Method and drug for treating viral pneumonia

Abstract

To provide a method and a drug for treating viral pneumonia.SOLUTION: A pharmaceutical composition comprises a therapeutically effective amount of one or more compounds selected from the group consisting of a component of plasminogen activation pathway, a compound capable of activating plasminogen, a compound mimicking the activity of plasminogen or plasmin, a compound capable of upregulating the expression of plasminogen or an activator of plasminogen, an analog of plasminogen, an analog of plasmin, an analog of tPA or uPA, and an antagonist of fibrinolysis inhibitor.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for preventing or treating viral pneumonia, e.g., coronavirus-infected pneumonia, and complications thereof, comprising administering to a subject an effective amount of a component of the plasminogen activation pathway or a related compound thereof, e.g., plasminogen, to ameliorate pulmonary inflammation, increase blood oxygen saturation, treat pneumonia, and prevent pulmonary fibrosis and other associated complications. [Background technology]

[0002] Coronaviruses (CoVs) are enveloped, single-stranded, positive-sense RNA viruses belonging to the Coronaviridae family. According to genotype, they can be divided into four genera: Alpha Coronavirus (α-CoV), Beta Coronavirus (β-CoV), Gamma Coronavirus (γ-CoV), and Delta Coronavirus (δ-CoV), of which α-CoV and β-CoV coronaviruses are more susceptible to mammals (CUI J, Li F, SHI Z L. Origin and evolution of pathogenic coronaviruses [J]. Nature reviews. Micorbiology, 2019, 17(3): 181-192).

[0003] Severe acute respiratory syndromes coronavirus (SARS-CoV), or β-CoV, can cause severe acute respiratory syndromes (SARS), a disease characterized primarily by atypical pneumonia. A new human coronavirus (hCoVEMC / 2012) similar to SARS-CoV that emerged in the Middle East and South Korea can cause Middle East respiratory syndrome (MERS) in humans. In 2013, the World Health Organization named the pathogen Middle East respiratory syndrome coronavirus (MERS-CoV). The disease resembles SARS, but is more likely to cause other complications, such as liver and kidney failure, resulting in a higher mortality rate in patients.

[0004] The SARS virus has a wide range of tissue tropism, invades various tissues and organs, and causes damage to various tissues and organs. Patients exhibit pulmonary congestion, hemorrhage, edema, severe diffuse alveolar damage, hyaline membrane formation, alveolar epithelial hyperplasia, inflammatory infiltration of pulmonary interstitial mononuclear cells, loss of interalveolar cells, and pulmonary interstitial fibrosis.

[0005] The 2019 novel coronavirus belongs to the β genus of coronaviruses and was officially named 2019-nCoV (also referred to as COVID-19 in this application) by the WHO on January 12, 2020. Its genetic characteristics are significantly different from those of SARS-CoV and MERS-CoV, and are more similar to the bat SARS-like coronaviruses bat-SL-CoV ZC45 and bat-SL-CoV ZXC21 (CHEN Y, LIU Q, GUO D. Emerging coronaviruses: genome structure, replication, and pathogenesis [J]. Journal of medical virology, 2020).

[0006] 2019-nCoV is highly contagious and humans are generally susceptible to infection, mainly manifesting as lower respiratory tract infection symptoms such as dry cough, fever, and dyspnea. In severe cases, it can even cause acute respiratory distress syndrome (ARDS) or sepsis (Clinical management of severe acute respiratory infection when novel coronavirus (2019-nCoV) infection is suspected: interim guidance. 28 January 2020. WHO / nCoV / Clinical / 2020.2).

[0007] COVID-19 pneumonia can be divided into three stages: early, advanced, and severe. In the early stage of the disease, the lesions are limited and appear as single or multiple ground-glass opacities (GGOs), nodules, spots or sheets; in the advanced stage, the lesions progress, the lesions increase and expand in scope, GGOs coexist with consolidation or streak opacities, and there is some consolidation or bronchial trabecular thickening within the architectural distortion opacities; in the severe stage, there are diffuse lesions in both lungs, extensive exudation and consolidation of the lung parenchyma, mainly consolidation opacities, and there is also distortion of the lung architecture, bronchiectasis, and subsegmental atelectasis, and in severe cases, it is "white lung" (Shi Heshui, Han Xiaoyu, Fan Yanqing, Liang Bo, Yang Fan, Han Ping, Zheng Chuansheng, Clinical characteristics and imaging manifestations of pneumonia caused by novel coronavirus (2019-nCoV) infection. Journal of Clinical Radiology. https: / / doi.org / 10.13437 / j.cnki.jcr.20200206.002). Summary of the Invention

[0008] The present invention has discovered that plasminogen can significantly improve the pulmonary ventilation function, increase blood oxygen saturation, and improve the symptoms of dyspnea in patients with pneumonia (including viral infection pneumonia such as 2019-nCoV viral pneumonia) and pulmonary fibrosis, thereby treating diseases associated with pneumonia and pulmonary fibrosis.

[0009] Specifically, the present invention relates to the following: In one aspect, the present application relates to a method of preventing and treating pneumonia comprising administering to a subject a therapeutically effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

[0010] In one aspect, the present application relates to the use of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolysis inhibitors in the preparation of a medicament for preventing and treating pneumonia.

[0011] In one aspect, the present application relates to the use of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors, to prevent and treat pneumonia.

[0012] In one aspect, the present application relates to an agent for preventing and treating pneumonia comprising one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

[0013] 2. The method, use or agent according to item 1, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogues containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA and uPA.

[0014] 3. The method, use or medicament according to item 1, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, alpha2-antiplasmin or alpha2-macroglobulin, such as an antibody.

[0015] 4. The method, use, or agent according to any one of Items 1 to 3, wherein the pneumonia is bacterial pneumonia, viral pneumonia, mycoplasma pneumonia, chlamydia pneumonia, fungal pneumonia, or rickettsia pneumonia.

[0016] 5. The method, use, or medicament according to any one of items 1 to 3, wherein the pneumonia is pneumonia caused by a non-infectious agent. In some embodiments, the pneumonia is caused by radiation. In some embodiments, the pneumonia is caused by inhalation of toxic gases, for example, dust deposits such as haze. In some embodiments, the pneumonia is caused by an autoimmune disease (e.g., systemic sclerosis). In some embodiments, the pneumonia is caused by an allergy, such as asthma. In some embodiments, the pneumonia is caused by a toxic compound (e.g., monocrotaline or paraquat).

[0017] Item 6. The method, use, or medicament according to item 4, wherein the pneumonia is coronavirus-infected pneumonia.

[0018] 7. The method, use, or agent according to claim 6, wherein the pneumonia is 2019-nCoV infectious pneumonia.

[0019] In some embodiments, the pneumonia is 2019-nCoV infectious pneumonia, and the plasminogen provides one or more of: reduced lung tissue damage, reduced pulmonary inflammatory response, reduced fibrin deposition in the lungs, improved lung function, increased blood oxygen saturation, reduced blood pressure in a subject with 2019-nCoV infectious pneumonia, improved cardiac function in a subject with 2019-nCoV infectious pneumonia, and improved general physical condition in a subject with 2019-nCoV infectious pneumonia.

[0020] 8. The method, use, or medicament according to any one of items 1 to 7, wherein the compound has one or more effects selected from the group consisting of reducing inflammation in lung tissue, reducing inflammatory exudation in lung tissue, reducing fibrin deposition in lung tissue, reducing fibrosis in lung tissue, reducing apoptosis in lung tissue, improving ventilatory function, and increasing blood oxygen saturation.

[0021] 9. The method, use or agent according to any one of items 1 to 8, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2 and still has plasminogen activity, such as proteolytic activity, lysine-binding activity or proteolytic activity and lysine-binding activity.

[0022] 10. The method, use, or agent of any one of paragraphs 1 to 8, wherein the plasminogen comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the plasminogen active fragment shown in sequence 14, and has plasminogen proteolytic activity or lysine binding activity. In some embodiments, the plasminogen comprises the plasminogen active fragment shown in sequence 14, and has plasminogen proteolytic activity.

[0023] 11. The method, use, or agent according to any one of paragraphs 1 to 8, wherein the plasminogen is a conservatively substituted variant of the plasminogen shown in sequence 2. In some embodiments, the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or variants thereof that retain plasminogen activity. In some embodiments, the plasminogen comprises or consists of the amino acid sequence shown in sequence 2, 4, 6, 8, 10, 12, or 14.

[0024] 12. The method, use or agent according to any one of items 1 to 8, wherein the plasminogen is natural or synthetic human plasminogen, or a variant or fragment which still retains plasminogen activity.

[0025] 13. The method, use, or medicament according to any one of paragraphs 1 to 12, wherein the compound is used in combination with one or more other treatment methods or medicaments. In some embodiments, the other treatment method is extracorporeal assisted breathing, such as mechanical ventilation.

[0026] 14. The method, use, or medicament according to item 13, wherein the other drug is one or more selected from antiviral drugs, antibiotics, immunomodulatory drugs, hormonal drugs (e.g., steroid hormones), vaccines, and disease-associated neutralizing antibodies.

[0027] 15. The method, use, or medicament according to any one of items 1 to 14, wherein the compound is administered by one or more routes or methods selected from the group consisting of nasal inhalation, aerosol inhalation, nasal drops, ear drops, eye drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intramuscular, and intrarectal.

[0028] In any one of the above embodiments of the present application, the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12 and still have plasminogen activity, such as proteolytic activity, lysine-binding activity, or proteolytic activity and lysine-binding activity. In some embodiments, the plasminogen is a protein in which 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is added, deleted, and / or substituted based on sequence 2, 6, 8, 10 or 12 and still has plasminogen activity.

[0029] In some embodiments, the plasminogen is a protein that includes plasminogen activity fragments and still has plasminogen activity. In some embodiments, the plasminogen is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, δ-plasminogen, or variants thereof that retain plasminogen activity. In the above embodiments, the plasminogen is natural or synthetic human plasminogen, or variants or fragments that still retain plasminogen activity. In some embodiments, the plasminogen is an orthologue of human plasminogen from a primate or rodent, or variants or fragments that still retain plasminogen activity. In some embodiments, the amino acids of the plasminogen are shown in sequences 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human natural plasminogen.

[0030] In some embodiments, the subject is a human. In some embodiments, the subject is deficient or lacking in plasminogen. In some embodiments, the deficient or lacking is congenital, inherited and / or localized.

