WO2021170099A1 - 一种预防和治疗血压异常病症的方法和药物 - Google Patents

一种预防和治疗血压异常病症的方法和药物 Download PDF

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WO2021170099A1
WO2021170099A1 PCT/CN2021/078200 CN2021078200W WO2021170099A1 WO 2021170099 A1 WO2021170099 A1 WO 2021170099A1 CN 2021078200 W CN2021078200 W CN 2021078200W WO 2021170099 A1 WO2021170099 A1 WO 2021170099A1
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plasminogen
blood pressure
mice
plasmin
group
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PCT/CN2021/078200
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English (en)
French (fr)
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李季男
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泰伦基国际有限公司
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Priority to KR1020227032536A priority Critical patent/KR20220143913A/ko
Priority to EP21760438.8A priority patent/EP4112069A4/en
Priority to CA3169325A priority patent/CA3169325A1/en
Priority to CN202180012694.9A priority patent/CN115551534A/zh
Priority to JP2022548090A priority patent/JP2023514819A/ja
Priority to US17/802,280 priority patent/US20230139956A1/en
Publication of WO2021170099A1 publication Critical patent/WO2021170099A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin

Definitions

  • the invention relates to a method and medicine for treating abnormal blood pressure and its complications.
  • Hypertension is one of the most frequently-occurring diseases in the world, and its incidence is increasing year by year in China. Hypertension leads to complications of the heart, brain, kidneys and other internal organs, leading to disability and death. The hazard of hypertension is that it can cause diseases of multiple organs and systems such as the heart, brain, and kidneys, such as stroke, the most common complication. Secondly, hypertension leads to related heart damage, including myocardial hypertrophy, coronary atherosclerosis, arrhythmia and heart failure. Hypertension is often accompanied by kidney damage and peripheral vascular disease. Hypertension develops to the middle and late stages, and retinopathy can occur. Diabetes is also one of the common complications of hypertension. Research on the prevention and treatment of hypertension has attracted the attention of scholar from all over the world. At the same time, effective prevention and treatment of the complications of hypertension can significantly reduce the disability and mortality of patients.
  • the present invention finds that plasminogen can significantly reduce hypertension, and at the same time improve tissue and organ damage, fibrosis and dysfunction caused by hypertension, which opens up a new way for the prevention and treatment of hypertension and its related diseases and complications.
  • the present application relates to components of the plasminogen activation pathway, such as methods, uses and drugs for plasminogen to prevent and treat abnormal blood pressure disorders (including hypertension and hypotension).
  • This application proves through research that components of the plasminogen activation pathway, such as plasminogen, can promote the blood pressure of subjects suffering from hypertension or hypotension to return to normal, and at the same time can reduce and alleviate abnormal blood pressure. (Such as high blood pressure or low blood pressure) caused by tissues and organs, such as heart, lung, kidney and liver tissue structure damage and functional damage.
  • this application involves the following items:
  • a method for preventing or treating hypertension comprising administering to a subject suffering from hypertension an effective amount of one or more compounds selected from the group consisting of plasminogen activation pathway Substances, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.
  • the present application also relates to the use of one or more compounds selected from the following in the preparation of drugs for the prevention or treatment of hypertension, the one or more compounds selected from: plasminogen activation pathway Components, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin , Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolytic inhibitors.
  • plasminogen activation pathway Components compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin , Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs,
  • the present application also relates to drugs for preventing or treating hypertension comprising one or more compounds selected from the group consisting of components of the plasminogen activation pathway, Compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, can be upregulated Plasminogen or plasminogen activator-expressed compounds, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.
  • the present application also relates to the use of one or more compounds selected from the group to prevent or treat hypertensive disorders, the one or more compounds selected from the group consisting of components of the plasminogen activation pathway, which can directly Compounds that activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, and can upregulate fibrin Compounds expressed by lysinogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.
  • the one or more compounds selected from the group consisting of components of the plasminogen activation pathway, which can directly Compounds that activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, and can upregulate fibrin Compounds expressed by lys
  • the component of the plasminogen activation pathway is selected from the group consisting of plasminogen, recombinant human plasmin, Lys-plasminogen, Glu -Plasminogen, plasmin, plasminogen and plasmin variants and the like containing one or more kringle domains and protease domains of plasminogen and plasmin Substances, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasmin Enzyme, delta-plasmin, plasminogen activator, tPA and uPA.
  • any one of items 1 to 5 wherein the compound has one or more effects selected from the group consisting of: lowering the blood pressure of subjects suffering from hypertensive disorders, and lowering the blood pressure of subjects suffering from hypertensive disorders.
  • Subject's serum angiotensin II level adjust the subject's ACE or ACE2 level, reduce tissue and organ damage caused by hypertension, promote the repair of damaged tissues and organs, reduce tissue and organ fibrosis, and promote the elimination of free radicals.
  • plasminogen comprises the plasminogen active fragment shown in sequence 14 and has the proteolytic activity of plasminogen.
  • plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen or their variants that retain the proteolytic activity of plasminogen.
  • plasminogen is natural or synthetic human plasminogen, or it retains the proteolytic activity or lysine binding activity of plasminogen Variants or fragments of.
  • any one of items 1-16 wherein the compound is administered in the form of nasal cavity inhalation, nebulization inhalation, nasal drops, ear drops or eye drops, intravenously, intraperitoneally, subcutaneously , Intracranial, intrathecal, intraarterial, intrarectal and/or intramuscular administration.
  • the present invention also relates to the following items:
  • this application relates to a method for preventing or treating abnormal blood pressure or abnormal blood pressure conditions, comprising administering an effective amount of one or more compounds selected from the following group to a subject suffering from abnormal blood pressure conditions: fibrinolytic Components of the zymogen activation pathway, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, mimic plasminogen or plasminogen Enzyme activity compounds, compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and fibrinolytic inhibitors Antagonist.
  • fibrinolytic Components of the zymogen activation pathway compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, mimic plasminogen or plasminogen Enzyme activity compounds, compounds capable of up-regulating the expression of plasminogen or plasmin
  • the present application also relates to the use of one or more compounds selected from the group consisting of: plasminogen in the preparation of drugs for preventing or treating abnormal blood pressure or abnormal blood pressure disorders.
  • Components of the activation pathway compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating the upstream components of the plasminogen activation pathway, one of which mimics plasminogen or plasmin Active compounds, compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonism of fibrinolytic inhibitors Agent.
  • the present application also relates to drugs for preventing or treating abnormal blood pressure or abnormal blood pressure conditions comprising one or more compounds selected from the group consisting of: plasminogen activation pathway Components, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin , Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolytic inhibitors.
  • plasminogen activation pathway Components compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin , Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or u
  • the present application also relates to the use of one or more compounds selected from the group to prevent or treat abnormal blood pressure or abnormal blood pressure disorders, the one or more compounds selected from: components of plasminogen activation pathway , Compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, Compounds that up-regulate the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.
  • components of plasminogen activation pathway Compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, Compounds that up-regulate the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analog
  • Abnormal blood pressure or abnormal blood pressure conditions described in the present application include hypertension or hypertension conditions with blood pressure higher than normal and hypotension or hypotension conditions with blood pressure lower than normal. Therefore, the present application relates to methods, uses, and medicaments for using one or more of the above-mentioned compounds to restore the hypertension of a subject suffering from a hypertensive disorder or hypotension of a subject suffering from a hypotensive disorder to a normal level.
  • the component of the plasminogen activation pathway is selected from the group consisting of plasminogen, recombinant human plasmin, Lys-plasminogen, Glu -Plasminogen, plasmin, plasminogen and plasmin variants and the like containing one or more kringle domains and protease domains of plasminogen and plasmin Substances, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasmin Enzyme, delta-plasmin, plasminogen activator, tPA and uPA.
  • the abnormal blood pressure disorder includes tissue and organ damage or complications caused by the abnormal blood pressure disorder.
