组，统计差异显著(*表示P<0.05)，且与空白对照组相近。说明纤溶酶原能够缓解多发性硬化症模型小鼠抑郁行为。
图11边界区静息时间百分率＝边界区静息时间/总观察时间。结果显示，空白对照组小鼠具有一定的边界区静息时间百分率，约为57.8％，；溶 媒组小鼠边界区静息时间百分率明显降低，约为49.3％，给药组小鼠边界区静息时间百分率约为58.4％，明显大于溶媒组，统计差异显著(*表示P<0.05))，且与空白对照组相近。说明纤溶酶原能够一定程度地缓解多发性硬化症模型小鼠抑郁行为。
图12给予纤溶酶原34天后多发性硬化症模型小鼠三箱实验第二阶段陌生小鼠2接触范围内静息时间百分率统计结果。静息时间百分率＝静息时间/总观察时间。三箱社交测试实验第二阶段结果显示，空白对照组小鼠具有一定的陌生小鼠2接触范围内的静息时间百分率，约为13.7％；溶媒组明显降低，约为10.6％；给药组约为16.1％，明显高于溶媒组，且统计差异接近显著(P＝0.075)。说明纤溶酶原能够改善多发性硬化症模型小鼠的社交记忆。
图13给予纤溶酶原6天后多发性硬化症模型模型大鼠旷场实验边界区运动路程百分率统计结果。边界区运动路程为旷场实验测试时间内边界区运动轨迹长度。结果显示，空白对照组具有一定的边界区运动路程百分率，约为91.1％；溶媒组明显增加，约为93.6％；给药组约为88.1％，明显低于溶媒组，且统计差异显著(*表示P<0.05)。说明纤溶酶原能够缓解多发性硬化症模型小鼠的抑郁行为。图14给予纤溶酶原6天后多发性硬化症模型大鼠旷场实验中心区运动路程百分率统计结果。结果显示，空白对照组具有一定的中心区路程百分率，约为8.9％；溶媒组明显降低，约为6.4％；给药组约为11.9％，明显高于溶媒组，且统计差异显著(*表示P<0.05)。说明纤溶酶原能够缓解多发性硬化症模型大鼠的焦虑行为。
图15给药纤溶酶原20天后多发性硬化症模型小鼠高架十字迷宫测试开放臂进入百分率统计结果。结果显示，空白对照组小鼠具有一定的开放臂进入百分率，约为14.9％；溶媒组小鼠开放臂进入百分率明显增加，约为23.0％；给药组小鼠开放臂进入百分率约为14.5％，明显少于溶媒组，统计差异显著(P＝0.015)，且与空白对照组相近。说明纤溶酶原能够一定程度缓解多发性硬化症模型小鼠焦虑行为。
图16给药纤溶酶原20天后多发性硬化症模型小鼠高架十字迷宫测试闭合臂进入百分率统计结果。结果显示，空白对照组小鼠具有一定的闭合臂进入百分率，约为34.4％；溶媒组小鼠闭合臂进入百分率明显降低，约 为28.1％；给药组小鼠闭合臂进入百分率约为37.1％，明显大于溶媒组，统计差异显著(P＝0.007)，且与空白对照组相近。说明纤溶酶原能够一定程度缓解多发性硬化症模型小鼠焦虑抑郁行为。图17给予纤溶酶原27天后多发性硬化症模型小鼠高架十字迷宫测试闭合臂运动路程统计结果。闭合臂运动总路程为高架十字迷宫实验测试时间内小鼠闭合臂运动总路程。结果显示，空白对照组小鼠具有一定的闭合臂运动总路程，溶媒组小鼠闭合臂运动总路程明显增加，给药组小鼠闭合臂运动总路程明显少于溶媒组，统计差异显著(*表示P<0.05)，且与空白对照组相近。说明纤溶酶原能够一定程度缓解多发性硬化症模型小鼠焦虑抑郁行为。图18给予纤溶酶原27天后多发性硬化症模型小鼠高架十字迷宫测试闭合臂静息时间百分率统计结果。结果显示，空白对照组小鼠具有一定的闭合臂休息时间百分率，约为51.8％；溶媒组小鼠闭合臂休息时间百分率明显降低，约为36.8％；给药组小鼠闭合臂休息时间百分率约为50.1％，明显大于溶媒组，统计差异显著(*表示P<0.05)，且与空白对照组相近。说明纤溶酶原能够一定程度缓解多发性硬化症模型小鼠焦虑抑郁行为。
发明详述
纤维蛋白溶解系统(Fibrinolytic system)也称纤溶系统，为参与纤维蛋白溶解(纤溶)过程的一系列化学物质组成的系统，主要包括纤维蛋白溶解酶原(纤溶酶原)、纤溶酶、纤溶酶原激活物、纤溶抑制剂。纤溶酶原激活物包括组织型纤溶酶原激活物(t-PA)和尿激酶型纤溶酶原激活物(u-PA)。t-PA是一种丝氨酸蛋白酶，由血管内皮细胞合成。t-PA激活纤溶酶原，此过程主要在纤维蛋白上进行；尿激酶型纤溶酶原激活物(u-PA)由肾小管上皮细胞和血管内皮细胞产生，可以直接激活纤溶酶原而不需要纤维蛋白作为辅因子。纤溶酶原(PLG)由肝脏合成，当血液凝固时，PLG大量吸附在纤维蛋白网上，在t-PA或u-PA的作用下，被激活为纤溶酶，促使纤维蛋白溶解。纤溶酶(PL)是一种丝氨酸蛋白酶，作用如下：降解纤维蛋白和纤维蛋白原；水解多种凝血因子Ⅴ、Ⅷ、Ⅹ、Ⅶ、Ⅺ、Ⅱ等；使纤溶酶原转变为纤溶酶；水解补体等。纤溶抑制物：包括纤溶酶原激活物抑制剂(PAI)和α2抗纤溶酶(α2-AP)。PAI主要有PAI-1和PAI-2两种形式，能特异性与t-PA以1：1比例结合，从而使其失活，同 时激活PLG。α2-AP由肝脏合成，与PL以1：1比例结合形成复合物，抑制PL活性；FⅩⅢ使α2-AP以共价键与纤维蛋白结合，减弱了纤维蛋白对PL作用的敏感性。体内抑制纤溶系统活性的物质：PAI-1，补体C1抑制物；α2抗纤溶酶；α2巨球蛋白。
本发明的术语“纤维蛋白溶酶原激活途径的组分”涵盖：
1.纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、微纤溶酶原(micro-plasminogen)、delta-纤溶酶原；它们的变体或类似物；
2.纤维蛋白溶酶以及它们的变体或类似物；和
3.纤维蛋白溶酶原激活剂，例如tPA和uPA以及包含一个或多个tPA或uPA的结构域(如一个或多个kringle结构域和蛋白水解结构域)的tPA或uPA变体和类似物。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式，以及通过添加、删除和/或取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸、仍然具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA活性的蛋白质。例如，纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”包括通过例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个保守性氨基酸取代获得的这些蛋白质的突变变体。
本发明的“纤溶酶原变体”涵盖与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性的蛋白质。例如本发明的“纤溶酶原变体”可以是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。具体地，本发明纤溶酶原变体包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式，以及通过保守性氨基酸取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1- 20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸获得的这些蛋白质的突变变体。
本发明的纤溶酶原可以为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体，例如序列2、6、8、10或12所示的纤溶酶原，例如序列2所示的人天然纤溶酶原。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“类似物”包括分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用的化合物。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和“类似物”涵盖包含一个或多个结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和“类似物”。例如，纤维蛋白溶酶原的“变体”和“类似物”涵盖包含一个或多个纤溶酶原结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶原变体和类似物，例如小纤维蛋白溶酶原(mini-plasminogen)。纤维蛋白溶酶的“变体”和“类似物”涵盖包含一个或多个纤维蛋白溶酶结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶“变体”和“类似物”，例如小纤维蛋白溶酶(mini-plasmin)和δ-纤维蛋白溶酶(delta-plasmin)。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的“变体”或“类似物”是否分别具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的活性，或者是否分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用可以通过本领域已知方法进行检测，例如，通过基于酶谱法(enzymography)、ELISA(酶联免疫吸附测定)和FACS(荧光激活细胞分选方法)通过激活的纤维蛋白溶酶活性水平来衡量，例如可以参照选自如下文献中记载的方法测量：Ny，A.，Leonardsson，G.，Hagglund，A.C，Hagglof，P.，Ploplis，V.A.，Carmeliet，P.and Ny，T.(1999).Ovulation inplasminogen-deficient mice.Endocrinology 140，5030-5035；Silverstein RL,Leung LL,Harpel PC,Nachman RL(November 1984)."Complex formation of platelet thrombospondin with plasminogen.Modulation of activation by tissue activator".J.Clin.Invest.74(5):1625–33；Gravanis I,Tsirka SE(February  2008)."Tissue-type plasminogen activator as a therapeutic target in stroke".Expert Opinion on Therapeutic Targets.12(2):159–70；Geiger M,Huber K,Wojta J,Stingl L,Espana F,Griffin JH,Binder BR(Aug 1989)."Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo".Blood.74(2):722–8.