[0031] In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier and plasminogen for use in the aforementioned methods. In some embodiments, the kit may be a prophylactic or therapeutic kit comprising (i) plasminogen for use in the aforementioned methods, and (ii) means for delivering the plasminogen to the subject. In some embodiments, the means is a syringe or a vial. In some embodiments, the kit further comprises a label or a protocol for instructing administration of the plasminogen to the subject to perform any of the aforementioned methods.

[0032] In some embodiments, the article of manufacture comprises a container containing a label and (i) plasminogen or a pharmaceutical composition containing plasminogen for use in the aforementioned methods, the label providing instructions for administering the plasminogen or composition to the subject to perform any of the aforementioned methods.

[0033] In some embodiments, the kit or article of manufacture further comprises one or more additional components or containers that contain other agents.

[0034] In some embodiments of the methods, the plasminogen is administered systemically or locally, preferably by one or more routes or methods selected from nasal inhalation, aerosol inhalation, nasal drops, ear drops, eye drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intramuscular, and intrarectal. In some embodiments of the methods, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In some embodiments of the methods, the plasminogen is administered at a daily dose of 0.0001-2000 mg / kg, 0.001-800 mg / kg, 0.01-600 mg / kg, 0.1-400 mg / kg, 1-200 mg / kg, 1-100 mg / kg, 10-100 mg / kg (calculated per kilogram of body weight), or 0.0001-2000 mg / cm. 2 , 0.001-800mg / cm 2 , 0.01-600mg / cm 2 , 0.1-400mg / cm 2 , 1-200mg / cm 2 , 1-100mg / cm 2 , 10-100mg / cm 2 (calculated per square centimeter of body surface area) and is preferably administered one or more times, preferably at least daily.

[0035] The present invention expressly covers all combinations of the technical features belonging to the embodiments of the present invention, and the technical configurations after the combinations are expressly disclosed in this application as if the above technical configurations were separately expressly disclosed. Moreover, the present invention also expressly covers the combinations between each embodiment and their elements, and the technical configurations after the combinations are expressly disclosed in this specification. [Brief description of the drawings]

[0036] [Figure 1] Figure 1A-C shows representative photographs of Sirius red staining of lungs after administration of plasminogen to monocrotaline-induced pulmonary arterial hypertension model mice for 28 days. A is the blank control group, B is the solvent PBS-administered control group (abbreviated as solvent group in this application), and C is the plasminogen-administered group (abbreviated as administration group in this application). As a result, there was essentially no collagen deposition in the lungs of mice in the blank control group, and collagen deposition in the lung tissues of mice in the plasminogen group (marked by arrows) was significantly less than that in the solvent PBS-administered control group. This indicates that plasminogen can significantly reduce pulmonary fibrosis in monocrotaline-induced pulmonary arterial hypertension model mice. [Diagram 2] Figure 2A-C shows representative photographs of pulmonary Sirius red staining after 21 days of administration of plasminogen to bleomycin-induced systemic sclerosis model mice. A is the solvent PBS-administered control group, B is the plasminogen-administered group, and C is the quantitative analysis result. As a result, in the bleomycin-induced systemic sclerosis mouse model, the degree of pulmonary fibrosis (marked with arrows) in the solvent PBS-administered group mice was higher than that in the plasminogen-administered group, the morphology of the alveolar wall in the plasminogen-administered group mice was close to normal, the level of inflammatory cells was significantly reduced, and the degree of fibrosis was significantly lower than that in the solvent PBS-administered group, and the difference was statistically significant (* indicates P<0.05). [Diagram 3]Figure 3A-B shows the results of Sirius red staining of the lungs after plasminogen was administered to paraquat-induced poisoning mice for 14 days. A is the control group administered with solvent PBS, and B is the plasminogen-administered group. As a result, the deposition of collagen fibers in the plasminogen-administered mice was significantly less than that in the control group administered with solvent PBS. This indicates that plasminogen can reduce pulmonary fibrosis caused by paraquat poisoning. [Figure 4] Figure 4A-D shows the results of Sirius red staining of the lungs in LPS-induced pneumonia model mice after administration of plasminogen for 14 days. A is the blank control group, B is the solvent PBS-administered control group, C is the plasminogen-administered group, and D is the quantitative analysis result. The results showed that there was a certain amount of collagen deposition in the lung tissue of the blank control group (marked with an arrow), collagen deposition was significantly increased in the lung tissue of the mice in the solvent group, and collagen deposition in the lung tissue of the mice in the administration group was significantly less than that of the mice in the solvent group, and the difference was statistically significant (* represents P<0.05). This indicates that plasminogen can reduce collagen deposition in the lungs of pneumonia model mice and ameliorate pulmonary fibrosis caused by pneumonia. [Diagram 5] Figure 5A-C shows the results of Sirius red staining of the lungs in LPS-induced pneumonia model mice after pre-administration of plasminogen for 3 days. A is the blank control group, B is the solvent PBS-administered control group, and C is the treatment group. As a result, there was a certain amount of collagen deposition in the lung tissue of the blank control group (marked with an arrow), collagen deposition was significantly increased in the lung tissue of the mice in the solvent group, and collagen deposition in the lung tissue of the mice in the treatment group was significantly less than that of the mice in the solvent group. This indicates that pre-administration of plasminogen can reduce collagen deposition in the lungs of pneumonia model mice and improve pulmonary fibrosis caused by pneumonia. [Figure 6]Figure 6 shows the results of detecting total protein in the lung lavage fluid of LPS-induced pneumonia model mice after administration of plasminogen for 7 days. As a result, there was a certain amount of total protein in the lung lavage fluid of the blank control group, the total protein level in the lung lavage fluid of the solvent group was significantly higher than that of the blank control group (*** represents P<0.001), and the total protein level in the lung lavage fluid of the treatment group was significantly lower than that of the solvent group, and the statistical difference was close to significance (P=0.052). This indicates that plasminogen can reduce the total protein level in the lung lavage fluid of pneumonia model mice. [Figure 7] Figure 7 shows the results of immunohistochemical staining of pulmonary fibrin in LPS-induced pneumonia model mice after administration of plasminogen for 3 days. A is the solvent-administered control group, and B is the administration group. As a result, the level of fibrin deposition in the lung tissue of the solvent group mice was significantly higher than that of the administration group. This indicates that plasminogen can reduce pulmonary fibrin deposition in pneumonia model mice. [Figure 8] Figure 8 shows high-resolution CT images of typical COVID-19 patients. The patient ID is displayed on the left. Column A shows chest CT images before plasminogen inhalation. Column B shows chest CT images after plasminogen inhalation of the corresponding patients. Column C shows chest CT images of five patients reexamined 5 days after ultrasound examination B. Black arrows and boxes indicate abnormalities. After 2-3 doses of human plasminogen, the number, extent, and density of lung lesions in the five patients were reduced and even partially disappeared, and the patchy or punctate "ground-glass" shadows were significantly reduced or absorbed. This indicates that aerosol inhalation of human plasminogen can rapidly ameliorate lung damage caused by COVID-19 infection. [Figure 9] Figure 9 shows the heart rate monitoring results before and after plasminogen inhalation in ordinary, severe, and critical COVID-19 patients. Statistical analysis showed that after plasminogen administration, the average heart rates of ordinary, severe, and critical patients all showed a downward trend, and in general patients, there was a statistical difference between before and after administration (P<0.05). Detailed Description of the Invention

[0037] The fibrinolytic system, also called the fibrinolytic system, is a system of a series of chemicals involved in the process of fibrinolysis (fibrinolysis), mainly including plasminogen (PLG), plasmin, plasminogen activators, and fibrinolysis inhibitors. Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA is a serine protease and is synthesized by vascular endothelial cells. t-PA activates plasminogen, a process that mainly occurs in fibrin. Urokinase-type plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells and can directly activate plasminogen without the need for fibrin as a cofactor. Plasminogen (PLG) is synthesized in the liver. When blood clots, PLG is adsorbed in large quantities to the fibrin net and activated to plasmin by the action of t-PA or u-PA to promote fibrinolysis. Plasminase (PL) is a serine protease that degrades fibrin and fibrinogen, hydrolyzes various coagulation factors V, VIII, X, VII, XI, II, etc., converts plasminogen to plasmin, hydrolyzes complement, etc. Fibrinolysis inhibitors include plasminogen activator inhibitor (PAI) and α2-antithyplasmin (α2-AP). There are two main forms of PAI, PAI-1 and PAI-2, which can specifically bind to t-PA in a 1:1 ratio, thereby inactivating it and activating PLG at the same time. α2-AP is synthesized in the liver and binds to PL in a 1:1 ratio to form a complex, thereby inhibiting PL activity. FXIII covalently binds α2-AP to fibrin, thereby weakening the susceptibility of fibrin to PL. Substances that inhibit the activity of the fibrinolysis system in vivo include PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin.

[0038] As used herein, the term "a component of the plasminogen activation pathway" refers to a 1. Plasminogen, Lys-plasminogen, Glu-plasminogen, micro-plasminogen, delta-plasminogen, their variants or analogs; 2. Plasmin and its variants or analogues; and 3. Plasminogen activators, including, for example, tPA and uPA, as well as tPA or uPA variants and analogs that contain one or more domains of tPA or uPA, such as one or more of the kringle domain and the proteolytic domain.

[0039] The above "variants" of plasminogen, plasmin, tPA and uPA include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as proteins having, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid added, deleted and / or substituted and still having plasminogen activity, plasmin activity, tPA or uPA activity. For example, a "variant" of plasminogen, plasmin, tPA or uPA includes, for example, mutants of these proteins resulting from substitution of 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative amino acids.

[0040] The "plasminogen variant" of the present invention covers a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12 and still having plasminogen activity. For example, the "plasminogen variant" of the present invention can be a protein in which 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid is added, deleted and / or substituted based on sequence 2, 6, 8, 10 or 12 and still has plasminogen activity. In particular, the plasminogen variants of the present invention include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as mutants of these proteins obtained by, for example, conservative substitution of 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids.

[0041] The plasminogen of the present invention may be an orthologue of human plasminogen derived from a primate or rodent, or a variant which still retains plasminogen activity, such as the plasminogen shown in sequences 2, 6, 8, 10 or 12, e.g., the human native plasminogen shown in sequence 2.

[0042] The above-mentioned "analogs" of plasminogen, plasmin, tPA and uPA include compounds that provide substantially the same effect as plasminogen, plasmin, tPA or uPA, respectively.