  • the tissue organ damage or complication is heart, brain, lung, liver, kidney, or blood vessel damage or complication.
  • the tissue and organ damage is tissue and organ structural damage (for example, normal tissue structure change) or tissue and organ function damage (for example, tissue and organ function decline).
  • the abnormal blood pressure disorder includes a hypertension disorder or a hypotension disorder.
  • the complication caused by the abnormal blood pressure disorder is a complication caused by a hypertension disorder, including arrhythmia, heart failure, coronary heart disease, cerebral hemorrhage, cerebral thrombosis, cerebral infarction, hypertension Blood pressure nephropathy, renal failure, uremia, liver cirrhosis, pulmonary hypertension, pulmonary fibrosis or microthrombosis.
  • a hypertension disorder including arrhythmia, heart failure, coronary heart disease, cerebral hemorrhage, cerebral thrombosis, cerebral infarction, hypertension Blood pressure nephropathy, renal failure, uremia, liver cirrhosis, pulmonary hypertension, pulmonary fibrosis or microthrombosis.
  • the complication caused by the abnormal blood pressure disorder is a complication caused by a hypotension disorder, including insufficient blood supply to tissues and organs caused by a hypotension disorder, such as insufficient blood supply to the heart, angina pectoris, shock, and blood supply to the brain Insufficiency, syncope, cerebral infarction, insufficient blood supply to the kidneys, oliguria, proteinuria, and renal insufficiency.
  • a hypotension disorder including insufficient blood supply to tissues and organs caused by a hypotension disorder, such as insufficient blood supply to the heart, angina pectoris, shock, and blood supply to the brain Insufficiency, syncope, cerebral infarction, insufficient blood supply to the kidneys, oliguria, proteinuria, and renal insufficiency.
  • any one of items 1 to 5 wherein the compound has one or more effects selected from the group consisting of: lowering the blood pressure of subjects suffering from hypertensive disorders, and lowering the blood pressure of subjects suffering from hypertensive disorders.
  • the subject s serum angiotensin II level, adjust the subject’s ACE or ACE2 level, increase the blood pressure of the subject suffering from hypotension, promote the subject suffering from hypertension, or suffer from hypotension.
  • plasminogen and sequence 2 have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% % Or 99% sequence identity, and has plasminogen proteolytic activity or lysine binding activity.
  • plasminogen comprises the plasminogen active fragment shown in sequence 14 and has the proteolytic activity of plasminogen.
  • plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen or their variants that retain the proteolytic activity of plasminogen.
  • plasminogen is natural or synthetic human plasminogen, or it retains the proteolytic activity or lysine binding activity of plasminogen Variants or fragments of.
  • any one of items 1-16 wherein the compound is administered in the form of nasal cavity inhalation, nebulization inhalation, nasal drops, ear drops or eye drops, intravenously, intraperitoneally, subcutaneously , Intracranial, intrathecal, intraarterial, intrarectal and/or intramuscular administration.
  • One aspect of the present invention also relates to plasminogen, plasminogen-containing drugs, and drug combinations for preventing or treating abnormal blood pressure disorders (such as hypertension disorders or hypotension disorders) and related injuries or complications in a subject Materials, kits, products.
  • abnormal blood pressure disorders such as hypertension disorders or hypotension disorders
  • One aspect of the present invention also relates to plasminogen, plasminogen-containing drugs, pharmaceutical compositions, kits, and products.
  • the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, and the sequence 2, 6, 8, 10 or 12. %, 98%, or 99% sequence identity, and still have plasminogen activity.
  • the plasminogen is added, deleted and/or substituted 1-100, 1-90, 1-80, 1-70 on the basis of sequence 2, 6, 8, 10 or 12. , 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1 -3, 1-2, 1 amino acid, and still have proteolytic activity or lysine binding activity protein.
  • the plasminogen is a protein that contains proteolytic activity or lysine-binding activity fragments and still has proteolytic activity or lysine-binding activity.
  • the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen or their retention Variants of proteolytic activity or lysine binding activity.
  • the plasminogen is natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • the plasminogen is a human plasminogen ortholog from a primate or rodent or a variant or fragment thereof that still retains proteolytic activity or lysine binding activity .
  • the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is natural human plasminogen.
  • the subject is a human. In some embodiments, the subject lacks or lacks plasminogen. In some embodiments, the deficiency or deletion is congenital, secondary, and/or local.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and plasminogen used in the aforementioned methods.
  • the kit may be a prophylactic or therapeutic kit comprising: (i) plasminogen for the aforementioned method and (ii) for delivery of the plasminogen to the The means of the subject.
  • the member is a syringe or vial.
  • the kit further includes a label or instructions for use that instructs the plasminogen to be administered to the subject to perform any of the foregoing methods.
  • the article of manufacture comprises: a container containing a label; and a pharmaceutical composition comprising (i) plasminogen or plasminogen used in the foregoing method, wherein the label indicates that the plasminogen
  • a pharmaceutical composition comprising (i) plasminogen or plasminogen used in the foregoing method, wherein the label indicates that the plasminogen
  • the prolysozyme or composition is administered to the subject to perform any of the aforementioned methods.
  • the kit or article of manufacture further includes one or more additional components or containers that contain other drugs.
  • the plasminogen is administered systemically or locally, preferably by the following routes: nasal inhalation, nebulization inhalation, nasal drops or eye drops, intravenous, peritoneal Plasminogen is administered intra-, subcutaneously, intracranially, intrathecally, intraarterially, intrarectally, and/or intramuscularly for treatment.
  • the plasminogen is administered in combination with an appropriate polypeptide carrier or stabilizer.
  • the plasminogen is at a dose of 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg per day /kg, 10-100mg/kg (calculated per kilogram of body weight) or 0.0001-2000mg/cm2, 0.001-800mg/cm2, 0.01-600mg/cm2, 0.1-400 mg/cm2, 1-200mg/cm2, 1-100mg /cm2, 10-100mg/cm2 (calculated per square centimeter of body surface area) dose administration, preferably repeated at least once, preferably at least daily administration.
  • the present invention clearly covers all combinations of technical features belonging to the embodiments of the present invention, and the technical solutions after these combinations have been clearly disclosed in this application, just as the above-mentioned technical solutions have been separately and clearly disclosed.
  • the present invention also clearly covers the combinations between the various embodiments and their elements, and the technical solutions after the combination are clearly disclosed herein.
  • Figures 1A-1B show the blood pressure measurement results of 15-16 weeks old diabetic hypertension model mice given plasminogen for 21 days.
  • Figure 1A shows the systolic blood pressure
  • Figure 1B shows the average blood pressure.
  • the results showed that the blood pressure of the mice in the PBS control group did not change significantly after the administration for 21 days, while the systolic blood pressure and the mean blood pressure of the mice in the plasminogen group were significantly lower than those in the vehicle PBS control. Group, and the statistical difference is significant (* means P ⁇ 0.05, ** means P ⁇ 0.01). It shows that plasminogen can significantly reduce the hypertension of diabetic mice aged 15-16 weeks.
  • Figures 2A-2B show the blood pressure measurement results of 25-26 weeks old diabetic hypertension model mice given plasminogen for 21 days.
  • Figure 2A is the systolic blood pressure
  • Figure 2B is the average blood pressure.
  • the results showed that the systolic blood pressure of the mice in the PBS control group did not change significantly after the administration for 21 days, while the systolic blood pressure of the mice in the plasminogen group had begun to decrease significantly after 7 days of administration, which was significantly lower than that of the administration.
  • the vehicle PBS control group, and the statistical difference was significant (P 0.019); 14 and 21 days after the administration, the two groups of mice were also significantly different.
  • Figure 3 shows the detection results of serum angiotensin II in 15-16 week old diabetic hypertension model mice given plasminogen for 28 days. The results showed that 28 days after the administration of plasminogen, the serum angiotensin II level in the plasminogen group was significantly lower than that in the vehicle PBS control group. It shows that plasminogen can reduce the serum angiotensin II level in diabetic hypertension model mice, thereby improving hypertension.