在本发明的一些实施方案中，本发明的“纤维蛋白溶酶原激活途径的组分”为纤溶酶原，选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的保守突变变体或其片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的保守突变变体或其片段。在一些实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。在一些实施方案中，所述纤溶酶原是如序列2所示的人天然纤溶酶原。
“能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物”指能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的任何化合物，例如tPA、uPA、链激酶、沙芦普酶、阿替普酶、瑞替普酶、替奈普酶、阿尼普酶、孟替普酶、拉诺替普酶、帕米普酶、葡激酶。
本发明“纤溶抑制剂的拮抗剂”为拮抗、减弱、封闭、阻止纤溶抑制剂作用的化合物。所述纤溶抑制剂例如PAI-1、补体C1抑制物、α2抗纤溶酶和α2巨球蛋白。所述拮抗剂例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抗体，或阻断或下调例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白表达的反义RNA或小RNA，或占据PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的结合位点但无PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白功能的化合物”，或封闭PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的结合结构域和/或活性结构域的化合物。
纤溶酶是纤溶酶原激活系统(PA系统)的关键组分。它是一种广谱的蛋白酶，能够水解细胞外基质(ECM)的几个组分，包括纤维蛋白、明胶、纤连蛋白、层粘连蛋白和蛋白聚糖。此外，纤溶酶能将一些金属蛋白酶前体(pro-MMPs)激活形成具有活性的金属蛋白酶(MMPs)。因此纤溶酶被认为是胞外蛋白水解作用的一个重要的上游调节物。纤溶酶是由纤溶酶原通过两种生理性的PAs：组织型纤溶酶原激活剂(tPA)或尿激酶型纤溶酶原激活剂(uPA)蛋白水解形成的。由于纤溶酶原在血浆和其他体液中相对水平较高，传统上认为PA系统的调节主要通过PAs的合成和活性水平实现。PA系统组分的合成受不同因素严格调节，如激素、生长因子和细胞因子。此外，还存在纤溶酶和PAs的特定生理抑制剂。纤溶酶的主要抑制剂是α2-抗纤溶酶(α2-antiplasmin)。PAs的活性同时被uPA和tPA的纤溶酶原激活剂抑制剂-1(PAI-1)抑制以及主要抑制uPA的溶酶原激活剂抑制剂-2(PAI-2)调节。某些细胞表面具有直接水解活性的uPA特异性细胞表面受体(uPAR)。
人纤溶酶原是一个单链糖蛋白，由791个氨基酸组成，分子量约为92kDa。纤溶酶原主要在肝脏合成，大量存在于胞外液中。血浆中纤溶酶原含量约为2μM。因此纤溶酶原是组织和体液中蛋白质水解活性的一个巨大的潜在来源。纤溶酶原存在两种分子形式：谷氨酸-纤溶酶原(Glu-plasminogen)和赖氨酸-纤溶酶原(Lys-plasminogen)。天然分泌和未裂解形式的纤溶酶原具有一个氨基末端(N-末端)谷氨酸，因此被称为谷氨酸-纤溶酶原。然而，在纤溶酶存在时，谷氨酸-纤溶酶原在Lys76-Lys77处水解成为赖氨酸-纤溶酶原。与谷氨酸-纤溶酶原相比，赖氨酸-纤溶酶原与纤维蛋白具有更高的亲和力，并可以更高的速率被PAs激活。这两种形式的纤溶酶原的Arg560-Val561肽键可被uPA或tPA切割，导致二硫键连接的双链蛋白酶纤溶酶的形成。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringles，羧基末端部分包含蛋白酶结构域。一些kringles含有介导纤溶酶原与纤维蛋白及其抑制剂α2-AP特异性相互作用的赖氨酸结合位点。最新发现一个纤溶酶原为38kDa的片段，其中包括kringles1-4，是血管生成的有效抑制剂。这个片段被命名为血管抑素，可通过几个蛋白酶水解纤溶酶原产生。
纤溶酶的主要底物是纤维蛋白，纤维蛋白的溶解是预防病理性血栓形成的关键。纤溶酶还具有对ECM几个组分的底物特异性，包括层粘连蛋白、纤连蛋白、蛋白聚糖和明胶，表明纤溶酶在ECM重建中也起着重要作用。间接地，纤溶酶还可以通过转化某些蛋白酶前体为活性蛋白酶来降解ECM的其他组分，包括MMP-1，MMP-2，MMP-3和MMP-9。因此，有人提出，纤溶酶可能是细胞外蛋白水解的一个重要的上游调节器。此外，纤溶酶具有激活某些潜在形式的生长因子的能力。在体外，纤溶酶还能水解补体系统的组分并释放趋化补体片段。
“纤溶酶”是存在于血液中的一种非常重要的酶，能将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体。
“纤溶酶原”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源纤溶酶原氨基酸序列(序列4)计算由810个氨基酸组成，分子量约为90kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的纤溶酶原包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基Cys103-Cys181，Kringle2包括残基Glu184-Cys262，Kringle3包括残基Cys275-Cys352，Kringle4包括残基Cys377-Cys454，Kringle5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是人天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。Delta-纤溶酶原(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶域(也称蛋白酶结构域(protease domain，PD))，有文献报道了delta-纤溶酶原的氨基酸序列(序列8)，编码该氨基酸序列的cDNA序列如序列7所示。小纤溶酶原(Mini-plasminogen)由Kringle5和丝氨酸蛋白 酶域组成，有文献报道其包括残基Val443-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，其氨基酸序列如序列10所示，编码该氨基酸序列的cDNA序列如序列9所示。而微纤溶酶原(Micro-plasminogen)仅含有丝氨酸蛋白酶结构域，有文献报道其氨基酸序列包括残基Ala543-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，也有专利文献CN102154253A报道其序列包括残基Lys531-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，在本专利申请中微纤溶酶原序列参考专利文献CN102154253A，其氨基酸序列如序列12所示，编码该氨基酸序列的cDNA序列如序列11所示。
全长纤溶酶原的结构也描述在Aisina等(Aisina R B,Mukhametova L I.Structure and function of plasminogen/plasmin system[J].Russian Journal of Bioorganic Chemistry,2014,40(6):590-605)的文章中。在该文章中，Aisina等描述纤溶酶原包括Kringle1、2、3、4、5结构域和丝氨酸蛋白酶结构域(也称蛋白酶结构域(protease domain，PD))，其中，Kringles负责纤溶酶原与低分子量和高分子量的配体结合(即赖氨酸结合活性)，导致纤溶酶原转变成一个更加开放的构型，从而更容易被活化；蛋白酶结构域(PD)为残基Val562-Asn791，tPA和UPA特异性切割纤溶酶原的Arg561-Val562位活化键，从而使纤溶酶原形成纤溶酶，因此，蛋白酶结构域(PD)是赋予纤溶酶原蛋白水解活性的区域。