[0043] The above "variants" and "analogs" of plasminogen, plasmin, tPA and uPA cover "variants" and "analogs" of plasminogen, plasmin, tPA and uPA that contain one or more domains (e.g., one or more kringle domains and a proteolytic domain). For example, "variants" and "analogs" of plasminogen cover plasminogen variants and analogs, such as mini-plasminogen, that contain one or more plasmin domains (e.g., one or more kringle domains and a proteolytic domain). "Variants" and "analogs" of plasmin cover plasmin "variants" and "analogs" such as mini-plasmin and delta-plasmin that contain one or more plasmin domains (e.g., one or more kringle domains and a proteolytic domain).

[0044] Whether the above-mentioned "variants" or "analogs" of plasminogen, plasmin, tPA or uPA have the activity of plasminogen, plasmin, tPA or uPA, respectively, or whether they exert substantially the same effect as plasminogen, plasmin, tPA or uPA, respectively, can be measured by the level of activated plasmin activity using methods known in the art, such as enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS (fluorescence-activated cell sorting). For example, the measurement can be performed with reference to the methods described in the following documents:Ny,A.,Leonardsson,G.,Hagglund,A.C,Hagglof,P.,Ploplis,V.A.,Carmeliet,P. and Ny,T. (1999). Ovulation 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。

[0045] In some embodiments of the present invention, the "component of the plasminogen activation pathway" of the present invention is a plasminogen selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, δ-plasminogen, or a variant thereof that retains plasminogen activity. In some embodiments, the plasminogen is natural or synthetic human plasminogen, or a conservative mutant or fragment thereof that still retains plasminogen activity. In some embodiments, the plasminogen is an orthologue of human plasminogen from a primate or rodent, or a conservative mutant or fragment thereof that still retains plasminogen activity. In some embodiments, the amino acid sequence of the plasminogen is shown in sequence 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human native plasminogen. In some embodiments, the plasminogen is human native plasminogen shown in sequence 2.

[0046] "A compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway" refers to any compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway, including, for example, tPA, uPA, streptokinase, saruplase, alteplase, reteplase, tenecteplase, anistreplase, monteplase, lanoteplase, pamiteplase, and staphylokinase.

[0047] The "antagonist of fibrinolysis inhibitors" of the present invention is a compound that antagonizes, weakens, blocks, or prevents the action of fibrinolysis inhibitors, such as PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin. Said antagonists are antibodies to PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or for example antisense RNA or miniRNA that blocks or downregulates the expression of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that occupy the binding site of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin but do not have the function of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that block the binding and / or activity domains of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0048] Plasmin is an important component of the plasminogen activation system (PA system). It is a broad-spectrum protease that can hydrolyze several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans. Plasmin can also activate some pro-matrix metalloproteinases (pro-MMPs) to become active matrix metalloproteinases (MMPs). Thus, plasmin is an important upstream regulator of extracellular proteolysis. Plasmin is formed by hydrolysis of two physiological PAs proteins, tissue-type plasminogen activator (tPA) or urokinase plasminogen activator (uPA), from plasminogen. Plasminogen is present at relatively high relative levels in plasma and other body fluids, and traditionally, it has been believed that the regulation of the PA system is mainly achieved by the synthesis and activity levels of PAs. The synthesis of PA system components is tightly regulated by different factors, such as hormones, growth factors, and cytokines. In addition, there are specific physiological inhibitors of plasmin and PAs. The main inhibitor of plasmin is α2-antiplasmin. The activity of PAs is simultaneously inhibited by plasminogen activator inhibitor-1 (PAI-1) of uPA and tPA, and is regulated by plasminogen activator inhibitor-2 (PAI-2), which mainly inhibits uPA. Some cells have a uPA-specific cell surface receptor (uPAR) with direct hydrolytic activity.

[0049] Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. Plasminogen is synthesized mainly in the liver and is present in large amounts in extracellular fluids. The plasminogen content in plasma is approximately 2 μM. Thus, plasminogen is a large potential source of proteolytic activity in tissues and body fluids. There are two molecular forms of plasminogen: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). The naturally secreted and undegraded form of plasminogen has one amino-terminal (N-terminal) glutamic acid and is therefore called glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys77. Compared to glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate. The Arg560-Val561 peptide bond of the two forms of plasminogen is cleaved by uPA or tPA, resulting in the formation of the disulfide-linked double-chain protease plasmin. The amino-terminal part of plasminogen contains five homologous tricycles, the so-called kringles, and the carboxyl-terminal part contains the protease domain. Some kringles contain a lysine-binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor α2-AP. The most recently discovered fibrin-plasminogen fragment of 38 kDa, kringlel-4, is a potent inhibitor of angiogenesis. This fragment, named angiostatin, is generated from several protease-hydrolyzed plasminogens.

[0050] The main substrate for plasmin is fibrin, and fibrin lysis is a key factor in preventing the formation of pathological thrombi. [Plasmin also has substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycans, and gelatin, indicating that plasmin plays an important role in ECM remodeling. Indirectly, plasmin also degrades other components of the ECM by converting several protease precursors, including MMP-1, MMP-2, MMP-3, and MMP-9, into active proteases. Thus, it may be proposed that plasmin is an important upstream regulator of extracellular protein hydrolysis. Plasmin also has the ability to activate several latent forms of growth factors. In vitro, plasmin can also hydrolyze components of the complement system to release chemotactic complement fragments.

[0051] "Plasmin" is a very important enzyme present in blood, which hydrolyzes fibrin clots into fibrin degradation products and D-dimers.

[0052] "Plasminogen" is the proenzyme form of plasmin, which, based on the sequence in swiss prot, is calculated as the amino acid sequence of natural human plasminogen including the signal peptide (Sequence 4), and consists of 810 amino acids, has a molecular weight of about 90 kD, is synthesized mainly in the liver, and is a glycoprotein that can circulate in the blood, and the cDNA sequence encoding said amino acid sequence is as shown in Sequence 3. Full-sized plasminogen contains seven domains: a serine protease domain located at the C-terminus, a Pan Apple (PAp) domain located at the N-terminus, and five Kringle domains (Kringle 1-5). With reference to the sequences in swiss prot, the signal peptide comprises residues Met1-Gly19, Pap comprises residues Glu20-Val98, Kringle1 comprises residues Cys103-Cys181, Kringle2 comprises residues Glu184-Cys262, Kringle3 comprises residues Cys275-Cys352, Kringle4 comprises residues Cys377-Cys454, and Kringle5 comprises residues Cys481-Cys560. According to NCBI data, the serine protease domain comprises residues Val581-Arg804.

[0053] Glu-plasminogen is a natural full-size human plasminogen, and consists of 791 amino acids (not including a signal peptide consisting of 19 amino acids). The cDNA sequence encoding this sequence is as shown in sequence 1, and its amino acid sequence is as shown in sequence 2. In vivo, Lys-plasminogen is formed by hydrolysis at the 76th-77th amino acid positions of Glu-plasminogen, and is shown, for example, in sequence 6, and the cDNA sequence encoding this amino acid sequence is shown in sequence 5. Delta-plasminogen (δ-plasminogen) is a fragment of full-size plasminogen lacking the Kringle2-Kringle5 structure, and contains only Kringle1 and a serine protease domain (also called the protease domain (PD)). There is a literature that reports the amino acid sequence of δ-plasminogen (sequence 8), and the cDNA sequence encoding this amino acid sequence is shown, for example, in sequence 7. Mini-plasminogen consists of Kringle5 and serine protease domains, and the literature reports residues Val443-Asn791 (starting with the Glu residue of the Glu-plasminogen sequence without the signal peptide), and its amino acid sequence is as shown in sequence 10, and the cDNA sequence encoding this amino acid sequence is as shown in sequence 9. However, literature reports that microplasminogen only contains a serine protease domain, and its amino acid sequence is residues Ala543-Asn791 (the Glu residue of the Glu-plasminogen sequence without the signal peptide is the starting amino acid), and patent document CN102154253A discloses that it contains residues Lys531-Asn791 (the Glu residue of the Glu-plasminogen sequence without the signal peptide is the starting amino acid), and in this patent application, the sequence of microplasminogen can be referred to patent document CN102154253A, and its amino acid sequence is as shown in sequence 12, and the cDNA sequence encoding the amino acid sequence is as shown in sequence 11.

[0054] The structure of full-length plasminogen is also described in a paper by Aisina et al. (Aisina RB, Mukhametova L I. Structure and function of plasminogen / plasmin system[J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6):590-605). According to the above mentioned article by Aisina et al., plasminogen contains Kringle 1, 2, 3, 4, 5 domains and a serine protease domain (also called protease domain (PD)), Kringles are responsible for binding plasminogen to low and high molecular weight ligands (i.e., lysine binding activity), which results in the conversion of plasminogen to a more open configuration and makes it more accessible to activation, and the protease domain (PD) is residues Val562-Asn791, and tPA and UPA specifically cleave the Arg561-Val562 activating bond of plasminogen, thereby allowing plasminogen to form plasmin. Thus, the protease domain (PD) is the region that confers the proteolytic activity of plasminogen.

[0055] In the present invention, "plasmin", "fibrin plasmin", and "fibrous protein plasmin" can be used interchangeably and have the same meaning. "Plasminogen", "fibrin plasminogen", and "fibrous protein plasminogen" can be used interchangeably and have the same meaning.

[0056] In this application, the term "insufficiency" of plasminogen refers to the content or activity of plasminogen in the subject's body being lower than that of a normal person and being sufficiently low to affect the normal physiological function of the subject. The term "deficiency" of plasminogen refers to the content or activity of plasminogen in the subject's body being significantly lower than that of a normal person, and thus the activity or expression is so minimal that normal physiological function can only be maintained by external supply.

[0057] Those skilled in the art can understand as follows. All technical configurations of plasminogen in the present invention can be applied to plasmin, so the technical configurations described in the present invention cover plasminogen and plasmin. In the circulation process, plasminogen is in a closed inactive conformation, but when it binds to a thrombus or cell surface, it becomes active plasmin with an open conformation under the mediation of plasminogen activator (PA). The active plasmin further hydrolyzes fibrin clots into fibrin degradation products and D-dimers, thereby dissolving the thrombus. Among them, the PAp domain of plasminogen contains an important epitope that maintains plasminogen in an inactive closed conformation, but the KR domain can bind to lysine residues on receptors and substrates. Several types of enzymes as plasminogen activators have already been known, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hageman factor), etc.