  • Figures 4A-4B show representative pictures of H&E staining of the heart of 25-26 weeks old diabetic hypertension model mice given plasminogen for 28 days.
  • Fig. 4A is the control group given the vehicle PBS
  • Fig. 4B is the group given the plasminogen.
  • the results showed that compared with the vehicle PBS control group ( Figure 4A), the plasminogen group ( Figure 4B) mice cardiomyocytes were tighter and arranged more regularly.
  • Figures 5A-5B show representative pictures of renal SR staining in 25-26 week old diabetic hypertension model mice given plasminogen for 28 days.
  • Fig. 5A is the control group given vehicle PBS
  • Fig. 5B is the group given plasminogen.
  • Fig. 6 Results of detection of serum SOD levels in angiotensin II-induced hypertension model mice 14 days after administration of plasminogen.
  • the results showed that the serum of the blank control group had a certain level of SOD, the serum SOD level of the vehicle PBS control group was significantly reduced, and the serum SOD level of the plasminogen group was significantly higher than that of the vehicle PBS control group, and the statistical difference was significant (* means P ⁇ 0.05). It shows that plasminogen can enhance the body's ability to scavenge free radicals.
  • FIG. 7A-7C Angiotensin II-induced hypertension model mice are given plasminogen for 14 days after the kidney Sirius red staining representative pictures.
  • Fig. 7A is the blank control group
  • Fig. 7B is the vehicle PBS control group
  • Fig. 7C is the plasminogen group.
  • the results showed that there was no obvious collagen fiber deposition in the kidney of the blank control group, and the collagen fiber deposition in the plasminogen group (marked by the arrow) was significantly less than that in the solvent PBS control group. It shows that plasminogen can significantly reduce the renal fibrosis in hypertensive model mice induced by angiotensin II, and improve the renal pathology caused by hypertension.
  • Fig. 8A-8C Angiotensin II-induced hypertension model mice 14 days after administration of plasminogen, representative pictures of Sirius red staining of the heart.
  • Fig. 8A is a blank control group
  • Fig. 8B is a vehicle PBS control group
  • Fig. 8C is a plasminogen group.
  • the results showed that there was no obvious collagen fiber deposition in the heart of the blank control group, and the collagen fiber deposition in the plasminogen group (marked by the arrow) was significantly less than that in the solvent PBS control group. It shows that plasminogen can significantly reduce the heart fibrosis in hypertensive model mice induced by angiotensin II, and improve the heart disease caused by hypertension.
  • FIG. 9A-9C Monocrotaline-induced pulmonary hypertension model mice 28 days after administration of plasminogen, representative pictures of lung Sirius red staining.
  • Fig. 9A is a blank control group
  • Fig. 9B is a vehicle PBS control group
  • Fig. 9C is a plasminogen group.
  • the results showed that the lungs of mice in the blank control group had almost no collagen deposition, and the deposition of collagen in the lung tissues of the mice in the plasminogen group (marked by the arrow) was significantly less than that in the PBS control group. It shows that plasminogen can significantly reduce the fibrosis of the lungs of mice with pulmonary hypertension induced by monocrotaline.
  • Fig. 10A-10C Lilyine-induced pulmonary hypertension model mice 28 days after administration of plasminogen, representative pictures of Sirius red staining of the heart.
  • Fig. 10A is a blank control group
  • Fig. 10B is a vehicle PBS control group
  • Fig. 10C is a plasminogen group.
  • the results showed that there was almost no collagen deposition in the hearts of mice in the blank control group, and the collagen deposition in the hearts of the mice in the plasminogen group (marked by the arrow) was significantly less than that in the PBS control group. It shows that plasminogen can significantly reduce the fibrosis of the heart of pulmonary hypertension model mice induced by monocrotaline.
  • FIGS 11A-11C Monocrotaline-induced pulmonary hypertension model mice 28 days after administration of plasminogen, representative pictures of Sirius red staining in the kidneys.
  • Fig. 11A is a blank control group
  • Fig. 11B is a vehicle PBS control group
  • Fig. 11C is a plasminogen group.
  • the results showed that there was almost no collagen deposition in the kidney of the blank control group, and the collagen deposition in the kidney of the plasminogen group (marked by the arrow) was significantly less than that in the PBS control group. It shows that plasminogen can significantly reduce the fibrosis of the kidney in mice with pulmonary hypertension induced by monocrotaline.
  • FIGS 12A-12C Monocrotaline-induced pulmonary hypertension model mice 28 days after administration of plasminogen, representative pictures of liver Sirius red staining.
  • Fig. 12A is a blank control group
  • Fig. 12B is a vehicle PBS control group
  • Fig. 12C is a plasminogen group.
  • the results showed that the mice in the blank control group had almost no collagen deposition in the liver, and the liver collagen deposition in the plasminogen group (marked by the arrow) was significantly less than that in the vehicle PBS control group. It shows that plasminogen can significantly reduce the fibrosis of the liver in mice with pulmonary hypertension induced by monocrotaline.
  • Figure 13 The results of blood pressure measurement in renal atrophy model mice given plasminogen for 14 days. The results showed that the systolic blood pressure and mean blood pressure of the plasminogen group and the blank control group were significantly higher than the blood pressure of the PBS control group, and there was a statistically significant difference between the plasminogen group and the PBS control group (* means P ⁇ 0.05). It shows that plasminogen can promote the normal level of hypotension caused by renal atrophy.
  • FIG 14 Angiotensin II-induced hypertension model mice were given plasminogen for 5 days and measured results of mean blood pressure and systolic blood pressure. The results showed that the mice in the blank control group had a certain level of mean blood pressure and systolic blood pressure, the mean blood pressure and systolic blood pressure of the mice in the vehicle group were significantly increased, and the mean blood pressure and systolic blood pressure of the mice in the administration group were significantly lower than those in the vehicle group. And the statistical difference is significant (* means P ⁇ 0.05, ** table P ⁇ 0.01). It shows that plasminogen can reduce blood pressure in hypertensive model mice.
  • FIG 16 Angiotensin II-induced hypertension model mice were given plasminogen for 7 days of serum ACE2 level detection results. The results showed that there was a certain level of ACE2 in the blood of the blank control group, the ACE2 level in the blood of the vehicle group was significantly higher than that of the blank control group, and the ACE2 level in the blood of the administration group was significantly lower than that of the vehicle group. And the statistical difference is extremely significant (** means P ⁇ 0.01). It is suggested that plasminogen can promote the decrease of serum ACE2 level in AngII-induced hypertension model mice.
  • Figure 18 Test results of systolic blood pressure (high pressure) and diastolic blood pressure (low pressure) during medication. The results showed that the patient's systolic blood pressure dropped to 141mmHg from the 6th day after the medication, and the blood pressure fluctuated unstable (normal and abnormal alternation) during the 11th day; after the 12th day, the blood pressure dropped and remained normal and stable within a week. Shows that plasminogen can reduce blood pressure in hypertensive patients.
  • Figure 19 Results of systolic blood pressure (high pressure) and diastolic blood pressure (low pressure) during medication. The results showed that the patient's blood pressure was 135/54mmHg on the 13th day after the medication, and the later period was normal and stable, and antihypertensive drugs were basically not needed. Shows that plasminogen can treat high blood pressure.
  • Fig. 20 Test results of systolic blood pressure (high pressure) and diastolic blood pressure (low pressure) during medication. The results showed that the diastolic blood pressure reached a normal value after the medication. It used to be lower than 60mmHg, and the blood pressure was normal and stable during the treatment; after the treatment, the antihypertensive drug was halved, and the blood pressure was basically controlled at 130-140/64-76mmHg. It shows that plasminogen can promote the normalization of blood pressure in patients with hypertension, and can reduce the dosage of antihypertensive drugs.