本发明的“纤溶酶”与“纤维蛋白溶酶”、“纤维蛋白溶解酶”可互换使用，含义相同；“纤溶酶原”与“纤溶酶原”、“纤维蛋白溶解酶原”可互换使用，含义相同。
在本申请中，所述纤溶酶原“缺乏”的含义或活性为受试者体内纤溶酶原的含量比正常人低，低至足以影响所述受试者的正常生理功能；所述纤溶酶原“缺失”的含义或活性为受试者体内纤溶酶原的含量显著低于正常人，甚至活性或表达极微，只有通过外源提供才能维持正常生理功能。
本领域技术人员可以理解，本发明纤溶酶原的所有技术方案适用于纤溶酶，因此，本发明描述的技术方案涵盖了纤溶酶原和纤溶酶。在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时， 在纤溶酶原激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性纤溶酶。具有活性的纤溶酶可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中纤溶酶原的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为纤溶酶原激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”是指具有与底物靶序列中赖氨酸结合的活性(赖氨酸结合活性)、或发挥蛋白水解功能的活性(蛋白水解活性)、或蛋白水解活性和赖氨酸结合活性的片段。本发明涉及纤溶酶原的技术方案涵盖了用纤溶酶原活性片段代替纤溶酶原的技术方案。在一些实施方案中，本发明所述的纤溶酶原活性片段包含纤溶酶原的丝氨酸蛋白酶结构域或由纤溶酶原的丝氨酸蛋白酶结构域组成，优选，本发明所述的纤溶酶原活性片段包含序列14、或与序列14具有至少80％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列或由序列14、或与序列14具有至少80％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列组成。在一些实施方案中，本发明所述的纤溶酶原活性片段包含选自Kringle 1、Kringle 2、Kringle 3、Kringle 4、Kringle 5中一个或多个的区域，或由选自Kringle 1、Kringle 2、Kringle 3、Kringle 4、Kringle 5中一个或多个的区域组成。在一些实施方案中，本发明所述的纤溶酶原包括含有上述纤溶酶原活性片段的蛋白质。
目前，对于血液中纤溶酶原及其活性测定方法包括：对组织纤溶酶原激活剂活性的检测(t-PAA)、血浆组织纤溶酶原激活剂抗原的检测(t-PAAg)、对血浆组织纤溶酶原活性的检测(plgA)、血浆组织纤溶酶原抗原的检测(plgAg)、血浆组织纤溶酶原激活剂抑制物活性的检测、血浆组织纤溶酶原激活剂抑制物抗原的检测、血浆纤维蛋白溶酶-抗纤维蛋白溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤溶酶原活性成 正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤溶酶原包括人的天然纤溶酶原，还包括来源于不同物种的、具有纤溶酶原活性的纤溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性，碱性，疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸，蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤溶酶原是指从其天然环境分离和/或回收的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原会纯化(1)至大于90％、大于95％、或大于98％的纯度(按重量计)，如通过Lowry法所确定的，例如超过99％(按重量计)，(2)至足以通过使用旋转杯序列分析仪获得N端或内部氨基酸序列的至少15个残基的程度，或(3)至同质性，该同质性是通过使用考马斯蓝或银染在还原性或非还原性条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定的。分离的纤溶酶原也包括通过生物工程技术从重组细胞制备，并通过至少一个纯化步骤分离的纤溶酶原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指任何长度的氨基酸的聚合形式，其可以包括遗传编码的和非遗传编码的氨基酸，化学或生物化学修饰的或衍生化的氨基酸，和具有经修饰的肽主链的多肽。该术 语包括融合蛋白，包括但不限于具有异源氨基酸序列的融合蛋白，具有异源和同源前导序列(具有或没有N端甲硫氨酸残基)的融合物；等等。
关于参照多肽序列的“氨基酸序列同一性百分数(％)”定义为在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式实现，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员能决定用于比对序列的适宜参数，包括对所比较序列全长实现最大对比需要的任何算法。然而，为了本发明的目的，氨基酸序列同一性百分数值是使用序列比较计算机程序ALIGN-2产生的。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：
分数X/Y乘100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基的数目，且其中Y是B中的氨基酸残基的总数。应当领会，在氨基酸序列A的长度与氨基酸序列B的长度不相等的情况下，A相对于B的％氨基酸序列同一性会不等于B相对于A的％氨基酸序列同一性。除非另有明确说明，本文中使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
如本文中使用的，术语“治疗”指获得期望的药理和/或生理效果。所述效果可以是完全或部分预防疾病或其症状的发生、发作，部分或完全减轻疾病和/或其症状，和/或部分或完全治愈疾病和/或其症状，包括：(a)预防疾病在受试者体内发生或发作，所述受试者可以具有疾病的素因，但是尚未诊断为具有疾病；(b)抑制疾病，即阻滞其形成；和(c)减轻疾病和/或其症状，即引起疾病和/或其症状消退或消失。
术语“个体”、“受试者”和“患者”在本文中可互换使用，指哺乳动物，包括但不限于鼠(大鼠、小鼠)、非人灵长类、人、犬、猫、有蹄动物(例如马、牛、绵羊、猪、山羊)等。
“治疗有效量”或“有效量”指在对哺乳动物或其它受试者施用以治疗疾病时足以实现对疾病的所述预防和/或治疗的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)的量。“治疗有效量”会根据所使用的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)、要治疗的受试者的疾病和/或其症状的严重程度以及年龄、体重等而变化。
本发明纤溶酶原的制备
纤溶酶原可以从自然界分离并纯化用于进一步的治疗用途，也可以通过标准的化学肽合成技术来合成。当通过化学合成多肽时，可以经液相或固相进行合成。固相多肽合成(SPPS)(其中将序列的C末端氨基酸附接于不溶性支持物，接着序贯添加序列中剩余的氨基酸)是适合纤溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤溶酶原。用于固相合成的技术描述于Barany和Solid-Phase Peptide Synthesis；第3-284页于The Peptides:Analysis,Synthesis,Biology.第2卷：Special Methods in Peptide Synthesis,Part A.,Merrifield,等J.Am.Chem.Soc.,85:2149-2156(1963)；Stewart等,Solid Phase Peptide Synthesis,2nd ed.Pierce Chem.Co.,Rockford,Ill.(1984)；和Ganesan A.2006 Mini Rev.Med Chem.6:3-10和Camarero JA等2005 Protein Pept Lett.12:723-8中。简言之，用其上构建有肽链的功能性单元处理小的不溶性多孔珠。在偶联/去保护的重复循环后，将附接的固相游离N末端胺与单个受N保护的氨基酸单元偶联。然后，将此单元去保护，露出可以与别的氨基酸附接的新的N末端胺。肽保持固定在固相上，之后将其切掉。