[0058] "Plasminogen active fragment" refers to an active fragment that binds to lysine in the target sequence of a substrate (lysine binding activity), or an active fragment that exerts a proteolytic function (proteolytic activity), or a fragment that has both proteolytic activity and lysine binding activity. The technical configuration of the present invention related to plasminogen includes a technical configuration in which plasminogen is replaced with a plasminogen active fragment. In some embodiments, the plasminogen active fragment of the present invention comprises the serine protease domain of plasminogen or consists of the serine protease domain of plasminogen, and preferably, the plasminogen active fragment of the present invention comprises sequence 14 or comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with sequence 14, or consists of an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with sequence 14. In some embodiments, the plasminogen active fragments of the present invention comprise one or more domains selected from Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5, or consist of one or more domains selected from Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5. In some embodiments, the plasminogen of the present invention comprises a protein comprising the above plasminogen active fragments.

[0059] At present, the methods for measuring plasminogen and its activity in blood include tissue plasminogen activator activity (t-PAA), plasma tissue plasminogen activator antigen (t-PAAg), plasma tissue plasminogen activity (plgA), plasma tissue plasminogen antigen (plgAg), plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator inhibitor antigen, and plasma plasmin-antiplasmin complex (PAP). The most common method is the chromogenic substrate method: streptokinase (SK) and luminescent substrate are added to the plasma of the subject, and the PLG in the subject's plasma becomes PLM under the action of SK, which acts on the luminescent substrate, and then measured by a spectrophotometer, and the increase in absorbance is directly proportional to the activity of plasminogen. In addition, immunochemical methods, gel electrophoresis, immunoturbidimetry, radial immunodiffusion, etc. can also be used to measure plasminogen activity in blood.

[0060] "Ortholog" refers to a homologue between different species, including both protein and DNA homologues, also called a direct lineage gene. It specifically refers to a protein or gene that is evolved from the same ancestral gene between different species. The plasminogen of the present invention includes human native plasminogen, and further includes orthologs or orthologs of plasminogens with plasminogen activity derived from different species.

[0061] A "conservative substitution variant" is one in which a specified amino acid residue is modified but does not change the overall conformation and function of a protein or enzyme, including but not limited to those that substitute an amino acid in the amino acid sequence of a parent protein with an amino acid of similar properties (e.g., acidic, alkaline, hydrophobic, etc.). Amino acids with similar properties are known. For example, arginine, histidine, and lysine are hydrophilic alkaline amino acids and can be substituted for each other. Similarly, isoleucine is a hydrophobic amino acid and can be substituted by leucine, methionine, or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may vary. For example, they have a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variant" further includes polypeptides or enzymes with an amino acid identity of 60% or more based on the BLAST or FASTA algorithm, preferably 75% or more, most preferably 85% or more, and most preferably 90% or more, and have the same or essentially similar properties or functions compared to the natural or parent protein or enzyme.

[0062] "Isolated" plasminogen is plasminogen protein that has been separated and / or recovered from its natural environment. In some embodiments, the plasminogen is (1) purified to greater than 90%, greater than 95%, or greater than 98% purity (by weight), e.g., greater than 99% (by weight), as determined by the Lowry method; (2) purified to at least 15 residues of N-terminal or internal amino acid sequence as determined by a spinning cup sequencer; or (3) purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions with Coomassie brilliant blue or silver staining. Isolated plasminogen includes plasminogen produced from recombinant cells by bioengineering techniques and separated by at least one further purification step.

[0063] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having modified peptide backbones. The terms also include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions containing leader sequences of heterologous and homologous origin (with or without an N-terminal methionine residue); and the like.

[0064] The definition of "percentage of amino acid sequence identity" of a reference peptide sequence is the percentage of amino acid residues in a candidate sequence that are the same as those in a reference polypeptide sequence, after introducing gaps as necessary to achieve the maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example by publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm that achieves maximum comparison requirements for the full size of the sequences to be compared. However, for the purposes of the present invention, the percentage of amino acid sequence identity is obtained by the sequence comparison computer software ALIGN-2.

[0065] When comparing amino acid sequences by using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (or alternatively, a given amino acid sequence A that has or contains a certain % amino acid sequence identity to, with, or for a given amino acid sequence B) is calculated as follows: Fraction X / Y×100

[0066] where X is the number of amino acid residues that the sequence alignment program ALIGN-2 evaluates as identical and matching in its alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood as follows: if the length of amino acid sequence A and the length of amino acid sequence B are not equal, then the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise stated, all % amino acid sequence identity values ​​used in this text are as described in the previous paragraph and are obtained by the ALIGN-2 computer program.

[0067] As used herein, the term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect may be completely or partially preventing the onset or development of a disease or its symptoms, or partially or completely reducing the disease and / or its symptoms, and / or partially or completely curing the disease and / or its symptoms. It also includes: (a) preventing a disease from occurring in a subject, where the subject has the causative agent for the disease but has not been diagnosed with the disease; (b) inhibiting the disease or inhibiting its formation; and (c) attenuating a disease and / or its symptoms, i.e., causing a decrease or disappearance of the disease and / or its symptoms.

[0068] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0069] A "therapeutically effective amount" or "effective amount" is an amount of a component of the plasminogen activation pathway or its related compound (e.g., plasminogen) that is capable of achieving said prevention and / or treatment of a disease when administered to a mammal or other subject and used to treat the disease. A "therapeutically effective amount" will vary according to the component of the plasminogen activation pathway or its related compound (e.g., plasminogen) used, the severity of the disease and / or symptoms, and the age, weight, etc., of the subject to be treated.

[0070] Preparation of the Plasminogen of the Invention For therapeutic use, plasminogen may be isolated and purified from nature or may be synthesized by standard chemical peptide synthesis techniques. When polypeptides are synthesized by chemical methods, the synthesis may be performed in liquid phase or solid phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support and the remaining amino acids in the sequence are added in order, is suitable for the chemical synthesis of plasminogen. Various forms of SPPS, e.g., Fmoc and Boc, can be used to synthesize plasminogen. The techniques used for solid-phase synthesis are described in: Barany and Solid-Phase Peptide Synthesis; pages 3-284, The Peptides: Analysis, Synthesis, Biology. Volume 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6: 3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12: 723-8. Briefly, small insoluble porous beads are treated with functional units on which the peptide chain is built. After repeated coupling / deprotection cycles, the free N-terminal amine of the attached solid phase is coupled with a single N-protected amino acid unit, which is then deprotected to expose a new N-terminal amine for linking with another amino acid. The peptide remains immobilized on the solid phase and is then excised.

[0071] The plasminogen of the present invention is produced by standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector and operably linked to control sequences in the expression vector. Expression control sequences include, but are not limited to, a promoter (e.g., a naturally associated promoter or a heterologous promoter), a signal sequence, an enhancer element, and a transcription termination sequence. Control of expression can be a eukaryotic promoter system in a vector, which is transformed or transfected into a eukaryotic host cell (e.g., COS or CHO cells). Once the vector is introduced into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and for collection and purification of the plasminogen.

[0072] Suitable expression vectors usually replicate in the host cell either free or as an integral part of the host cell chromosomal DNA. Usually, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) which is useful for detecting those cells transformed in vitro with the desired DNA sequence.

[0073] Escherichia coli is an example of a prokaryotic host cell for cloning a polynucleotide encoding a compound of interest. Other suitable microbial hosts include bacilli, such as Bacillus subtilis and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can be generated, which usually contain expression control sequences (e.g., origins of replication) compatible with the host cell. There are also many known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the phage lambda-derived promoter system. Promoters generally control expression and may further include ribosome binding site sequences, etc., to initiate transcription and translation when controlling gene sequences, as needed.

[0074] Other microorganisms, such as yeast, can also be used for expression. Yeast (e.g., Saccharomyces (S. cerevisiae) and Pichia are examples of suitable yeast host cells, in which suitable carriers include expression control sequences (e.g., promoters), origins of replication, termination sequences, etc., as necessary. Exemplary promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes for maltose and galactose utilization.

[0075] In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in in vitro cell culture) can also be used to express and produce the anti-Tau antibodies of the present invention (e.g., polynucleotides encoding such anti-Tau antibodies). See, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for use in these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and required processing information sites, such as ribosome binding sites, RNA cleavage sites, polyadenosine oxidation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from rabbit immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0076] Once synthesized (chemically or recombinantly), the plasminogen according to the invention can be purified by standard procedures in the art, such as ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, etc. The plasminogen is essentially pure, e.g., at least about 80% to 85% pure, at least about 85%-90% pure, at least about 90%-95% pure, or 98%-99% pure or even more pure, e.g., free of contaminants, such as cellular debris, large molecules other than the desired product, etc.

[0077] Drug combinations A component of the plasminogen activation pathway or related compound (e.g., plasminogen) of the desired purity is mixed with an optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)) to form a lyophilized formulation or aqueous solution to obtain a therapeutic combination. Acceptable carriers, excipients, and stabilizers are non-toxic to subjects at the required dosages and concentrations, and further include buffers such as, for example, phosphates, citrates, and other organic acids. Antioxidants include ascorbyl methionine; preservatives (e.g., octadecyl dimethylbenzyl ammonium chloride; hexamethylenediamine chloride; benzalkonium chloride). chloride), benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parahydroxybenzoic acid esters, such as methyl or propyl parahydroxybenzoic acid ester; pyrocatechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptides (those having less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates include glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, fucose or sorbitol; salt forming counterions, such as sodium; metal complexes (such as zinc-protein complexes); and / or non-ionic surfactants, such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). A preferred lyophilized anti-VEGF antibody formulation is described in WO97 / 04801, incorporated herein by reference.

[0078] The combinations of the present invention may contain one or more active compounds as required for the particular condition requiring treatment, preferably those having complementary activities and no adverse effects on each other.

[0079] The plasminogen of the present invention can be encapsulated in microcapsules made, for example, by aggregation techniques or interfacial polymerization, can be incorporated, for example, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or can be incorporated into hydroxymethylcellulose or gel microcapsules and poly(methyl methacrylate) microcapsules in crude emulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0080] The plasminogen activation pathway components or related compounds (e.g., plasminogen) of the present invention to be administered to the body must be sterile, which can be readily achieved by filtration through sterile filtration membranes, prior to or after lyophilization and recombination.