  • Figure 21A-21B systolic blood pressure (high pressure) and diastolic blood pressure (low pressure) test results in the morning (21A) and evening (221B) during the medication period of patients.
  • the blood pressure began to drop sooner or later after the fifth day of medication, and the pressure difference decreased.
  • the patient complained that he had not seen blood pressure drop below 140/75mmHg under the condition of taking antihypertensive drugs. This is the first time that the blood pressure has improved.
  • the blood pressure was maintained at about 136/73mmHg in the morning and evening after the sixth day of medication. On the seventh day, the blood pressure remained stable in the morning and evening, and the blood pressure was around 130/70mmHg. Shows that plasminogen can improve the symptoms of hypertension in patients.
  • Figure 22 systolic blood pressure (high pressure) and diastolic blood pressure (low pressure) during medication.
  • the results showed that the blood pressure showed an upward trend after medication, and normal blood pressure appeared for the first time on the third day of medication.
  • the blood pressure began to be normal on the 9th day after the medication, and the blood pressure remained normal all the time, and the blood pressure remained at 95/70mmHg after stopping the medication. Shows that plasminogen can promote blood pressure recovery in patients with hypotension.
  • Figure 23 systolic blood pressure (high pressure) and diastolic blood pressure (low pressure) test results during medication. The results showed that the patient's mental state improved after the medication, and the blood pressure reached normal on the second day after the medication, and reached about 110/70mmHg a week later. It shows that the administration of plasminogen can promote the recovery of blood pressure in patients with hypotension.
  • Fibrinolytic system also known as fibrinolytic system, is a system composed of a series of chemical substances involved in the process of fibrinolysis (fibrinolysis), mainly including fibrinolytic enzyme (plasminogen) and plasmin , Plasminogen activator, fibrinolysis inhibitor.
  • Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA).
  • t-PA tissue-type plasminogen activator
  • u-PA urokinase-type plasminogen activator
  • t-PA activates plasminogen
  • fibrin urokinase-type plasminogen activator
  • u-PA urokinase-type plasminogen activator
  • PLG Plasminogen
  • Plasminase is a serine protease, which has the following functions: degrades fibrin and fibrinogen; hydrolyzes a variety of coagulation factors V, VIII, X, VII, XI, II, etc.; turns plasminogen into fibrinolysis Enzymes; hydrolysis of complement, etc.
  • Fibrinolytic inhibitors including plasminogen activator inhibitor (PAI) and ⁇ 2 antiplasmin ( ⁇ 2-AP).
  • PAI mainly has two forms, PAI-1 and PAI-2, which can specifically bind to t-PA in a ratio of 1:1 to inactivate it and activate PLG at the same time.
  • ⁇ 2-AP is synthesized by the liver and combined with PL in a ratio of 1:1 to form a complex, inhibiting PL activity; FXIII makes ⁇ 2-AP covalently bond with fibrin, reducing the sensitivity of fibrin to PL.
  • Substances that inhibit the activity of the fibrinolytic system in the body PAI-1, complement C1 inhibitor; ⁇ 2 anti-plasmin; ⁇ 2 macroglobulin.
  • component of the plasminogen activation pathway covers:
  • Plasminogen activators such as tPA and uPA, and tPA or uPA variants and analogs containing one or more domains of tPA or uPA (such as one or more kringle domains and proteolytic domains) .
  • variants of plasminogen, plasmin, tPA and uPA include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as by addition, deletion and/or substitution such as 1- 100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, Proteins of 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid that still have plasminogen, plasmin, tPA or uPA activity.
  • variants of plasminogen, plasmin, tPA, and uPA include those by, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1- 45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative Mutant variants of these proteins obtained by amino acid substitutions.
  • the "plasminogen variant” of the present invention encompasses sequences 2, 6, 8, 10 or 12 that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99 % Sequence identity, and still have proteolytic activity or lysine binding activity protein.
  • the "plasminogen variant” of the present invention can be added, deleted and/or substituted 1-100, 1-90, 1-80, 1- on the basis of sequence 2, 6, 8, 10 or 12. 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid, and still have proteolytic activity or lysine binding activity protein.
  • the plasminogen variants of the present invention include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as through conservative amino acid substitutions such as 1-100, 1-90, 1-80, 1- 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, Mutant variants of these proteins obtained from 1-3, 1-2, 1 amino acids.
  • the plasminogen of the present invention may be a human plasminogen ortholog from a primate or rodent or a variant that still retains proteolytic activity or lysine binding activity, such as sequence 2, 6 , 8, 10 or 12, such as human natural plasminogen shown in sequence 2.
  • plasminogen, plasmin, tPA, and uPA include compounds that provide substantially similar effects to plasminogen, plasmin, tPA, or uPA, respectively.
  • variants and analogs of plasminogen, plasmin, tPA and uPA encompass fibers comprising one or more domains (for example, one or more kringle domains and proteolytic domains)
  • variants and analogs encompass fibers comprising one or more domains (for example, one or more kringle domains and proteolytic domains)
  • variants and analogs encompass plasminogen comprising one or more plasminogen domains (eg, one or more kringle domains and proteolytic domains) Variants and analogs, such as mini-plasminogen.
  • Variants and “analogs” of plasmin encompass plasmin "variants” that include one or more plasmin domains (eg, one or more kringle domains and proteolytic domains) And “analogs” such as mini-plasmin and delta-plasmin.
  • plasminogen, plasmin, tPA or uPA have the activity of plasminogen, plasmin, tPA or uPA respectively, or whether they provide the same
  • the substantially similar effects of plasminogen, plasmin, tPA or uPA can be detected by methods known in the art, for example, by methods based on enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS ( Fluorescence-activated cell sorting method) is measured by the level of activated plasmin activity, for example, it can be measured with reference to a method selected from the following documents: Ny, A., Leonardsson, G., Hagglund, AC, Hagglof, P.
  • the "component of plasminogen activation pathway" of the present invention is plasminogen, selected from Glu-plasminogen, Lys-plasminogen, and microplasmin Pro, microplasminogen, delta-plasminogen or their variants that retain proteolytic activity or lysine binding activity.
  • the plasminogen is natural or synthetic human plasminogen, or a conservative mutant variant or fragment thereof that still retains proteolytic activity or lysine binding activity.
  • the plasminogen is a human plasminogen ortholog from a primate or rodent or a conservative mutant variant that still retains proteolytic activity or lysine binding activity Or fragments thereof.
  • the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is natural human plasminogen.
  • the plasminogen is human natural plasminogen as shown in sequence 2.
  • a compound capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway refers to a compound capable of directly activating plasminogen or by activating plasminogen Any compound that activates upstream components of the pathway and indirectly activates plasminogen, such as tPA, uPA, streptokinase, saruplase,reteplase, reteplase, tenecteplase, aniplase, Monteplase, Lanoteplase, Pamideplase, Staphylokinase.
  • the "antagonist of the fibrinolysis inhibitor" of the present invention is a compound that antagonizes, weakens, blocks, and prevents the action of the fibrinolysis inhibitor.
  • the fibrinolysis inhibitors are, for example, PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, and ⁇ 2 macroglobulin.
  • the antagonist such as PAI-1, complement C1 inhibitor, ⁇ 2 anti-plasmin or ⁇ 2 macroglobulin antibody, or blocking or down-regulating such as PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin Antisense RNA or small RNA expressed by globulin, or occupy the binding site of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin but without PAI-1, complement C1 inhibitor, ⁇ 2 anti-fibrosis Compounds that function as lysozyme or ⁇ 2 macroglobulin", or compounds that block the binding domain and/or active domain of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin.
  • Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease that can hydrolyze several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycan. In addition, plasmin can activate some metalloprotease precursors (pro-MMPs) to form active metalloproteases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis. Plasmin is formed by proteolysis of plasminogen through two physiological PAs: tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA).
  • tPA tissue-type plasminogen activator
  • uPA urokinase-type plasminogen activator
  • PAs Due to the relatively high levels of plasminogen in plasma and other body fluids, it is traditionally believed that the regulation of the PA system is mainly achieved through the synthesis and activity levels of PAs.