可以使用标准重组方法来生产本发明的纤溶酶原。例如，将编码纤溶酶原的核酸插入表达载体中，使其与表达载体中的调控序列可操作连接。表达调控序列包括但不限于启动子(例如天然关联的或异源的启动子)、信号序列、增强子元件、和转录终止序列。表达调控可以是载体中的真核启动子系统，所述载体能够转化或转染真核宿主细胞(例如COS或CHO细胞)。 一旦将载体掺入合适的宿主中，在适合于核苷酸序列的高水平表达及纤溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆主题抗体编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(enterobacteriaceae)，诸如沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物种。在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子系统，色氨酸(trp)启动子系统，beta-内酰胺酶启动子系统，或来自噬菌体λ的启动子系统。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
在微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达并生成本发明的抗-Tau抗体(例如编码主题抗-Tau抗体的多核苷酸)。参见Winnacker,From Genes to Clones,VCH Publishers,N.Y.,N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起点，启动子和增强子(Queen等,Immunol.Rev.89:49(1986))，以及必需的加工信息位点，诸如核糖体结合位点，RNA剪接位点，多聚腺苷酸化位点，和转录终止子序列。 合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛乳头瘤病毒、巨细胞病毒等衍生的启动子。参见Co等,J.Immunol.148:1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀，亲和柱，柱层析，高效液相层析(HPLC)，凝胶电泳等来纯化本发明所述的纤溶酶原。该纤溶酶原是基本上纯的，例如至少约80％至85％纯的，至少约85％至90％纯的，至少约90％至95％纯的，或98％至99％纯的或更纯的，例如不含污染物，所述污染物如细胞碎片，除目标产物以外的大分子，等等。
药物配制剂
可以通过将具有所需纯度的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)与可选的药用载体，赋形剂，或稳定剂(Remington's Pharmaceutical Sciences,16版，Osol,A.ed.(1980))混合形成冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。优选冻干的抗-VEGF抗体配制剂在WO 97/04801中描述，其包含在本文中作为参考。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。
本发明的纤溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送系统(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980)。
用于体内给药的本发明的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J.Biomed.Mater.Res.,15:167-277(1981)；Langer,Chem.Tech.,12:98-105(1982))或聚(乙烯醇)，聚交酯(美国专利3773919,EP 58,481)，L-谷氨酸与γ乙基-L-谷氨酸的共聚物(Sidman，等，Biopolymers 22:547(1983))，不可降解的乙烯－乙烯乙酸酯(ethylene-vinyl acetate)(Langer，等，出处同上)，或可降解的乳酸-羟基乙酸共聚物如Lupron DepotTM(由乳酸-羟基乙酸共聚物和亮氨酰脯氨酸(leuprolide)乙酸酯组成的可注射的微球体)，以及聚D-(-)-3-羟丁酸。聚合物如乙烯-乙酸乙烯酯和乳酸-羟基乙酸能持续释放分子100天以上，而一些水凝胶释放蛋白的时间却较短。可以根据相关机理来设计使蛋白稳定的合理策略。例如，如果发现凝聚的机理是通过硫代二硫键互换而形成分子间S-S键，则可通过修饰巯基残基、从酸性溶液中冻干、控制湿度、采用合适的添加剂、和开发特定的聚合物基质组合物来实现稳定。
给药和剂量
可以通过不同方式，例如通过鼻腔吸入、雾化吸入、滴鼻液或滴眼液，静脉内，腹膜内、皮下、颅内、鞘内、动脉内(例如经由颈动脉)、肌内、直肠给药来实现本发明药物组合物的施用。
用于胃肠外施用的制备物包括无菌水性或非水性溶液、悬浮液和乳剂。非水性溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油，和可注射有机酯，如油酸乙酯。水性载体包括水、醇性/水性溶液、乳剂或悬浮液， 包括盐水和缓冲介质。胃肠外媒介物包含氯化钠溶液、林格氏右旋糖、右旋糖和氯化钠、或固定油。静脉内媒介物包含液体和营养补充物、电解质补充物，等等。也可以存在防腐剂和其他添加剂，诸如例如，抗微生物剂、抗氧化剂、螯合剂、和惰性气体，等等。
医务人员会基于各种临床因素确定剂量方案。如医学领域中公知的，任一患者的剂量取决于多种因素，包括患者的体型、体表面积、年龄、要施用的具体化合物、性别、施用次数和路径、总体健康、和同时施用的其它药物。本发明包含纤溶酶原的药物组合物的剂量范围可以例如为每天约0.0001至2000mg/kg，或约0.001至500mg/kg(例如0.02mg/kg，0.25mg/kg，0.5mg/kg，0.75mg/kg，10mg/kg，50mg/kg等等)受试者体重。例如，剂量可以是1mg/kg体重或50mg/kg体重或在1-50mg/kg的范围，或至少1mg/kg。高于或低于此例示性范围的剂量也涵盖在内，特别是考虑到上述的因素。上述范围中的中间剂量也包含在本发明的范围内。受试者可以每天、隔天、每周或根据通过经验分析确定的任何其它日程表施用此类剂量。例示性的剂量日程表包括连续几天0.01-100mg/kg。在本发明的药物施用过程中需要实时评估治疗效果和安全性。
制品或药盒
本发明的一个实施方案涉及一种制品或药盒，其包含纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)。所述制品优选包括一个容器，标签或包装插页。适当的容器有瓶子，小瓶，注射器等。容器可由各种材料如玻璃或塑料制成。所述容器含有组合物，所述组合物可有效治疗本发明的疾病或病症并具有无菌入口(例如所述容器可为静脉内溶液包或小瓶，其含有可被皮下注射针穿透的塞子的)。所述组合物中至少一种活性剂为纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)。所述容器上或所附的标签说明所述组合物用于治疗本发明所述病症。所述制品可进一步包含含有可药用缓冲液的第二容器，诸如磷酸盐缓冲的盐水，林格氏溶液以及葡萄糖溶液。其可进一步包含从商业和使用者角度来看所需的其它物质，包括其它缓冲液，稀释剂，过滤物，针和注射器。此外，所述制品包含带有使用说明的包装插页，包括例如指示所述组 合物的使用者将包含纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)的组合物以及治疗伴随的疾病的其它药物给药患者。
实施例
以下实施例中使用的人纤溶酶原来自捐赠者血浆，基于如下文献描述的方法：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 Apr；235:1005-10，并进行工艺优化，从人血浆中纯化所得从人捐赠者血浆纯化获得，其中人纤维蛋白溶酶原单体>98％。