[0081] The plasminogen activation pathway components or related compounds (e.g., plasminogen) of the present invention can be prepared in sustained release formulations. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers having a defined shape and containing the glycoprotein, such as membranes or microcapsules. Examples of sustained release matrices include polyesters, aqueous gels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981); Langer, Chem. Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid (Sidman, et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al., same source as above), or degradable lactic acid-hydroxyacetic acid copolymers, e.g., Lupron. Depot™ (injectable microspheres composed of lactic acid-hydroxyacetic acid copolymer and leuprolide acetate), and poly D-(-)-3-hydroxybutyrate. Polymers such as ethylene-ethyl acetate and lactic acid-hydroxyacetic acid can release molecules sustainedly for over 100 days, but some aqueous gels release proteins for a shorter period of time. Rational strategies can be designed to stabilize proteins depending on the mechanism involved. For example, if the aggregation mechanism is the exchange of sulfur disulfide bonds to form intermolecular SS bonds, stabilization can be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions.

[0082] Dosage and dosage Administration of the pharmaceutical compositions of the present invention can be achieved by different modes, such as nasal inhalation, aerosol inhalation, nasal or eye drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), intramuscular, and intrarectal administration.

[0083] Preparations for use in parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolytic replenishers, and the like. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, inert gases, and the like.

[0084] A medical practitioner may determine a dosage regimen based on various clinical factors. For example, as is known in the medical arts, the dosage regimen for any given patient is determined by several factors, including the patient's body type, body surface area, age, the specific compound being administered, sex, frequency and route of administration, overall health, and other drugs being administered at the same time. The dosage range of the plasminogen-containing pharmaceutical composition of the present invention may be, for example, about 0.0001-2000 mg / kg of subject body weight daily, or about 0.001-500 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 10 mg / kg, 50 mg / kg, etc.). For example, the dosage may be 1 mg / kg body weight or 50 mg / kg body weight or in the range of 1-50 mg / kg, or at least 1 mg / kg. Doses higher or lower than this illustrative range are also covered, particularly when the above factors are taken into account. Intermediate doses within the above range are also within the scope of the present invention. Subjects may receive such doses daily, every other day, weekly, or according to any schedule determined by empirical analysis. An exemplary dose schedule is 0.01-100 mg / kg for several 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.

[0085] Product or drug kit One embodiment of the invention relates to an article of manufacture or drug kit comprising a component of the plasminogen activation pathway or its associated compound (e.g., plasminogen). The article of manufacture preferably comprises a container, a label or a protocol. Suitable containers are bottles, vials, syringes, etc. The containers can be made of a variety of materials, such as glass or plastic. The container contains a composition, the composition is effective to treat a disease or condition of the invention, and has a sterile inlet (e.g., the container is an intravenous infusion pack or vial and includes a stopper that can be pierced by a hypodermic needle). At least one activator in the composition is a component of the plasminogen activation pathway or its associated compound (e.g., plasminogen). A label on or attached to the container describes the composition as being used to treat a condition of the invention. The article of manufacture may further comprise a second container containing a pharmaceutical buffer, the pharmaceutical buffer including, for example, phosphate buffered saline, Ringer's solution, and glucose solution. It may further comprise other materials as may be required from a commercial and user perspective, i.e., other buffers, diluents, filters, needles, and syringes. The product also includes a protocol with instructions for use, e.g., instructing a user of the composition to administer a composition of a component of the plasminogen activation pathway or its related compounds (e.g., plasminogen) and other drugs associated with the treatment of a disease to a patient. EXAMPLES

[0086] The human plasminogen used in the following examples was derived from the plasma of a human donor and has been described in the following publications: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry, 1965, 240(1):541-550; Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J Biol Chem. 1976 Jun 25; 251(12):3693-9; HAGAN JJ, ABLONDI FB, DE RENZO EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 Based on the method described in Apr;235:1005-10, the process was optimized and purified from human donor plasma, in which human Lys-plasminogen (Lys-plasminogen) and Glu-plasminogen (Glu-plasminogen) were more than 98%. Example 1

[0087] Example 1 relates to the effect of plasminogen on reducing the level of pulmonary fibrosis in a mouse model of monocrotaline-induced pulmonary arterial hypertension. Twelve 12-week-old C57 male mice were taken, and their weights and blood pressures were measured. The mice were randomly divided into two groups according to their blood pressure: 4 mice in the blank control group and 8 mice in the model group. The mice in the blank control group were administered 100 μl of saline via tail vein injection, and the mice in the model group were administered 60 mg / kg / mouse of monocrotaline via tail vein injection for three consecutive days. The mice were fed a normal diet. [3~4]After 3 days, blood pressure was measured, and the model mice were randomly divided into two groups according to blood pressure: a PBS-treated control group and a plasminogen-treated group, each of which had 4 mice. Plasminogen was administered to the mice in the plasminogen-treated group at 1 mg / 0.1 ml / mouse / day via tail vein injection, and the same amount (same volume) of PBS solution was administered to the mice in the PBS-treated control group via tail vein injection for 28 consecutive days. No administration was administered to the mice in the blank control group. The day when administration began after modeling was set as day 1, and on the 29th day, the mice were sacrificed, and the lungs were collected and fixed in 4% paraformaldehyde fixative for 24 hours. The fixed lung tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and then embedded in paraffin. The tissue sections were 3 μm thick, and the sections were deparaffinized, rehydrated, and washed once with water. After staining with 0.1% Sirius Red saturated with picric acid for 30 minutes, the sections were rinsed with running water for 2 minutes, stained with hematoxylin for 1 minute, rinsed with running water, fractionated with 1% hydrochloric acid alcohol, blued with ammonia water, rinsed with running water, dried, and then sealed in neutral rubber. The sections were observed under an optical microscope at 200x magnification.

[0088] Monocrotaline is a bipyrrole alkaloid that can be converted by P450 monooxygenase in the liver and then reach the lungs via the blood circulation, where it can cause irreversible damage to pulmonary vascular tissue. Pulmonary vascular endothelial cells are thought to be the target cells of monocrotaline, and damage to endothelial cells plays an important role in the process of pulmonary vascular remodeling. [28,29] . As a result, there was essentially no collagen deposition in the lungs of mice in the blank control group (Figure 1A), and the collagen deposition (marked by arrows) in the lung tissues of mice in the plasminogen group (Figure 1C) was significantly less than that in the solvent PBS-treated control group (Figure 1B). This indicates that plasminogen can significantly reduce lung fibrosis in monocrotaline-induced pulmonary arterial hypertension model mice. Example 2

[0089] Example 2 relates to the fact that plasminogen reduces pulmonary fibrosis in mice with systemic sclerosis. Seventeen 12-week-old C57 male mice were randomly divided into two groups: 11 mice in the control group administered with PBS and 6 mice in the plasminogen-administered group. The day the experiment began was recorded as day 0, and the mice were weighed and divided into groups. From day 1, mice were administered bleomycin subcutaneously at 0.1 mg / 0.1 ml / mouse / day to induce systemic sclerosis. [5] After 2 days, plasminogen or PBS was administered for 21 consecutive days to model the mice. Plasminogen was administered to the mice in the plasminogen group at 1 mg / 0.1 ml / mouse / day via tail vein injection, and the same amount of PBS solution was administered to the mice in the PBS control group. On the 22nd day, the mice were sacrificed, and the lung tissue was collected and fixed in 4% paraformaldehyde fixative for 24 hours. After fixation, the lung tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The tissue sections were 3 μm thick, and the sections were deparaffinized, rehydrated, and washed once with water. After staining with 0.1% Sirius Red saturated with picric acid for 30 minutes, the sections were rinsed with running water for 2 minutes, stained with hematoxylin for 1 minute, rinsed with running water, fractionated with 1% hydrochloric acid alcohol, blued with ammonia water, rinsed with running water, dried, and then sealed in neutral rubber. The sections were observed under an optical microscope at 200x magnification. The results of the study showed that in a bleomycin-induced systemic sclerosis mouse model, the degree of collagen fibrosis (marked by arrows) observed under a microscope in the solvent PBS-treated group (Figure 2A) was higher than that in the plasminogen-treated group (Figure 2B), the morphology of the alveolar walls in the lungs of the plasminogen-treated mice was close to normal, inflammatory cells were significantly reduced, and the degree of fibrosis was significantly lower than that in the solvent PBS-treated group, and the difference was statistically significant (Figure 2C). This indicates that plasminogen can effectively reduce fibrosis in the lung tissue of bleomycin-induced systemic sclerosis mice. Example 3

[0090] Example 3 relates to the fact that plasminogen reduces pulmonary fibrosis in paraquat-intoxicated mice. Twelve C57 mice aged 9-10 weeks were randomly divided into two groups: a control group administered with PBS, and a group administered with lasminogen, each consisting of six mice. The mice in both groups were given a single intraperitoneal injection of paraquat solution at 15 mg / kg body weight, after which treatment was started immediately, and this was recorded as day 1. [6] Plasminogen was administered to mice in the plasminogen group at 1 mg / 0.1 ml / mouse / day via tail vein injection, and the same amount of PBS solution was administered to mice in the vehicle PBS control group. The administration cycle was 14 days, and on the 15th day, the mice were sacrificed, and lung tissue was collected and fixed in 4% neutral formalin fixative for 24 hours. The fixed lung tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The tissue sections were 3 μm thick, and the sections were deparaffinized, rehydrated, and washed once with water. After staining with 0.1% Sirius Red for 60 minutes, they were rinsed with running water, stained with hematoxylin for 1 minute, rinsed with running water, and then blued by fractionation with 1% hydrochloric acid alcohol and ammonia water, rinsed with running water, dried, mounted, and the sections were observed under an optical microscope at 200x magnification. Paraquat is a fast-acting herbicide that is highly toxic to humans, and once in the human body, it can cause damage to the lungs, liver, kidneys, etc. Lung damage manifests as early alveolar epithelial cell damage, symptoms such as intraalveolar hemorrhage and edema, and later intraalveolar and interstitial fibrosis. [6] Currently, there is no cure for paraquat poisoning, and the mortality rate is nearly 100%. Sirius red staining showed that the deposition of collagen fibers in the lungs of mice administered plasminogen (Figure 3B) was significantly less than that in the control group administered solvent PBS (Figure 3A), indicating that plasminogen can reduce pulmonary fibrosis caused by paraquat poisoning. Example 4