  • the synthesis of PA system components is strictly regulated by different factors, such as hormones, growth factors and cytokines.
  • the main inhibitor of plasmin is ⁇ 2-antiplasmin ( ⁇ 2-antiplasmin).
  • the activity of PAs is regulated by both uPA and tPA's plasminogen activator inhibitor-1 (PAI-1) and mainly inhibits uPA's lysinogen activator inhibitor-2 (PAI-2).
  • PAI-1 tPA's plasminogen activator inhibitor-1
  • PAI-2 mainly inhibits uPA's lysinogen activator inhibitor-2
  • Certain cell surfaces have uPA-specific cell surface receptors (uPAR) with direct hydrolytic activity.
  • Plasminogen is a single-chain glycoprotein consisting of 791 amino acids and a molecular weight of approximately 92kDa. Plasminogen is mainly synthesized in the liver and exists in large amounts in the extracellular fluid. Plasminogen content in plasma is about 2 ⁇ M. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids. Plasminogen exists in two molecular forms: Glu-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved form of plasminogen has an amino terminal (N-terminal) glutamate and is therefore called glutamate-plasminogen.
  • glutamate-plasminogen is hydrolyzed at Lys76-Lys77 to lysine-plasminogen.
  • lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate.
  • the Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide bond-linked double-chain protease plasmin.
  • the amino terminal part of plasminogen contains five homologous three loops, so-called kringles, and the carboxy terminal part contains the protease domain.
  • Some kringles contain lysine binding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor ⁇ 2-AP.
  • Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycan and gelatin, indicating that plasmin also plays an important role in ECM reconstruction.
  • plasmin can also degrade other components of ECM by converting certain protease precursors into active proteases, including MMP-1, MMP-2, MMP-3 and MMP-9. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis.
  • plasmin has the ability to activate certain latent forms of growth factors. In vitro, plasmin can also hydrolyze components of the complement system and release chemotactic complement fragments.
  • Pulminin is a very important enzyme present in the blood, which can hydrolyze fibrin clots into fibrin degradation products and D-dimers.
  • “Plasminogen” is the zymogen form of plasmin. According to the sequence in swiss prot, it is composed of 810 amino acids and the molecular weight is about 90kD, a glycoprotein mainly synthesized in the liver and able to circulate in the blood. The cDNA sequence encoding this amino acid sequence is shown in sequence 3. The full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle1-5).
  • PAp Pan Apple
  • Kringle1 includes residues Cys103-Cys181
  • Kringle2 includes residues Glu184-Cys262
  • Kringle3 includes residues Cys275-Cys352
  • Kringle4 Including residues Cys377-Cys454
  • Kringle5 includes residues Cys481-Cys560.
  • the serine protease domain includes residues Val581-Arg804.
  • Glu-plasminogen is a human natural full-length plasminogen, composed of 791 amino acids (without the signal peptide of 19 amino acids).
  • the cDNA sequence encoding this sequence is shown in sequence 1, and its amino acid sequence is shown in sequence 2 shown.
  • Lys-plasminogen formed by hydrolysis from amino acids 76-77 of Glu-plasminogen.
  • sequence 6 the cDNA sequence encoding this amino acid sequence is as shown in sequence 5.
  • Delta-plasminogen ( ⁇ -plasminogen) is a fragment of the full-length plasminogen without the Kringle2-Kringle5 structure, and only contains Kringle1 and serine protease domain (also called protease domain (PD)).
  • Mini-plasminogen (Mini-plasminogen) is composed of Kringle5 and serine protease domain. It has been reported in the literature that it includes residues Val443-Asn791 (with the Glu residue of the Glu-plasminogen sequence without signal peptide as the starting amino acid). ), its amino acid sequence is shown in sequence 10, and the cDNA sequence encoding the amino acid sequence is shown in sequence 9.
  • Micro-plasminogen (Micro-plasminogen) only contains the serine protease domain.
  • plasminogen includes Kringle 1, 2, 3, 4, and 5 domains and serine protease domains (also called protease domains (protease domain, PD)), among which Kringles is responsible for plasmin Proto binds to low-molecular-weight and high-molecular-weight ligands (ie, lysine binding activity), which causes plasminogen to transform into a more open configuration, which is easier to be activated; the protease domain (PD) is residue Val562 -Asn791, tPA and UPA specifically cleave the Arg561-Val562 activation bond of plasminogen, thereby allowing plasminogen to form plasmin. Therefore, the protease domain (PD) confers plasminogen proteolytic activity area.
  • Plasin can be used interchangeably and have the same meaning;
  • plasminogen is equivalent to “plasminogen” and “plasminogen” “Can be used interchangeably and have the same meaning.
  • the meaning or activity of the "deficiency" of plasminogen means that the content of plasminogen in the subject is lower than that of a normal person, and is low enough to affect the normal physiological function of the subject;
  • the meaning or activity of "deletion" of plasminogen is that the content of plasminogen in the subject is significantly lower than that of normal people, even the activity or expression is minimal, and normal physiological functions can only be maintained through external sources.
  • plasminogen of the present invention covers both plasminogen and plasmin.
  • plasminogen activator PA
  • PA plasminogen activator
  • the active plasmin can further hydrolyze the fibrin clot into fibrin degradation products and D-dimers, and then dissolve the thrombus.
  • the PAp domain of plasminogen contains important determinants that maintain plasminogen in an inactive closed conformation, while the KR domain can bind to lysine residues present on the receptor and substrate.
  • a variety of enzymes that can act as plasminogen activators are known, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hager Man factor) and so on.
  • the technical scheme of the present invention related to plasminogen covers the technical scheme of replacing plasminogen with active fragments of plasminogen.
  • the active fragment of plasminogen according to the present invention comprises or consists of the serine protease domain of plasminogen, preferably, the plasminogen according to the present invention
  • the original active fragment contains sequence 14, or an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identical to sequence 14, or is composed of sequence 14, or has at least 80% identity with sequence 14. %, 90%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the active plasminogen fragment of the present invention comprises a region selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, Kringle 5, or is selected from Kringle 1, Kringle 2.
  • Kringle 3, Kringle 4, Kringle 5 are composed of one or more areas.
  • the plasminogen of the present invention includes a protein containing the above-mentioned active fragment of plasminogen.
  • the methods for measuring plasminogen and its activity in blood include: the detection of tissue plasminogen activator activity (t-PAA), the detection of plasma tissue plasminogen activator antigen (t-PAAg), Detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator inhibition Detection of substance antigens, plasma plasmin-antiplasmin complex detection (PAP).
  • t-PAA tissue plasminogen activator activity
  • t-PAAg the detection of plasma tissue plasminogen activator antigen
  • plgA Detection of plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma tissue plasminogen activator inhibition
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the tested plasma, the PLG in the tested plasma is transformed into PLM under the action of SK, and the latter acts on The chromogenic substrate is subsequently measured with a spectrophotometer, and the increase in absorbance is proportional to the activity of plasminogen.
  • SK streptokinase
  • immunochemical methods, gel electrophoresis, immunoturbidimetry, radioimmuno-diffusion methods, etc. can also be used to determine the plasminogen activity in the blood.
  • orthologs or orthologs refer to homologs between different species, including both protein homologs and DNA homologs, and are also called orthologs and vertical homologs. It specifically refers to proteins or genes in different species that evolved from the same ancestor gene.
  • the plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs derived from different species that have plasminogen activity.
  • Constant substitution variant refers to a given amino acid residue that changes but does not change the overall conformation and function of the protein or enzyme. This includes, but is not limited to, those with similar characteristics (such as acidic, basic, hydrophobic, etc.) Amino acids replace amino acids in the amino acid sequence of the parent protein. Amino acids with similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and can be interchanged. Similarly, isoleucine is a hydrophobic amino acid and can be replaced by leucine, methionine or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different.