实施例1纤溶酶原促进己酮草酰双腙诱导脱髓鞘模型小鼠胼胝体髓鞘的再生
取8周龄C57雄性小鼠20只，随机分为2组，空白对照组6只，模型组14只。空白对照组小鼠饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组小鼠饲喂0.2％双环己酮草酰双腙(cuprizone)模型饲料(南通特洛菲饲料科技有限公司)，饲喂6周，诱导小鼠髓鞘脱落模型 [1]。6周后模型组小鼠根据体重再次随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各7只。给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组以相同方式给予相同体积的PBS，空白对照组小鼠不做注射处理，连续给药14天。给药期间所有小鼠饲喂正常维持饲料。开始给药定为第1天，第15天解剖小鼠取脑于4％多聚甲醛固定，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。脑组织冠状切片厚度为3μm，脱蜡至水后，用髓鞘染色液进行LFB染色。梯度酒精脱水，二甲苯透明，中性树胶封片。在光学显微镜下，观察拍照。
结果显示，空白对照组(图1A)胼胝体髓鞘形态基本正常，给纤溶酶原组(图1C)胼胝体髓鞘阳性着色(箭头标识)明显多于给溶媒PBS对照组 (图1B)，且统计差异显著(图1D)(*表示P<0.05)。说明纤溶酶原能够促进己酮草酰双腙诱导脱髓鞘模型小鼠髓鞘的再生。
实施例2纤溶酶原促进己酮草酰双腙诱导脱髓鞘模型小鼠胼胝体PLP表达
取8周龄C57雄性小鼠20只，随机分为2组，空白对照组6只，模型组14只。空白对照组小鼠饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组小鼠饲喂0.2％己酮草酰双腙模型饲料(南通特洛菲饲料科技有限公司)，饲喂6周，诱导小鼠髓鞘脱落模型 [1]。6周后模型组小鼠根据体重再次随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各7只。给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组以相同方式给予相同体积的PBS，空白对照组小鼠不做注射处理，连续给药3天。给药期间所有小鼠饲喂正常维持饲料。开始给药定为第1天，第4天解剖小鼠取脑于4％多聚甲醛固定，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。脑组织冠状切片厚度为3μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.,USA)封闭1小时；时间到后，弃除羊血清液。兔源抗蛋白脂质蛋白(proteolipid protein，PLP)抗体(Abcam)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.,USA)显色，水洗3次后苏木素复染30秒，流水返蓝5分钟，然后PBS洗1次。梯度脱水透明并封片，切片在200倍光学显微镜下观察。
蛋白脂蛋白(Proteolipid Protein，PLP)是一种高度疏水的膜蛋白，是中枢神经系统中含量最多的髓磷脂 [2]。
结果显示，给纤溶酶原组(图2C)胼胝体PLP的表达(箭头标识)明显多于给溶媒PBS对照组(图2B)，与后者相比表达更加接近空白对照组小鼠(图2A)。说明纤溶酶原促进胼胝体PLP的表达，促进己酮草酰双腙诱导脱髓鞘模型小鼠髓鞘再生。
实施例3纤溶酶原促进己酮草酰双腙诱导脱髓鞘模型小鼠胼胝体神经丝蛋白(NFP)表达
取8周龄C57雄性小鼠20只，随机分为2组，空白对照组6只，模型组14只。空白对照组小鼠饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组小鼠饲喂0.2％己酮草酰双腙模型饲料(南通特洛菲饲料科技有限公司)，饲喂6周，诱导小鼠髓鞘脱落模型 [1]。6周后模型组小鼠根据体重再次随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各7只。给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组以相同方式给予相同体积的PBS，空白对照组小鼠不做注射处理，连续给药14天。给药期间所有小鼠饲喂正常维持饲料。开始给药定为第1天，第15天解剖小鼠取脑于4％多聚甲醛固定，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。脑组织冠状切片厚度为3μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.,USA)封闭1小时；时间到后，弃除羊血清液。兔源抗NF抗体(Abcam，ab207176)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.,USA)显色，水洗3次后苏木素复染30秒，流水返蓝5分钟，然后PBS洗1次。梯度脱水透明并封片，切片在200倍光学显微镜下观察。
神经丝蛋白(Neurofilament protein，NFP)是构成神经细胞轴突中间丝的蛋白质。其功能是提供弹性使神经纤维易于伸展和防止断裂，在维持细胞骨架、稳定细胞形态和轴突转运方面均有十分重要意义 [3]。
结果显示，给纤溶酶原组(图3C)小鼠胼胝体NFP的表达(箭头标识)明显多于给溶媒PBS对照组(图3B)，且统计差异显著(*为P<0.05)(图3D)，与给溶媒PBS对照组相比给纤溶酶原组胼胝体NFP的表达更加接近空白对照组(图3A)。说明纤溶酶原能够促进NFP的表达，从而促进神经纤维再生。
实施例4纤溶酶原促进多发性硬化症模型小鼠胼胝体神经丝蛋白的表达增加
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药35天，给药期间所有小鼠饲喂正常维持饲料。于第36天处死小鼠，取材脑组织于10％甲醛溶液中固定，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。脑组织冠状切片厚度为3μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.,USA)封闭1小时；时间到后，弃除羊血清液。兔源抗NFP抗体(Abcam，ab207176)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.,USA)显色，水洗3次后苏木素复染30秒，流水返蓝5分钟，然后PBS洗1次。梯度脱水透明并封片，切片在100倍光学显微镜下观察。
结果显示，空白对照组(图4A)胼胝体表达一定水平的NFP(箭头标识)，溶媒组(图4B)小鼠胼胝体NFP的表达明显减少，给药组(图4C)小鼠胼胝体NFP表达水平明显高于溶媒小鼠，且统计差异显著(*表示P<0.05)(图4D)。该结果提示纤溶酶原能够促进多发性硬化症模型小鼠胼胝体NFP的表达。
实施例5纤溶酶原促进多发性硬化症模型小鼠胼胝体蛋白脂蛋白的水平增加
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂维持饲料，模型组饲喂含有0.6％双环己酮草酰二腙 (CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症模型 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药35天，给药期间所有小鼠饲喂正常维持饲料(购自北京科澳协力有限公司)。于第36天处死小鼠，取材脑组织于10％甲醛溶液中固定，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。脑组织冠状切片厚度为3μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.,USA)封闭1小时；时间到后，弃除羊血清液。兔源抗PLP抗体(Abcam)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.,USA)显色，水洗3次后苏木素复染30秒，流水返蓝5分钟，然后PBS洗1次。梯度脱水透明并封片，切片在100倍光学显微镜下观察。