[0091] Example 4 relates to the effect of plasminogen on reducing collagen deposition in lung tissue of pneumonia model mice. Eighteen 6-8 week old C57 mice were taken and randomly divided into three groups according to body weight, with 6 mice each in the blank control group, vehicle group, and treatment group. After the vehicle and treatment group mice were anesthetized with 2% isoflurane, a pneumonia model was established by tracheal instillation of 1.5 mg / ml bacterial lipopolysaccharide solution (LPS) (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number: L8880) at 3 mg / kg body weight. [7] Two hours after LPS administration, plasminogen was administered to the mice in the treatment group at 1 mg / 0.1 ml / mouse / day via tail vein injection, and the same amount of solvent was administered to the mice in the vehicle group via tail vein injection for 14 consecutive days. On the 15th day, the mice were sacrificed, and lung tissue was taken and fixed in 4% neutral formalin fixative for 24 hours. After fixation, the lung tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The tissue sections were 3 μm thick, and the sections were deparaffinized, rehydrated, and washed once with water. After staining with 0.1% Sirius Red for 60 minutes, they were rinsed with running water, stained with hematoxylin for 1 minute, rinsed with running water, and then blued by fractionation with 1% hydrochloric acid alcohol and ammonia water, rinsed with running water, dried, mounted, and the sections were observed under an optical microscope at 200x magnification. As a result, there was a certain amount of collagen deposition in the lung tissue of the blank control group (Figure 4A) (marked by arrows), collagen deposition was significantly increased in the lung tissue of mice in the vehicle group (Figure 4B), and collagen deposition in the lung tissue of mice in the treatment group (Figure 4C) was significantly less than that of mice in the vehicle group, and the difference was statistically significant (* represents P<0.05) (Figure 4D). This indicates that plasminogen can reduce collagen deposition in the lungs of pneumonia model mice and improve pulmonary fibrosis caused by pneumonia. Example 5

[0092] Example 5 relates to the effect that pre-administration of plasminogen reduces collagen deposition in lung tissue of pneumonia model mice. Eighteen 6-8 week old C57 mice were taken and randomly divided into three groups according to body weight, with 6 mice each in the blank control group, the solvent group, and the treatment group. Plasminogen was administered to the treatment group mice at 1 mg / 0.1 ml / mouse / day via tail vein injection, and the same amount of solvent was administered to the solvent group mice via tail vein injection for three consecutive days. On the fourth day, the solvent group and treatment group mice were anesthetized with 2% isoflurane, and then a pneumonia model was established by instilling 1.5 mg / ml bacterial lipopolysaccharide solution (LPS) (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number: L8880) at 3 mg / kg body weight into the trachea. [7] On the ninth day, mice were sacrificed, and lung tissues were harvested and fixed in 4% neutral formalin fixative for 24 hours. After fixation, the lung tissues were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The tissue sections were 3 μm thick, and the sections were deparaffinized, rehydrated, and washed once with water. They were stained with 0.1% Sirius Red for 60 minutes, rinsed with running water, stained with hematoxylin for 1 minute, rinsed with running water, and blued with 1% hydrochloric acid alcohol and ammonia water by fractionation, rinsed with running water, dried, mounted, and the sections were observed under an optical microscope at 200x magnification. As a result, there was a certain amount of collagen deposition in the lung tissue of the blank control group (Figure 5A) (marked with an arrow), collagen deposition was significantly increased in the lung tissue of the mice in the vehicle group (Figure 5B), and collagen deposition in the lung tissue of the mice in the treatment group (Figure 5C) was significantly less than that of the mice in the vehicle group. This indicates that pre-administration of plasminogen can reduce collagen deposition in the lungs of pneumonia model mice and ameliorate pulmonary fibrosis caused by pneumonia. Example 6

[0093] Example 6 relates to the effect of plasminogen on reducing total protein levels in lung lavage fluid of pneumonia model mice. Eighteen 6-8 week old C57 mice were taken and randomly divided into three groups according to body weight, with 6 mice each in the blank control group, vehicle group, and treatment group. After the vehicle and treatment group mice were anesthetized with 2% isoflurane, a pneumonia model was established by tracheal instillation of 1.5 mg / ml bacterial lipopolysaccharide solution (LPS) (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number: L8880) at 3 mg / kg body weight. [7] Two hours after LPS administration, plasminogen was administered to the mice in the treatment group at 1 mg / 0.1 ml / mouse / day via tail vein injection, and the same amount of solvent was administered to the mice in the vehicle group via tail vein injection for 7 consecutive days. On the 8th day, all mice were sacrificed, and the lungs were dissected and weighed. The right lung bronchus was washed twice with PBS in total of 0.7 ml, and the liquid was collected and centrifuged at 700 g for 10 min at 4°C to collect the supernatant, and the total protein was detected using the bicinchoninic acid (BCA) method. As a result, there was a certain amount of total protein in the lung lavage fluid of the blank control group, the total protein level in the lung lavage fluid of the vehicle group was significantly higher than that of the blank control group (*** indicates P<0.001), and the total protein level in the lung lavage fluid of the treatment group was significantly lower than that of the vehicle group, and the statistical difference was close to significance (P=0.052) (Figure 6). This indicates that plasminogen can reduce the total protein level in the lung lavage fluid of pneumonia model mice. Example 7

[0094] Example 7 relates to the effect of plasminogen on reducing pulmonary fibrin deposition in pneumonia model mice. Twelve 6-8 week old C57 mice were taken and randomly divided into two groups according to body weight, six in the vehicle group and six in the treatment group. After the vehicle and treatment group mice were anesthetized with 2% isoflurane, a pneumonia model was established by tracheal instillation of 1.5 mg / ml bacterial lipopolysaccharide solution (LPS) (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number: L8880) at 3 mg / kg body weight. [7] Two hours after LPS administration, plasminogen was administered to the mice in the treatment group at 1 mg / 0.1 ml / mouse / day via tail vein injection, and the same volume of the solvent was administered to the mice in the vehicle group via tail vein injection for three consecutive days. On the fourth day, the mice were sacrificed, and the left lungs were harvested and fixed in 4% neutral formalin fixative for 24 hours. The fixed tissue samples were dehydrated in an alcohol gradient, permeabilized with xylene, and then embedded in paraffin. The coronal sections of the lung tissue were 3 μm thick, and the sections were deparaffinized, rehydrated, and washed once with water. They were fixed in citric acid for 30 minutes, cooled at room temperature for 10 minutes, and then gently rinsed with water. They were incubated in 3% hydrogen peroxide for 15 minutes, and the tissues were circled with a PAP marker. They were blocked in 10% sheep serum solution (Vector laboratories, Inc., USA) for 1 hour. After the time was up, the sheep serum solution was discarded. Rabbit anti-fibrin antibody (Abcam) was added and incubated overnight at 4°C, then washed twice with PBS for 5 minutes each time. Secondary antibody goat anti-rabbit IgG (HRP) antibody (Abcam) was incubated at room temperature for 1 hour, washed twice with PBS for 5 minutes each time. The sections were stained with DAB kit (Vector laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, blued with running water for 5 minutes, and washed once with PBS. The sections were dehydrated in a gradient, cleared, and mounted, and the sections were observed under a light microscope at 200x magnification. Fibrin is the main component of the hyaline membrane in the lungs during acute respiratory distress, which impedes the gas exchange function of the alveoli, causing a decrease in blood oxygen saturation and respiratory distress. [8] . As a result, the level of fibrin deposition in the lung tissue of mice in the vehicle group (Figure 7A) was significantly higher than that in the treatment group (Figure 7B). This indicates that plasminogen can reduce pulmonary fibrin deposition in pneumonia model mice.

[0095] The following examples further illustrate the practice of the present invention but are not intended to limit it.

[0096] All patients below took the medication voluntarily by signing an informed consent form, and approval was obtained from the hospital ethics committee.

[0097] The method and dosage were adjusted and increased or decreased depending on the condition and progress of the disease. The main administration methods were aerosol inhalation and intravenous injection. The concentration of human plasminogen purified from human donor blood administered by aerosol inhalation and intravenous injection was 5mg / ml in all cases, and saline was used as the solvent. Example 8

[0098] Example 8 concerns patients with pneumonia and pulmonary fibrosis. The patient was a 77-year-old man with cerebral infarction sequelae and pulmonary fibrosis. Before using this drug, he had developed pneumonia, and had a lot of phlegm, which was yellow and viscous, and difficult to cough. He needed to suction phlegm 25 to 26 times a day (24 hours), and was able to suction a large amount of phlegm each time. He was constantly inhaling oxygen (almost no deoxygenation was possible), and his breathing was a little rough. He had a laryngotracheotomy (metal sleeve). He had been bedridden for nearly half a year, and continued to suffer from lack of sleep and poor mental condition. Dosage regimen: 10 mg / dose, 3 times / day, by aerosol inhalation for the first 9 days, 10 mg / dose, 2 times / day, by aerosol inhalation on the 10th day, no drug administration on the 11th day, 10 mg by 1 aerosol inhalation on the 12th day, 10 mg by 1 aerosol inhalation on the 13th day, 10 mg by intravenous injection at the same time, and 15 mg by intravenous injection on the 14th day. Therapeutic effect: Medication on the first day: Symptoms improved, manifested as strong sputum, naturally discharged from the tracheotomy, and the number of sputum suctions decreased to 8 times (8 am to 8 pm). Medication on the second day: The first time the patient was deoxygenated and sat with hands supported for 20 minutes, blood oxygen saturation was 94%, and heart rate was 65. The second time the patient was deoxygenated and sat with hands supported for 10 minutes, blood oxygen saturation was 98%, and heart rate was 75. The third time the patient was deoxygenated and sat with hands supported for 20 minutes, blood oxygen saturation fluctuated between 94 and 98%, and heart rate was about 78. Sputum was stronger than the previous day, and suction was 6 times (8 am to 8 pm). From the 14th day of medication onwards, the patient spontaneously requested deoxygenation. After deoxygenation, the blood oxygen saturation was 97%, and at bedtime it was about 94%, and the deoxygenation time could reach more than 12 hours, so deoxygenation was basically possible. The sputum changed from a yellow, sticky, viscous substance before the drug administration to a white, thin, foamy substance, and changed from being mainly excreted with a sputum aspirator to being mostly excreted spontaneously. The above results indicate that plasminogen can effectively improve the symptoms of pneumonia in patients, including strong sputum, spontaneous sputum discharge, reduced number of sputum suctions, prolonged deoxygenation time, improved blood oxygen saturation, and improved sputum. Example 9