  • Constant substitution variants also include polypeptides or enzymes that are determined by BLAST or FASTA algorithms to have more than 60% amino acid identity. If it can reach more than 75%, it is better, preferably more than 85%, or even more than 90%. It is the best, and has the same or substantially similar properties or functions compared with the natural or parent protein or enzyme.
  • isolated plasminogen refers to plasminogen protein separated and/or recovered from its natural environment.
  • the plasminogen will be purified (1) to a purity (by weight) greater than 90%, greater than 95%, or greater than 98%, as determined by the Lowry method, for example, greater than 99% (By weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, which is achieved by using Coomassie blue or silver staining is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions.
  • the isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering technology and separated by at least one purification step.
  • polypeptide refers to polymerized forms of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived Modified amino acids, and polypeptides with modified peptide backbones.
  • the term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without an N-terminal methionine residue); and so on.
  • the “percent amino acid sequence identity (%)" with respect to the reference polypeptide sequence is defined as when gaps are introduced when necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity, the candidate sequence is Refers to the percentage of amino acid residues that are identical to amino acid residues in the polypeptide sequence.
  • the comparison for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment of the sequences, including any algorithm that achieves the maximum alignment requirements over the entire length of the sequence being compared. However, for the purposes of the present invention, the percent amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2.
  • the% amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or can be expressed as having or containing relative to, with, or against a given amino acid sequence)
  • a given amino acid sequence A) of a certain% amino acid sequence identity of B is calculated as follows:
  • X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's A and B alignment
  • Y is the total number of amino acid residues in B. It should be appreciated that in the case where the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% amino acid sequence identity of A relative to B may not be equal to the% amino acid sequence identity of B relative to A. Unless expressly stated otherwise, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.
  • the term "treatment” refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be to completely or partially prevent the occurrence and onset of the disease or its symptoms, partially or completely reduce the disease and/or its symptoms, and/or partially or completely cure the disease and/or its symptoms, including: (a) prevention of disease Occurs or attacks in a subject, the subject may have the cause of the disease, but has not yet been diagnosed as having the disease; (b) inhibit the disease, that is, block its formation; and (c) reduce the disease and/or its symptoms , That is, causing the disease and/or its symptoms to subside or disappear.
  • mice rats, mice
  • non-human primates humans
  • dogs and cats
  • Hoofed animals such as horses, cows, sheep, pigs, goats
  • “Therapeutically effective amount” or “effective amount” refers to a component or component of the plasminogen activation pathway that is sufficient to achieve the prevention and/or treatment of the disease when administered to a mammal or other subject to treat the disease
  • the amount of related compounds e.g. plasminogen.
  • the “therapeutically effective amount” will depend on the components of the plasminogen activation pathway used or related compounds (such as plasminogen), the severity of the disease and/or symptoms of the subject to be treated, and the age. , Weight, etc.
  • promoting the repair of damaged tissues and organs refers to promoting the repair of the structure and function of damaged tissues and organs, so as to restore the integrity and function of the damaged tissues and organs to normal as much as possible.
  • hypertension or “hypertensive disorder” refers to a state where the systemic artery pressure is higher than normal.
  • the diagnostic criteria for hypertension is systolic blood pressure ⁇ 18.7Kpa (140mmHg) or diastolic blood pressure ⁇ 12.0Kpa (90mmHg).
  • “Hypotension” or “hypotensive disorder” refers to a state in which the systemic arterial pressure is lower than normal. There is no uniform standard for the diagnosis of hypotension.
  • treating a subject with “hypertension” or “hypertensive disorder” includes “decreasing” or “decreasing” the hypertension of a subject suffering from “hypertension” or “hypertensive disorder”.
  • “Decrease” or “decrease” means that the blood pressure of the subject is decreased compared with the control without administration, or compared with the blood pressure of the subject before the administration, for example, the blood pressure of the subject tends to be normal or close to normal Or return to normal levels.
  • treating a subject with “hypotension” or “hypotension” includes “raising” the hypotension of a subject suffering from “hypotension” or “hypotension”.
  • the “increased” means that the blood pressure of the subject is increased compared with the control without administration, or compared with the blood pressure of the subject before the administration, for example, the blood pressure of the subject tends to be normal , Close to normal or return to normal level.
  • “Complication” refers to the occurrence of another disease or symptom caused by a disease in the development process, the latter is the complication of the former, that is, the occurrence of the latter disease is caused by the former disease, or In the course of disease diagnosis and treatment, the patient has another disease or several diseases related to this disease.
  • “Hypertensive complications” refer to complications caused by hypertension, including heart, brain, lung, liver, kidney or blood vessel damage caused by hypertension. Complications of heart damage caused by hypertension such as left ventricular hypertrophy, angina pectoris, myocardial infarction and heart failure; complications of brain tissue damage caused by hypertension such as cerebral hemorrhage, cerebral thrombosis, cerebral infarction, hemorrhagic stroke, ischemic stroke , Hypertensive encephalopathy; renal damage caused by hypertension, such as slow progressing arterioles nephrosclerosis, malignant arterioles nephrosclerosis, chronic renal failure, hypertensive nephropathy, renal failure, uremia. The most common complication of hypertension is cerebrovascular accident, followed by hypertensive heart disease, heart failure, and kidney failure. A rare but serious complication is aortic dissecting aneurysm.
  • “Hypotension complications” refer to complications caused by hypotension.
  • the common complications of hypotension are mostly caused by insufficient perfusion of important organs. If there is insufficient blood supply to the brain, it is often manifested as tinnitus, dizziness, etc., and severe cases may be complicated by cerebral infarction; if the heart has insufficient blood supply, it is mostly manifested as palpitation, shortness of breath, chest tightness, etc.; if the kidney has insufficient blood supply, it is mostly manifested as oliguria , Proteinuria, and renal insufficiency such as anuria in severe cases.
  • Plasminogen can be isolated from nature and purified for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When a polypeptide is synthesized chemically, it can be synthesized via a liquid phase or a solid phase.
  • Solid phase peptide synthesis (SPPS) (where the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence) is a suitable method for the chemical synthesis of plasminogen.
  • SPPS Solid phase peptide synthesis
  • Various forms of SPPS, such as Fmoc and Boc can be used to synthesize plasminogen.
  • the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. Then, the unit is deprotected, exposing a new N-terminal amine that can be attached to other amino acids.
  • the peptide remains immobilized on the solid phase, after which it is cut off.
  • Standard recombinant methods can be used to produce the plasminogen of the present invention.
  • a nucleic acid encoding plasminogen is inserted into an expression vector so that it is operably linked to the regulatory sequence in the expression vector.
  • Expression control sequences include, but are not limited to, promoters (such as naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression control can be a eukaryotic promoter system in a vector that can transform or transfect eukaryotic host cells (such as COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of plasminogen.
  • Suitable expression vectors are usually replicated in the host organism as an episome or as an integrated part of the host chromosomal DNA.
  • the expression vector contains a selection marker (for example, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate the transformation of the desired DNA sequence for exogenous use Those cells are tested.
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone a polynucleotide encoding a subject compound.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various pseudomonas. Genus (Pseudomonas) species.
  • expression vectors can also be produced, which usually contain expression control sequences compatible with the host cell (for example, an origin of replication).
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from bacteriophage lambda. Promoters usually control expression, optionally in the case of manipulating gene sequences, and have ribosome binding site sequences, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast such as S. cerevisiae
  • Pichia Pichia
  • suitable yeast host cells in which suitable vectors have expression control sequences (such as promoters), origins of replication, termination sequences, etc., as required.
  • suitable promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeasts are initiated by specifically including promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose.
  • mammalian cells e.g., mammalian cells cultured in an in vitro cell culture
  • the plasminogen of the present invention e.g., a polynucleotide encoding plasminogen.
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas.
  • Expression vectors used in these cells may contain expression control sequences such as an origin of replication, promoters and enhancers (Queen et al., Immunol. Rev.