结果显示，空白对照组(图5A)胼胝体表达一定水平的PLP(箭头标识)，溶媒组(图5B)小鼠胼胝体PLP的表达明显减少，给药组(图5C)小鼠胼胝体PLP的表达水平明显高于溶媒小鼠。该结果提示纤溶酶原能够促进多发性硬化症模型小鼠胼胝体PLP的表达。
实施例6纤溶酶原促进多发性硬化症模型小鼠胼胝体MBP水平增加
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症模型 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/ 天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药35天，给药期间所有小鼠饲喂正常维持饲料。于第36天处死小鼠，取材脑组织于10％甲醛溶液中固定，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。脑组织冠状切片厚度为3μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.,USA)封闭1小时；时间到后，弃除羊血清液。兔源抗髓鞘碱性蛋白(myelin basic protein，MBP)抗体(Abcam)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.,USA)显色，水洗3次后苏木素复染30秒，流水返蓝5分钟，然后PBS洗1次。梯度脱水透明并封片，切片在100倍光学显微镜下观察。
髓鞘碱性蛋白(MBP)是脊椎动物中枢神经系统少突细胞和周围神经系统雪旺细胞合成的一种强碱性膜蛋白.含有多种碱性氨基酸。
结果显示，空白对照组(图6A)胼胝体表达一定水平的MBP(箭头标识)，给药组(图6C)小鼠胼胝体MBP明显高于溶媒组(图6B)小鼠，且统计差异接近显著(P＝0.063)(图6D)。该结果提示纤溶酶原能够促进多发性硬化症模型小鼠胼胝体MBP的水平增加。
实施例7纤溶酶原促进多发性硬化症模型小鼠海马损伤炎症修复
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症模型 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药35天，给药期间所有小鼠饲喂正常维持饲料。于第36天处死小鼠， 取材脑组织于10％甲醛溶液中固定，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。脑组织冠状切片厚度为3μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.,USA)封闭1小时；时间到后，弃除羊血清液。兔源抗离子钙接头蛋白-1(Ionized calcium binding adaptor molecule-1,Iba-1)抗体(Abcam，ab178847)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.,USA)显色，水洗3次后苏木素复染30秒，流水返蓝5分钟，然后PBS洗1次。梯度脱水透明并封片，切片在200倍光学显微镜下观察拍照，拍照图片用Imaging-Pro软件进行阳性染色光密度分析。
离子钙接头蛋白-1(Ionized calcium binding adaptor molecule-1,Iba-1)是中枢神经系统中的小胶质细胞表面标志物。小胶质细胞做为中枢神经系统中的免疫细胞，在其病变或者损伤时快速感测神经障碍并被活化。活化的小胶质细胞在数量与形态上有显著的变化并迁移到损伤部位，发挥多种功能，例如吞噬死细胞、促炎性细胞因子产量增加等 [4]。
结果显示，空白对照组(图7A)小鼠海马存在一定量的小胶质细胞(箭头标识)，溶媒组(图7B)小鼠海马小胶质细胞量明显增加，但给药组小鼠海马小胶质细胞数量明显多于溶媒组(图7C),且统计差异显著(图7D)(*表示P<0.05,***表示P<0.001)。提示纤溶酶原能够促进多发性硬化症模型小鼠海马损伤炎症修复。
实施例8纤溶酶原促进多发性硬化症模型小鼠海马BDNF水平增加
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症模型 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠 开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药35天，给药期间所有小鼠饲喂正常维持饲料。于第36天处死小鼠，取材脑组织于10％甲醛溶液中固定，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。脑组织冠状切片厚度为3μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.,USA)封闭1小时；时间到后，弃除羊血清液。兔源抗脑源性神经营养因子(brain-derived neurotrophic factor，BDNF)抗体(BosterBio,PB9075)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.,USA)显色，水洗3次后苏木素复染30秒，流水返蓝5分钟，然后PBS洗1次。梯度脱水透明并封片，切片在400倍光学显微镜下观察拍照，拍照图片用Imaging-Pro软件进行阳性染色光密度分析。
成熟的脑源性神经营养因子(brain-derived neurotrophic factor，BDNF)及其受体广泛分布于中枢神经系统内，在中枢神经系统发育过程中，对神经元存活、分化、生长发育起重要作用，并能防止神经元受损伤死亡，改善神经元的病理状态，促进受损伤神经元再生及分化等生物效应，而且是成熟的中枢及周围神经系统的神经元维持生存及正常生理功能所必须 [5]。
结果显示，空白对照组(图8A)小鼠海马具有一定水平的BDNF(箭头标识)，溶媒组(图8B)小鼠海马BDNF水平增加，给药组小鼠海马BDNF水平明显高于溶媒组(图8C),且统计差异接近显著(图8D)(P＝0.095)。提示纤溶酶原能够促进多发性硬化症模型小鼠海马BDNF水平增加。
实施例9纤溶酶原促进多发性硬化症模型小鼠海马星形胶质细胞活性增加
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲 喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症模型 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药35天，给药期间所有小鼠饲喂正常维持饲料。于第36天处死小鼠，取材脑组织于10％甲醛溶液中固定，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。脑组织冠状切片厚度为3μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.,USA)封闭1小时；时间到后，弃除羊血清液。兔源抗胶质细胞纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)抗体(Abcam,ab4648)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.,USA)显色，水洗3次后苏木素复染30秒，流水返蓝5分钟，然后PBS洗1次。梯度脱水透明并封片，切片在200倍光学显微镜下观察拍照，拍照图片用Imaging-Pro软件进行阳性染色光密度分析。
胶质细胞纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)是星形胶质细胞中标志性的中间丝蛋白，参与细胞骨架构成并维持其张力强度 [6]。
结果显示，空白对照组(图9A)小鼠海马表达一定水平的GFAP(箭头标识)，溶媒组(图9B)小鼠海马GFAP表达减少，给药组小鼠海马GFAP表达明显高于溶媒组(图9C)，且统计差异接近显著(图9D)(P＝0.051)。提示纤溶酶原能够促进多发性硬化症模型小鼠海马GFAP表达增加，促进星形胶质细胞活性增加。
实施例10纤溶酶原缓解多发性硬化症模型小鼠抑郁行为