[0099] Example 9 concerns patients with pulmonary fibrosis and asthma. The patient was an 83-year-old woman who had suffered from pulmonary tuberculosis in her younger years and was diagnosed with pulmonary fibrosis and asthma about 20 years ago, which had gradually deteriorated.The main symptoms were decreased lung function, which could only support activities in bed and could not leave bed. Dosage regimen: 10 mg once daily by aerosol inhalation on day 1, 10 mg twice daily by aerosol inhalation on day 2, and 10 mg three times daily by aerosol inhalation on day 3. Treatment effect: After taking the medication, the patient could breathe consciously and smoothly, and could do more activities out of bed. The above results indicate that plasminogen can improve lung function in patients with pulmonary fibrosis and asthma. Example 10

[0100] Example 10 concerns asthma patients. The patient was a 55-year-old man with stable vital signs and clear consciousness. Chief complaint: No history of hypertension, heart disease, diabetes, etc. The patient had been taking trimetazidine, bayaspirin, ginkgo capsules, and deep-sea fish oil for many years and had suffered a cerebral infarction 3 years ago. The patient complained of poor quality sleep, no other discomfort, and asthma was observed after exercise. Dosage regimen: Treatment consisted of 14 doses over 22 days, with the drug administered once daily for the first 6 days, as an intravenous bolus injection of 150 mg each time, and then once every 2 days for the next 16 days, as an intravenous bolus injection of 150 mg each time. Therapeutic effect: The therapeutic effect was evaluated by scoring the patient's overall condition and asthma symptoms after exercise. On the first day, the patient's overall condition and asthma symptoms after exercise without medication were scored as 10, and the patient's overall condition and asthma symptoms after exercise the day before the injection were scored as 10. 10 is the most severe and 1 is the mildest. On the fourth day of administration, the overall condition score was 8, the asthma symptom score after exercise was 8, the patient's mental state was good, and the quality of sleep improved. As the administration time was extended, the patient's symptoms further improved. On the 22nd day of administration, the overall condition score was 5, the asthma symptom score after exercise was 3, the patient was not uncomfortable, the quality of sleep further improved, and the appetite improved. As can be seen from the above, plasminogen can improve the symptoms of asthma patients, such as general condition, asthma symptoms after exercise, mental state, sleep quality, and appetite. Example 11

[0101] Example 11 relates to non-5q SMA patients. The patient was a 40-month-old female (3 years and 4 months old) diagnosed with non-5q SMA (spinal muscular atrophy). The onset was at 6 months of age, and she was diagnosed with SMA at 1.5 years (18 months). Due to lung infections and difficulty in expelling sputum, the airway became blocked, her breathing weakened, and after intermittent use of a ventilator, she gradually became unable to leave the ventilator due to spontaneous respiratory failure, and used the ventilator for about 1.5 years. She lost her speech function, was unable to exercise, and her muscle strength was basically grade 0. Symptoms: Since the use of the ventilator, she received expectorants, expectorants, sputum suction devices, oxygen inhalation, nebulization (twice a day), and nasogastric feeding every day. The parameters of the ventilator used were: pressure 20; tidal volume about 120. There was almost no spontaneous breathing, oxygen flow rate 1-1.5 ml, sputum suction 15-20 times a day, irregular sputum suction at night (sputum suction damaged the airway mucosa and caused bleeding), blood oxygen detector showed that blood oxygen saturation was 95% or higher when oxygen was used (oxygen inhalation could not be discontinued), heart rate was 140 beats / min. Body temperature was above 37.5 ° C for a long time. Medication Regimen Initial treatment course (2 weeks): Aerosol inhalation, 10 mg, 3 times a day, combined with intravenous injection of 50-100 mg, administered once every 3 days. After completion of the first course of treatment, a second course of treatment was administered 57 days later. The second to fourth treatment courses (with a 2-week interval between each course): intravenous injection, 1 time / 3 days, dose 150-250 mg. Treatment effect First course of treatment: the ventilator parameters were not changed, spontaneous breathing increased and reached about 20-30% (about 2-3 spontaneous breaths occurred in 10 breaths), and blood oxygen saturation after medication could reach 98-99% if the oxygen flow rate was unchanged. The second treatment course: After 15 days of hospitalization, a tracheotomy was performed, and the patient was discharged and nursed at home. There was a large amount of sputum, which continued to overflow from the airway, and it was necessary to timely suction and remove the sputum. The pressure of the ventilator was reduced and adjusted from 18 at the time of discharge to 16, the tidal volume was 130 to 140, and the recovery of spontaneous breathing was good. Oxygen could be turned off, and oxygen was basically not required at night, blood oxygen saturation was above 95%, the heart rate was about 120 beats per minute, and it was even better from time to time, and the body temperature returned to normal. The third course of treatment: the ventilator pressure could be adjusted to 15 or 16, the tidal volume was between 140-150, the airway was more open, all were spontaneously breathing, the number of oxygen inhalations was small, and it was used occasionally during sputum suction. The normal heart rate was 120 beats / min, and no oxygen was required within 4-5 hours after administration, the heart rate was 100 beats / min, and the blood oxygen saturation was 99%. The patient's sputum volume decreased, the tracheotomy was basically free of sputum, and sputum suction was performed 5-6 times a day. The fourth treatment course: the ventilator parameters and spontaneous breathing conditions were the same as those in the third treatment course, the blood oxygen saturation was usually 97-99%, oxygen was basically not used, the frequency of sputum suction was reduced to 2-3 times a day, occasionally 5-6 times, and the body temperature was normal. The above results indicate that plasminogen can improve lung function in SMA patients, including improved blood oxygen saturation, improved spontaneous breathing function, patent airway, deoxygenation or occasional oxygen inhalation, and reduced sputum volume. Example 12

[0102] Example 12 concerns SMA type I patients. The patient was an 18-month-old male who was diagnosed with type I SMA at 6 months of age. At the time of diagnosis, the doctor informed him that the average survival time for patients with this type of disease was 2 years, but the family did not take any treatment measures. Pulmonary function symptoms: Blood oxygen was monitored 24 hours a day, blood oxygen saturation was 92-97%, there was little rise and fall of the chest when breathing, breathing swell was weak during sleep, there was phlegm, and phlegm could not be expelled spontaneously. There was a weak cry and no energy. Treatment method: aerosol inhalation (2-3 times / day) + intravenous injection (1 time / 3 days), treatment cycle was 5 courses (2 weeks per course), interval between each course was 2 weeks. Aerosol inhalation dose was 5-10 mg / time, intravenous injection dose was 50 mg. Treatment effect First course of treatment: On the second day of treatment, the blood oxygen saturation reached 97-98%, occasionally reaching 95-96%. The blood oxygen saturation became normal, respiratory function improved, sputum could be expelled with assistance, and the amount of sputum decreased. Second course of treatment: Blood oxygen saturation remained normal and breathing became stronger. Third treatment course: After aerosol inhalation, phlegm was discharged, there was a sound of phlegm in the morning, after crying, the sound of phlegm was loud, and phlegm could be discharged with assistance. 4th-5th treatment course: The effect continued, the patient's mental state was good, and there was no worsening of the condition such as lung infection or respiratory failure. As can be seen from the above, plasminogen can improve the lung function of patients with SMA type I, improve respiratory function, and increase blood oxygen saturation. After treatment, blood oxygen saturation reaches normal levels, assisted expectoration is possible, and there are no worsening symptoms such as lung infections or respiratory failure, and various symptoms of the patients can be improved. Example 13

[0103] Example 13 concerns patients with Corona Virus Disease 2019 (COVID-19). The patient was a 48-year-old man diagnosed with severe novel coronavirus pneumonia (2019-nCoV). In the intensive care unit, he was on a ventilator and monitored, with an oxygen concentration of 100% and a blood oxygen saturation of 80-90%. Vital signs: heart rate 92 beats / min, respirations 41 breaths / min, blood pressure 128 / 84 mmHg. Dosage regimen: aerosol inhalation, 10 mg / dose, given in two doses 5.5 hours apart. Medication Effects First dose: Before the dose, the blood oxygen saturation was 84%, the heart rate was 92 beats / min, the respiration rate was 41 breaths / min, and the blood pressure was 128 / 84 mmHg. One hour after the dose, the blood oxygen saturation was 90%, the heart rate was 83 breaths / min, the respiration rate was 37 breaths / min, and the blood pressure was 128 / 84 mmHg. Second dose: Before administration, blood oxygen saturation was 88%, heart rate was 81 beats / min, respiration was 39 breaths / min, and blood pressure was 120 / 83 mmHg. One hour after administration, blood oxygen saturation was 91%, heart rate was 70 breaths / min, respiration was 28 breaths / min, and blood pressure was 120 / 83 mmHg. There were no side effects before or after dosing, and the patient reported feeling better. As can be seen from the above, plasminogen can effectively increase blood oxygen saturation, slow breathing frequency, and improve lung function in patients with severe novel coronavirus pneumonia. Example 14

[0104] Example 14 concerns COVID-19 patients. The patient was a 47-year-old man diagnosed with severe novel coronavirus pneumonia (2019-nCoV). In the intensive care unit, he was on a ventilator and monitored, with an oxygen concentration of 100% and a blood oxygen saturation of 80-90%. Vital signs: temperature 37.5°C, heart rate 110 beats / min, respirations 37 breaths / min, blood pressure 139 / 94 mmHg. Dosage regimen: aerosol inhalation, 10 mg / dose, given in two doses 4 hours 50 minutes apart. Medication Effects First dose: Blood oxygen saturation before administration was 81%. Three hours after administration, blood oxygen saturation was 88-90%. Second dose: Before administration, blood oxygen saturation was 89-92%, heart rate was 91 beats / min, respiration was 31 breaths / min, and blood pressure was 130 / 87mmHg. One hour after administration, blood oxygen saturation was 91%, heart rate was 89 breaths / min, respiration was 27 breaths / min, and blood pressure was 130 / 87mmHg. There were no side effects before or after dosing, and the patient reported feeling better. As can be seen from the above, plasminogen can effectively improve blood oxygen saturation and improve respiratory frequency in patients with severe novel coronavirus pneumonia. Example 15

[0105] Example 15 relates to plasminogen lowering blood pressure in severe COVID-19 patients. The patient was a 46-year-old man with a blood oxygen saturation of 93% and a respiratory rate of 26 breaths per minute, and was diagnosed with severe COVID-19 according to the clinical classification criteria of the Novel Coronavirus Pneumonia Diagnosis and Treatment Program (Trial Version 6). The patient's blood pressure before treatment was 130 / 80 mmHg (systolic / diastolic pressure). Lyophilized human plasminogen powder was dissolved in sterile water at a concentration of 5 mg / ml and administered to patients by nebulization with a nebulizer, 10 mg twice a day. After six doses, the patient's blood pressure dropped from a previous 130 / 80 mmHg to 116 / 69 mmHg, suggesting that plasminogen can lower blood pressure in patients with severe COVID-19. Example 16