  • ribosome binding Site RNA splice site
  • polyadenylation site RNA splice site
  • transcription terminator sequence RNA splice site
  • suitable expression control sequences are promoters derived from white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149 (1992).
  • Plasminogen is substantially pure, for example at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure Or purer, for example free of contaminants such as cell debris, macromolecules other than plasminogen, etc.
  • a freeze-dried formulation can be formed by mixing plasminogen with the required purity with optional pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.ed. (1980)) Or aqueous solutions to prepare therapeutic formulations.
  • Acceptable carriers, excipients, and stabilizers are non-toxic to recipients at the dose and concentration used, and include buffers such as phosphate, citrate and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (such as Octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoic acid Esters such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues) ; Proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • the formulations of the present invention may also contain more than one active compound required for the specific condition to be treated, preferably those with complementary activities and no side effects between each other.
  • active compound required for the specific condition to be treated, preferably those with complementary activities and no side effects between each other.
  • the plasminogen of the present invention can be encapsulated in microcapsules prepared by techniques such as coacervation or interfacial polymerization, for example, can be placed in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules) or placed in hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a coarse emulsion.
  • colloidal drug delivery system e.g., liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules
  • hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a coarse emulsion.
  • the plasminogen of the present invention for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after freeze-drying and reformulation.
  • the plasminogen of the present invention can be used to prepare sustained-release preparations.
  • sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers having a certain shape and containing glycoproteins, such as films or microcapsules.
  • sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.
  • Polymers such as ethylene- Vinyl acetate and lactic-glycolic acid can continue to release molecules for more than 100 days, but some hydrogels release proteins for a short time.
  • a reasonable strategy for stabilizing the protein can be designed according to the relevant mechanism. For example, if the mechanism of aggregation is found to be The formation of intermolecular SS bonds through the exchange of thiodisulfide bonds can be stabilized by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions .
  • nasal inhalation aerosol inhalation
  • nasal drops or eye drops intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (for example, via the carotid artery), intrarectal, intramuscular , Rectal administration to achieve the administration of the pharmaceutical composition of the present invention.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, and so on. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, among others.
  • the dosage range of the plasminogen-containing pharmaceutical composition of the present invention can be, for example, about 0.0001 to 2000 mg/kg, or about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75mg/kg, 10mg/kg, 50mg/kg, etc.) the weight of the subject.
  • the dosage may be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Doses above or below this exemplified range are also covered, especially taking into account the factors mentioned above.
  • the intermediate dose in the above range is also included in the scope of the present invention.
  • the subject can administer such doses every day, every other day, every week, or according to any other schedule determined by empirical analysis.
  • An exemplary dosage schedule includes 0.01-100 mg/kg for consecutive days. During the administration of the drug of the present invention, it is necessary to evaluate the therapeutic effect and safety in real time.
  • One embodiment of the present invention relates to a product or kit comprising the plasminogen or plasmin of the present invention which can be used to treat hypertension and related disorders.
  • the article preferably includes a container, label or package insert. Suitable containers are bottles, vials, syringes, etc.
  • the container can be made of various materials such as glass or plastic.
  • the container contains a composition that can effectively treat the disease or condition of the present invention and has a sterile access (for example, the container may be an intravenous solution pack or a vial, which contains a stopper that can be penetrated by a hypodermic injection needle of). At least one active agent in the composition is plasminogen/plasmin.
  • the label on or attached to the container indicates that the composition is used to treat the hypertension and related disorders of the present invention.
  • the preparation may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further contain other substances required from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.
  • the article contains a package insert with instructions for use, including, for example, instructing the user of the composition to administer the plasminogen composition and other medications for the treatment of concomitant diseases to the patient.
  • the plasminogen used in all the following examples is human plasminogen, derived from donor plasma, based on the method described in the following documents: 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 and Glu-ins-plasminogens J Biol Chem.1976 Jun 25; 251(12):3693-9; HAGAN JJ, ABLONDI FB, DE RENZO EC.
  • db/db mice purchased from Nanjing Institute of Biomedicine, strain name BKS.Cg-Dock7m+/-Leprdb/JNju
  • db/db mice spontaneously develop hypertension in 11-14 weeks, and with the increase of week age, the blood pressure has a trend of continuous increase [24,25] .
  • mice Twelve db/db male mice aged 15-16 weeks were collected. On the day before the administration, the basal blood pressure was measured after weighing. According to the blood pressure, they were randomly divided into two groups, namely the vehicle PBS control group and the plasminogen group, with 6 rats in each group. The mice in the plasminogen group were given plasminogen by tail vein injection of 2mg/0.2ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS solution through the tail vein for 21 consecutive days. The start of administration was set as the first day, and blood pressure was measured on days 0, 8, 15, and 22. Each mouse was measured five times in a row, and the systolic blood pressure and average blood pressure of each measurement were recorded.
  • the systolic blood pressure and the average blood pressure are the average values of the data of the systolic blood pressure and the average blood pressure obtained by five measurements, respectively.
  • the calculation method of the average blood pressure is 1/3 of the systolic blood pressure + 2/3 of the diastolic blood pressure.
  • the blood pressure of mice was measured with a non-invasive blood pressure meter (MRBP-M01, IITC Life Science).
  • mice aged 25-26 weeks were collected. The day before the administration, the basal blood pressure was measured after weighing, and the blood pressure was randomly divided into two groups, 6 rats in the vehicle PBS control group and 7 rats in the plasminogen group.
  • the mice in the plasminogen group were given plasminogen by tail vein injection of 2mg/0.2ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS solution through the tail vein for 21 consecutive days.
  • the start of administration was set as the first day, and blood pressure was measured on days 0, 8, 15, and 22. Each mouse was measured five times in a row, and the systolic blood pressure and average blood pressure of each measurement were recorded.
  • the systolic blood pressure and the average blood pressure are the average values of the data of the systolic blood pressure and the average blood pressure obtained by five measurements, respectively.
  • the blood pressure of mice was measured with a non-invasive blood pressure meter (MRBP-M01, IITC Life Science).
  • mice Twelve db/db male mice aged 15-16 weeks were collected. On the day before the administration, the basal blood pressure was measured after weighing. According to the blood pressure, they were randomly divided into two groups, namely the vehicle PBS control group and the plasminogen group, with 6 rats in each group. The mice in the plasminogen group were given plasminogen by tail vein injection of 2mg/0.2ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS solution through the tail vein for 28 consecutive days. On the 29th day, the eyeballs were removed and blood was collected, and the supernatant was obtained by centrifugation. Following the instructions of the Angiotensin II detection kit (manufacturer: Wuhan Huamei Bioengineering Co., Ltd., article number: CSB-E04495m) to detect the serum angiotensin II level.
  • the Angiotensin II detection kit manufactured by Wuhan Huamei Bioengineering Co., Ltd.
  • mice aged 25-26 weeks were collected. The day before the administration, the basal blood pressure was measured after weighing, and the blood pressure was randomly divided into two groups, 6 rats in the vehicle PBS control group and 7 rats in the plasminogen group.
  • the mice in the plasminogen group were given plasminogen by tail vein injection of 2mg/0.2ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS solution through the tail vein for 28 consecutive days. After the mice were sacrificed on the 29th day, the hearts were taken and fixed in 4% paraformaldehyde fixative for 24 hours. The fixed tissue samples were dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the tissue section was 4 ⁇ m.
  • the sections were deparaffinized and rehydrated and stained with hematoxylin and eosin (H&E staining). After differentiation with 1% hydrochloric acid alcohol, the ammonia water turned blue and the alcohol gradient dehydration was mounted. The sections were observed under a 200X optical microscope.
  • mice cardiomyocytes were tighter and arranged more regularly. It shows that plasminogen can significantly improve the heart damage in diabetic hypertension model mice.