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲 喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性引证症 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药28天，给药期间所有小鼠饲喂正常维持饲料。第29天进行旷场实验。
多发性硬化患者中常会出现精神类症状，多表现为抑郁、易怒和脾气暴，部分患者出现欣快、兴奋、也可表现为淡漠、嗜睡、强哭强笑、反应迟钝、智能低下、重复语言、猜疑和被害妄想等。还可出现记忆力减退，注意力损害等认知功能障碍 [7]。
旷场实验
实验时，小鼠放入旷场(40x40x40cm)底面中心，同时进行摄像和计时，观察持续5分钟，每只小鼠进行3次实验。Smart系统是一个完整且方便使用的视频跟踪系统，用于评估实验动物的行为。它允许记录轨迹，活动，特定行为(如旋转，拉伸和饲养)和事件，并执行各种分析参数的计算。本实验使用Smart3.0系统记录分析小鼠的运动情况，参数包括总移动距离、边界静息时间率、中心区平均运动速度和边界平均运动速度。每次实验中采用70％酒精擦拭箱体防止嗅觉产生的偏好 [8]。
旷场实验的设计原理是基于小鼠的趋避性，指的是小鼠畏惧开
阔、未知、可能存在潜在危险的场所，因而其有“贴墙”活动的天性。趋避性是以小鼠在旷野周边区(四个角和四个边)的活动来评价的。从反映趋避性的周边区活动时间看，时间减少，说明小鼠更富有“冒险”倾向。在中央区活动时间显著增多，说明趋避性和焦虑(抑郁)水平较低。
总运动路程为旷场实验期间小鼠在各个区域的总运动路程。结果显示，空白对照组小鼠具有一定的总运动路程，溶媒组小鼠总运动路程明显增加，给药组小鼠总运动路程明显小于溶媒组，统计差异显著(*表示P<0.05)(图10)，且与空白对照组相近。说明纤溶酶原能够一定程度缓解多发性硬化症模型小鼠抑郁行为。
实施例11纤溶酶原缓解多发性硬化症模型小鼠抑郁行为
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药28天，给药期间所有小鼠饲喂正常维持饲料。于第29天进行旷场实验。
边界区静息时间百分率＝边界区静息时间/总观察时间。结果显示，空白对照组小鼠具有一定的边界区静息时间百分率，约为57.8％；溶媒组小鼠边界区静息时间百分率明显降低，约为49.3％，给药组小鼠边界区静息时间百分率约为58.4％，明显大于溶媒组，统计差异显著(*表示P<0.05)(图11)，且与空白对照组相近。说明纤溶酶原能够一定程度地缓解多发性硬化症模型小鼠抑郁行为。
实施例12纤溶酶原改善多发性硬化症模型小鼠社交记忆能力
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药34天，给药期间所有小鼠饲喂正常维持饲料。于第35天进行三箱社交测试实验。
三箱实验开始前，将小鼠放在行为测试室适应半个小时；用透明的玻璃树脂板将三个箱子隔开，并将测试实验小鼠放进中间的箱子里适应5分钟；将陌生小鼠1随机放进左侧或右侧箱子里的金属笼子里，另外一侧箱子的金属笼子空着；去掉隔开箱子的玻璃树脂板，使测试小鼠可以在三个箱子中自由活动十分钟；马上开始拍摄并记录下相关参数：第一阶段：1)实验小鼠与陌生小鼠1或空的金属笼之间的直接接触的次数以及持续时间，金属笼周围3-5cm定义为接触范围；2)实验小鼠进入每个箱子的次数以及持续的时间，当小鼠的头和四爪都进入一个箱子就认为它处在那个箱子中。第二阶段：在第二阶段实验中向空着的金属笼子中放入第二只陌生小鼠(陌生小鼠2)，然后记录十分钟，观察实验小鼠与陌生小鼠1和陌生小鼠2之间相互接触的时间和次数。正常小鼠会表现出社交行为，在一阶段的实验中，通常正常小鼠与陌生小鼠1交流的时间和次数明显多于空的金属笼。同时，小鼠还具有记忆力和“喜新厌旧”的特性，因此在第二阶段中，小鼠会更喜欢与之前素未抹面的陌生小鼠1交流，而不是已经沟通过十分钟的陌生小鼠1。
静息时间百分率＝静息时间/总观察时间。三箱社交测试实验第二阶段结果显示，空白对照组小鼠具有一定的陌生小鼠2接触范围内的静息时间百分率，约为13.7％；溶媒组明显降低，约为10.6％；给药组约为16.1％，明显高于溶媒组，且统计差异接近显著(P＝0.075)(图12)。说明纤溶酶原能够改善多发性硬化症模型小鼠的社交记忆。
实施例13纤溶酶原改善多发性硬化症模型大鼠抑郁行为
取3周龄的雄性大鼠26只，造模前进行称重，根据体重排除异常大鼠后所有大鼠随机分为2组，空白对照组8只大鼠，模型组18只大鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％CPZ的造模饲料，造模14天，建立多发性硬化症 [9]。同时所有大鼠开始饲喂正常维持饲料。造模完成后，所有大鼠进行旷场实验检测，根据检测结果对模型组大鼠进行分组，溶媒组7只大鼠，给药组8只大鼠。分组完成后，溶媒组及给药组大鼠开始给药，记为给药第1天，给药组大鼠按照35mg/kg尾静脉注射纤溶酶原，溶媒组大鼠尾静脉注射3.5ml/kg溶媒，连续给药6天，空白对照组大鼠不做给药处理。于给药第7 天进行旷场实验。
边界区路程为旷场实验测试时间内边界区运动轨迹长度。边界区路程百分率＝边界区路程/边界区与中心区路程之和。结果显示，空白对照组具有一定的边界区运动路程百分率，约为91.1％；溶媒组明显增加，约为93.6％；给药组约为88.1％，明显低于溶媒组，且统计差异显著(*表示P<0.05)(图13)。说明纤溶酶原能够改善多发性硬化症模型小鼠抑郁行为。
实施例14纤溶酶原缓解多发性硬化症模型大鼠抑郁行为
取3周龄的雄性大鼠26只，造模前进行称重，根据体重排除异常大鼠后所有大鼠随机分为2组，空白对照组8只大鼠，模型组18只大鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％CPZ的造模饲料，造模14天，建立多发性硬化症模型 [9]。同时所有大鼠开始饲喂正常维持饲料。造模完成后，所有大鼠进行旷场实验检测，根据检测结果对模型组大鼠进行分组，溶媒组7只大鼠，给药组8只大鼠。分组完成后，溶媒组及给药组大鼠开始给药，记为给药第1天，给药组大鼠按照35mg/kg尾静脉注射纤溶酶原，溶媒组大鼠尾静脉注射3.5ml/kg溶媒，连续给药6天，空白对照组大鼠不做给药处理。于给药第7天进行旷场实验。
中心区路程为旷场实验测试时间内中心区运动轨迹长度。中心区路程百分率＝中心区路程/边界区与中心区路程之和。结果显示，空白对照组具有一定的中心区路程百分率，约为8.9％；溶媒组明显降低，约为6.4％；给药组约为11.9％，明显高于溶媒组，且统计差异显著(*表示P<0.05)(图14)。说明纤溶酶原能够改善多发性硬化症模型大鼠的焦虑行为。
实施例15纤溶酶原缓解多发性硬化症模型小鼠焦虑抑郁行为
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症模型 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠 开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药20天，给药期间所有小鼠饲喂正常维持饲料。于第21天进行高架十字迷宫实验。
高架十字迷宫是利用动物对新异环境的探究特性和对高悬敞开臂的恐惧形成矛盾冲突行为来考察动物的焦虑状态。高架十字迷宫具有一对开臂和一对闭臂，啮齿类动物由于嗜暗性会倾向于在闭臂中活动，但出于好奇心和探究性又会在开臂中活动，在面对新奇刺激时，动物同时产生探究的冲动与恐惧，这就造成了探究与回避的冲突行为，从而产生焦虑心理。而抗焦虑药物能明显增加进入开臂的次数与时间，十字迷宫距离地面较高，相当于人站在峭壁上，使实验对象产生恐惧和不安心理。高架十字迷宫被广泛应用于新药开发/筛选/评价、药理学、毒理学、预防医学、神经生物学、动物心理学及行为生物学等多个学科的科学-研究和计算机辅助教学等领域，是医学院校与科研机构开展行为学研究尤其是焦虑抑郁研究的经典实验。
实验开始时将小鼠从中央格面向闭合臂放入迷宫，记录5分钟内的活动情况。观察指标包括：开放臂进入次数(必须有两只前爪进入臂内)，开放臂停留时间，闭合臂进入次数，闭合臂停留时间。计算开放臂停留时间比例，开放臂进入次数比例，高架十字迷宫中总进入次数。实验完成后将小鼠取出，将两臂清理干净，喷洒酒精除去气味。最后用动物行为学软件进行数据分析。