[0106] Example 16 relates to plasminogen reducing respiratory rate in severe COVID-19 patients. The patient was a 47-year-old man with a blood oxygen saturation of 93% and was diagnosed with severe COVID-19 according to the clinical classification criteria of the Novel Coronavirus Pneumonia Diagnosis and Treatment Program (Trial Version 6). The patient's respiratory rate before treatment was 45 breaths per minute. Lyophilized human plasminogen powder was dissolved in sterile water at a concentration of 5 mg / ml and administered to patients by nebulization with a nebulizer, 10 mg twice a day. After two doses, the patient's respiratory rate dropped from 45 breaths per minute to 37 breaths per minute, suggesting that plasminogen can reduce the respiratory rate in patients with severe COVID-19. Example 17

[0107] Example 17 relates to plasminogen lowering blood pressure in severe COVID-19 patients. The patient was a 48-year-old man with a blood oxygen saturation of 79% and a respiratory rate of 41 breaths per minute, and was diagnosed with severe COVID-19 according to the clinical classification criteria of the Novel Coronavirus Pneumonia Diagnosis and Treatment Program (Trial Version 6). The patient's blood pressure before treatment was 128 / 84 mmHg (systolic / diastolic pressure). Lyophilized human plasminogen powder was dissolved in sterile water at a concentration of 5 mg / ml and administered to patients by nebulization with a nebulizer, 10 mg twice a day. After five doses, the patient's blood pressure dropped from a previous 128 / 84 mmHg to 120 / 83 mmHg, suggesting that plasminogen can lower blood pressure in severe COVID-19 patients. Example 18

[0108] Example 18 relates to plasminogen reducing blood pressure and respiratory rate in severe COVID-19 patients. The patient was a 47-year-old man with a blood oxygen saturation of 82% and a respiratory rate of 37 breaths per minute, and was diagnosed with severe COVID-19 according to the clinical classification criteria of the Novel Coronavirus Pneumonia Diagnosis and Treatment Program (Trial Version 6). The patient's blood pressure before treatment was 139 / 94 mmHg (systolic / diastolic pressure). Lyophilized human plasminogen powder was dissolved in sterile water at a concentration of 5 mg / ml and administered to patients by nebulization with a nebulizer, 10 mg twice a day. After two doses, the patient's blood pressure dropped to 120 / 83 mmHg from a previous level of 139 / 94 mmHg, and his respiratory rate dropped to 27 breaths / min from 37 breaths / min before administration, suggesting that plasminogen can reduce blood pressure and respiratory rate in severe COVID-19 patients. Example 19

[0109] Example 19 relates to plasminogen improving the condition of COVID-19 patients. The study recruited 13 COVID-19 patients aged 30-78 years. According to the clinical classification criteria of the Novel Coronavirus Pneumonia Diagnosis and Treatment Program (Trial 6), the 13 patients included 5 common type patients, 6 severe type patients, and 2 critical type patients. The treatment was approved by the hospital's ethics committee. All patients signed informed consent. Human plasminogen freeze-dried powder was dissolved in sterile water at a concentration of 5 mg / ml and administered to patients by nebulization. Different doses of plasminogen were administered according to the severity of the disease. Patients with normal disease were administered 10 mg / dose once a day, while patients with severe and critical disease were administered 10 mg / dose twice a day. The basic information of the patients and the frequency of administration are shown in Table 1. High-resolution chest CT (Neusoft, NeuViz 16 Classic, China) scans were performed several days before and after administration. Real-time blood oxygen saturation and heart rate were monitored by a monitor (Mindary, iPM5, China), and blood oxygen saturation and heart rate were recorded 1 hour before and several hours after aerosol inhalation of human plasminogen.

[0110] Table 1. Overview of human plasminogen therapy in COVID-19 patients JPEG2025011148000001.jpg108138

[0111] Result 1: Plasminogen ameliorates lung injury in common COVID-19 patients CT scans of the five patients with ordinary type showed multiple patchy / punctate "ground-glass" shadows with unclear borders and uneven density in both lungs before administration, and the lesion area of ​​the mediastinal window showed sparse patchy shadows. The five patients were administered antibiotics and Chinese herbal medicines before administration of human plasminogen, but the density and extent of the "ground-glass" shadows in the lungs increased over time, indicating deterioration. After administration of human plasminogen 2 to 3 times, the number, extent, and density of the lung lesions in the five patients decreased and even partially disappeared, and the patchy or punctate "ground-glass" shadows were significantly reduced or absorbed (see Figure 8 and Table 2). This indicates that aerosol inhalation of human plasminogen can rapidly ameliorate lung damage caused by COVID-19 infection.

[0112] Table 2. High-resolution chest CT scan records of patients with common COVID-19 JPEG2025011148000002.jpg147144

[0113] Result 2: Plasminogen increases blood oxygen saturation in severe and critically ill COVID-19 patients During plasminogen administration, patients with severe or critical COVID-19 were infused with constant oxygen concentrations of 80% or 100%, respectively, via nasal cannula, and blood oxygen saturation was monitored under oxygen infusion conditions. As a result, after administration of human plasminogen, the blood oxygen saturation of five of the six patients increased by 1-4%, even though the blood oxygen saturation of the severe patients was normal. In two severe patients, the blood oxygen saturation of the patients increased from 79-82% before administration to approximately 91% just one hour after aerosol inhalation of human plasminogen and remained stable. After administration of plasminogen to one severe patient, the blood oxygen saturation decreased from 91% before administration to 89% (Table 3). These data indicate that plasminogen can generally improve the blood oxygen saturation levels of severe and critical COVID-19 patients, and the improving role is particularly significant for patients with particularly low blood oxygen saturation.

[0114] Table 3. Blood oxygen saturation values ​​in severe and critical COVID-19 patients JPEG2025011148000003.jpg85141

[0115] Result 3: Plasminogen can restore heart rate and reduce cardiac strain in COVID-19 patients Heart rate monitoring showed that after aerosol inhalation of human plasminogen, the heart rate of 8 of the 13 COVID-19 patients decreased by about 26 beats per minute, the heart rate increased in 2, and there was no significant change in 3 (Table 4). Statistical analysis showed that after plasminogen administration, the average heart rates of ordinary, severe, and critical patients all showed a tendency to decrease, and in ordinary patients, there was a statistical difference between before and after administration (P<0.05) (Figure 9). These results indicate that aerosol inhalation of human plasminogen can generally slow the high heart rate of COVID-19 patients and reduce the burden on the heart.

[0116] Table 4. Heart rate monitoring results of COVID-19 patients JPEG2025011148000004.jpg115141

[0117] In addition, all patients reported reduced chest tightness and easier breathing after aerosol inhalation of human plasminogen. Based on the above, aerosol inhalation of human plasminogen can reduce lung injury in COVID-19 patients, increase their blood oxygen saturation, reduce their heart rate, and improve their respiratory function. References: [1] Yongzhi G, Jinan L, Elin H, et al. Beneficial and Detrimental Effects of Plasmin(ogen) during Infection and Sepsis in Mice[J]. PLoS ONE, 2011, 6(9):e24774-. [2] Guo Y , Li J , Elin Hagstrom, et al. Protective effects of plasmin(ogen) in Staphylococcus aureus-induced arthritis[J]. Arthritis & Rheumatology, 2008, 58(3):764-772. [3]Rubin Tan, Jiansha Li,Chuanyu Wei1, Il-Kwon Kim, GAPDH is critical for superior efficacy of female bone marrow-derived mesenchymal stem cells on pulmonary hypertension, Cardiovascular Research (2013) 100, 19-27. [4]Jose G. Gomez-Arroyo,The monocrotaline model of pulmonary hypertension in perspective,Am J Physiol Lung Cell Mol Physiol 302: L363-L369, 2012. [5]Yosuke Kanno, En Shu, Hiroyuki Kanoh et al. The Antifibrotic Effect of a2AP Neutralization in Systemic Sclerosis Dermal Fibroblasts and Mouse Models of Systemic Sclerosis. J Invest Dermatol. 2016 Apr;136(4):762-9. [6] Ming-wei Liu, Rong Liu,Hai-ying Wu et al.Radix puerariae extracts ameliorate paraquat-induced pulmonary fibrosis by attenuating follistatin-like 1 and nuclear factor erythroid 2p45-related factor-2signalling pathways through downregulation of miRNA-21 expression. BMC Complementary and Alternative Medicine (2016) 16:11. [7] Franco R. D’Alessio. Mouse models of acute lung injury and ARDS[J]. 2018. [8] Ambrus CM, Choi TS, Cunnanan E, Eisenberg B, Staub HP, Weintraub DH, et al. Prevention of hyaline membrane disease with plasminogen. A cooperative study. Jama. 1977;237(17):1837-41. Sequence Listing Sequence 1: Array 2: EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHA HGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDNPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKCCQSWSSMTPHRHQKTPE NYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLI SPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Array 3: Array 4: MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMS GLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKCCQSWSSMT PHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFC GGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Array 5: Array 6: KVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQ CLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKK CSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPS SYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Array 7: Array 8: EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGI TCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFG MHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Array 9: Array 10: VRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEK SPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN Array 11: gccccttcatttgattgtgggaagcctcaagtggagccgaagaaatgtcctggaagggttgtaggggggtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgccccactgct tggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcacaccaagaagtgaatctcgaaccgcatgttcaggaaataagaagtgtctaggctgttcttggagcccacacgaaaagattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctg ccatccccaaattatgtggtcgctgaccggaccgaatgtttcatcactgggctggggagaaacccaaggtacttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatt tggccggaggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacatttacaaggatcacttcttggggtcttggctgtgcacgccccataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgagaaataattaa array12: APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN array13: gttgtaggggggtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgccccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctg ggtgcacaccaagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgct gaccggaccgaatgtttcatcactgggctggggagaaacccaaggtacttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctggggcatttggccgga ggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacatttacaaggatcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgaga array14: VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMR

Claims

[Claim 1] A pharmaceutical composition for preventing and treating pneumonia comprising one or more therapeutically effective amounts of compounds selected from components of the plasminogen activation pathway, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

Images