开放臂进入百分率＝开放臂进入总次数/开放臂与闭合臂进入总次数之和。结果显示，空白对照组小鼠具有一定的开放臂进入百分率，约为14.9％；溶媒组小鼠开放臂进入百分率明显增加，约为23.0％；给药组小鼠开放臂进入百分率约为14.5％，明显少于溶媒组，统计差异显著(P＝0.015)(图15)，且与空白对照组相近。说明纤溶酶原能够一定程度缓解多发性硬化症模型小鼠焦虑行为。
实施例16纤溶酶原缓解多发性硬化症模型小鼠焦虑行为
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成 后，空白对照组饲喂维持饲料，模型组饲喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症模型 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药20天，给药期间所有小鼠饲喂正常维持饲料(购自北京科澳协力有限公司)。于第21天进行高架十字迷宫实验。
闭合臂进入百分率＝闭合臂进入总次数/开放臂和闭合臂进入总次数。结果显示，空白对照组小鼠具有一定的闭合臂进入百分率，约为34.4％；溶媒组小鼠闭合臂进入百分率明显降低，约为28.1％；给药组小鼠闭合臂进入百分率约为37.1％，明显大于溶媒组，统计差异显著(P＝0.007)(图16)，且与空白对照组相近。说明纤溶酶原能够一定程度缓解多发性硬化症模型小鼠焦虑抑郁行为。
实施例17纤溶酶原缓解多发性硬化症模型小鼠焦虑抑郁行为
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症模型 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药27天，给药期间所有小鼠饲喂正常维持饲料。于第28天进行高架十字迷宫实验。
闭合臂运动总路程为高架十字迷宫实验测试时间内小鼠闭合臂运动总路程。结果显示，空白对照组小鼠具有一定的闭合臂运动总路程，溶媒组小鼠闭合臂运动总路程明显增加，给药组小鼠闭合臂运动总路程明显少于 溶媒组，统计差异显著(*表示P<0.05)(图17)，且与空白对照组相近。说明纤溶酶原能够一定程度地缓解多发性硬化症模型小鼠焦虑抑郁行为。
实施例18纤溶酶原缓解多发性硬化症模型小鼠焦虑抑郁行为
取C57小鼠雌性30只，造模前进行称重，根据体重排除异常小鼠后所有小鼠随机分为2组，空白对照组8只小鼠，模型组22只小鼠。分组完成后，空白对照组饲喂正常维持饲料(购自北京科澳协力有限公司)，模型组饲喂含有0.6％双环己酮草酰二腙(CPZ)(厂商：上海源叶生物科技有限公司，货号：S30349)的造模饲料，持续饲喂42天，诱导多发性硬化症模型 [1]。造模完成后，所有小鼠进行旷场实验检测，根据检测结果对模型组小鼠进行分组，溶媒组11只小鼠，给药组11只小鼠。分组完成后，所有小鼠开始给药，记为给药第1天，空白对照组小鼠和溶媒组小鼠按照0.1ml/只/天尾静脉注射溶媒，给药组小鼠按照1mg/只/天尾静脉注射纤溶酶原，连续给药27天，给药期间所有小鼠饲喂正常维持饲料。于第28天进行高架十字迷宫实验。
闭合臂休息时间百分率＝闭合臂休息时间/闭合臂运动时间与闭合臂休息时间之和。结果显示，空白对照组小鼠具有一定的闭合臂休息时间百分率，约为51.8％；溶媒组小鼠闭合臂休息时间百分率明显降低，约为36.8％；给药组小鼠闭合臂休息时间百分率约为50.1％，明显大于溶媒组，统计差异显著(*表示P<0.05)(图18)，且与空白对照组相近。说明纤溶酶原能够一定程度缓解多发性硬化症模型小鼠焦虑抑郁行为。
参考文献
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序列表
序列1：
Figure PCTCN2021075921-appb-000001
序列2：
Figure PCTCN2021075921-appb-000002
序列3：
Figure PCTCN2021075921-appb-000003
序列4：
Figure PCTCN2021075921-appb-000004
序列5：
Figure PCTCN2021075921-appb-000005
Figure PCTCN2021075921-appb-000006
序列6：
Figure PCTCN2021075921-appb-000007
序列7：
Figure PCTCN2021075921-appb-000008
序列8：
Figure PCTCN2021075921-appb-000009
序列9：
Figure PCTCN2021075921-appb-000010
序列10：
Figure PCTCN2021075921-appb-000011
序列11：
Figure PCTCN2021075921-appb-000012
序列12：
Figure PCTCN2021075921-appb-000013
序列13：
Figure PCTCN2021075921-appb-000014
序列14：
Figure PCTCN2021075921-appb-000015

Claims (14)

  1. 一种预防和治疗多发性硬化症的方法，包括给药受试者治疗有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
  2. 权利要求1所述的方法，其中所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro-plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。
  3. 权利要求1的方法，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抑制剂，例如抗体。
  4. 权利要求1-3任一项的方法，其中所述化合物具有选自如下一项或多项的活性：促进神经髓鞘的再生、促进髓鞘蛋白表达、促进神经组织NFP的表达、促进神经纤维再生、促进神经组织NFP的表达、提高神经组织MBP水平、增加神经组织小胶质细胞数量、促进神经组织炎症修复、促进神经组织星形胶质细胞活性、提高神经组织BDNF水平、促进神经组织GFAP表达、、改善受试者的社交行为能力、改善受试者的社交记忆能力、缓解受试者的抑郁行为、缓解受试者的焦虑行为。
  5. 权利要求1-4任一项的方法，其中所述化合物为纤溶酶原。
  6. 权利要求1-5任一项的方法，其中所述纤溶酶原与序列2具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且具有纤溶酶原的蛋白水解活性。
  7. 权利要求1-6任一项的方法，其中所述纤溶酶原与序列2具有至少 75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且具有纤溶酶原的赖氨酸结合活性。
  8. 权利要求1-6任一项的方法，所述纤溶酶原包含与序列14所示的纤溶酶原活性片段具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列、并且具有纤溶酶原的蛋白水解活性。
  9. 权利要求1-6任一项的方法，所述纤溶酶原为序列2所述纤溶酶原的保守取代变体。
  10. 权利要求1-6任一项的方法，所述纤溶酶原为天然或合成的人纤溶酶原。
  11. 权利要求1-10任一项的方法，其中所述化合物与一种或多种其他治疗方法或药物联合使用。
  12. 权利要求11的方法，其中所述其他治疗方法选自如下的一项或多项：手术治疗、细胞治疗(包括干细胞治疗)和物理治疗。
  13. 权利要求11的方法，其中所述其他药物选自如下的一项或多项：激素、免疫抑制剂、神经营养药物、抗菌素和抗病毒药物。
  14. 权利要求1-13任一项的方法，其中所述化合物通过选自如下的任一或多种方式或途径给药：鼻腔吸入、雾化吸入、滴鼻液、滴眼液、滴耳液、静脉内、腹膜内、皮下、颅内、鞘内、动脉内和肌肉内。
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CN114736948A (zh) * 2022-06-10 2022-07-12 深圳市帝迈生物技术有限公司 一种α2-抗纤溶酶活性测定试剂盒

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