  • mice aged 25-26 weeks were collected. The day before the administration, the basal blood pressure was measured after weighing, and the blood pressure was randomly divided into two groups, 6 rats in the vehicle PBS control group and 7 rats in the plasminogen group.
  • the mice in the plasminogen group were given plasminogen by tail vein injection of 2 mg/0.2 ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS solution through the tail vein for 28 consecutive days. After the mice were sacrificed on the 29th day, the kidneys were taken and fixed in 4% paraformaldehyde fixative for 24 hours. The fixed tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the tissue section is 3 ⁇ m, the section is deparaffinized to water and then washed once, stained with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed in running water for 2 minutes, hematoxylin stained for 1 minute, rinsed in running water, differentiated with 1% hydrochloric acid and alcohol, and ammonia is returned Blue, rinse with running water, seal with neutral gum after drying, and observe under a 200X optical microscope.
  • Sirius red staining can stain collagen for a long time.
  • Sirius red staining can specifically display collagen tissue.
  • mice A total of 14 Plg+/+ male mice at 21-22 weeks were taken to measure blood pressure and body weight.
  • the mice were randomly divided into 3 groups according to blood pressure and body weight. There were 4 blank control groups. 5 pieces.
  • the mice in the blank control group were injected subcutaneously with 0.1ml of normal saline, and the mice in the vehicle PBS control group and the plasminogen group were subcutaneously injected with angiotensin II 1mg/kg/mouse/day for 14 consecutive days [26] .
  • mice in the plasminogen group will be given plasminogen by tail vein injection of 1mg/0.1ml/mouse/day, and the mice in the vehicle PBS control group will be given plasminogen in the tail vein.
  • the same volume of PBS solution was injected and administered continuously for 14 days; the mice in the blank control group were not treated with administration.
  • the start of modeling and administration was set as the first day.
  • the eyeballs were removed and blood was collected, and the supernatant was obtained by centrifugation to detect the serum superoxide dismutase (SOD) level.
  • SOD detection adopts SOD detection kit (Nanjing Jiancheng Institute of Biological Engineering, article number A001-1), and the detection is carried out according to the method shown in the instructions.
  • SOD is an important enzyme system for the body to scavenge free radicals. It can scavenge superoxide anions (o2-), and o2- is the initiating free radical of oxygen free radicals. Studies have shown that SOD has a protective effect on hypertension, and the level of SOD in hypertensive patients is reduced. [27] .
  • Example 7 Plasminogen reduces renal fibrosis in mice with hypertension induced by angiotensin II
  • mice A total of 14 Plg+/+ male mice at 21-22 weeks were taken to measure blood pressure and body weight.
  • the mice were randomly divided into 3 groups according to blood pressure and body weight. There were 4 blank control groups. 5 pieces.
  • the mice in the blank control group were injected with 0.1ml of saline subcutaneously, and the mice in the vehicle PBS control group and the plasminogen group were subcutaneously injected with angiotensin II 1mg/kg/mouse/day, and the model was made by continuous subcutaneous injection for 14 days [26] .
  • Administer plasminogen or vehicle at the same time when the model is started.
  • mice in the plasminogen group will be given plasminogen by tail vein injection of 1mg/0.1ml/mouse/day, and the mice in the vehicle PBS control group will be given plasminogen in the tail vein.
  • the same volume of PBS solution was injected and administered continuously for 14 days; the mice in the blank control group were not treated with administration.
  • the start of modeling and administration was set as the first day. After the mice were sacrificed on the 15th day, the kidneys were taken and fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the tissue section is 3 ⁇ m, the section is deparaffinized to water and then washed once, stained with 0.1% Sirius Red saturated picric acid for 30 minutes, rinsed in running water for 2 minutes, stained with hematoxylin for 1 minute, rinsed in running water, differentiated with 1% hydrochloric acid alcohol, and ammonia return Blue, rinse with running water, seal with neutral gum after drying, and observe under a 200X optical microscope.
  • Example 8 Plasminogen alleviates cardiac fibrosis in angiotensin II-induced hypertensive model mice
  • mice Take 14 Plg +/+ male mice at 21-22 weeks to measure blood pressure and body weight.
  • the mice are divided into 3 groups randomly, 4 in the blank control group, the vehicle PBS control group and the plasminogen group 5 each.
  • the mice in the blank control group were injected with 0.1ml of saline subcutaneously, and the mice in the vehicle PBS control group and the plasminogen group were subcutaneously injected with angiotensin II 1mg/kg/mouse/day, and the model was made by continuous subcutaneous injection for 14 days [26] .
  • mice in the plasminogen group will be given plasminogen by tail vein injection of 1mg/0.1ml/mouse/day, and the mice in the vehicle PBS control group will be given plasminogen in the tail vein.
  • the same volume of PBS solution was injected and administered continuously for 14 days; the mice in the blank control group were not treated with administration.
  • the start of modeling and administration was set as the first day. After the mice were sacrificed on the 15th day, the hearts were taken and fixed in 4% paraformaldehyde fixative solution for 24 hours.
  • the fixed heart tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the tissue section is 3 ⁇ m, the section is deparaffinized to water and then washed once, stained with 0.1% Sirius Red saturated picric acid for 30 minutes, rinsed in running water for 2 minutes, stained with hematoxylin for 1 minute, rinsed in running water, differentiated with 1% hydrochloric acid alcohol, and ammonia return Blue, rinse with running water, seal with neutral gum after drying, and observe under a 200X optical microscope.
  • Example 9 Plasminogen alleviates monocrotaline-induced pulmonary fibrosis in a mouse model of pulmonary hypertension
  • mice Twelve 12-week C57 male mice were weighed and their blood pressure was measured, and they were randomly divided into 2 groups according to blood pressure, 4 mice in the blank control group, and 8 mice in the model group.
  • the mice in the blank control group were injected with 100 ⁇ l of normal saline in the tail vein, and the mice in the model group were injected with monocrotaline 60mg/kg/mouse in the tail vein for 3 consecutive days.
  • the mice had a normal diet [28,29] .
  • the blood pressure was measured after 3 days, and the model group mice were randomly divided into two groups according to the blood pressure, 4 mice each for the vehicle PBS control group and the plasminogen group.
  • mice in the plasminogen group were given plasminogen by tail vein injection of 1mg/0.1ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS solution through the tail vein for 28 consecutive days; blank control The mice in the group were not treated with administration.
  • the start of modeling and administration was set as the first day. After the mice were sacrificed on the 29th day, the lungs were taken and fixed in 4% paraformaldehyde fixative for 24 hours. The fixed lung tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the tissue section is 3 ⁇ m, the section is deparaffinized to water and then washed once, stained with 0.1% Sirius Red saturated picric acid for 30 minutes, rinsed in running water for 2 minutes, stained with hematoxylin for 1 minute, rinsed in running water, differentiated with 1% hydrochloric acid alcohol, and ammonia return Blue, rinse with running water, seal with neutral gum after drying, and observe under a 200X optical microscope.
  • Monocrotaline is a bipyrrole alkaloid. After being transformed by P450 monooxygenase in the liver, it reaches the lungs through the blood circulation and can cause irreversible damage to the pulmonary blood vessels. Pulmonary vascular endothelial cells are considered to be the target cells of monocrotaline, and endothelial cell damage plays a key role in the process of pulmonary vascular remodeling [28,29] .
  • Example 10 Plasminogen reduces monocrotaline-induced cardiac fibrosis in a mouse model of pulmonary hypertension
  • mice Twelve 12-week C57 male mice were weighed and their blood pressure was measured, and they were randomly divided into 2 groups according to blood pressure, 4 mice in the blank control group, and 8 mice in the model group.
  • the mice in the blank control group were injected with 100 ⁇ l of normal saline in the tail vein, and the mice in the model group were injected with monocrotaline 60mg/kg/mouse in the tail vein for 3 consecutive days.
  • the mice had a normal diet [28,29] .
  • the blood pressure was measured after 3 days, and the model group mice were randomly divided into two groups according to the blood pressure, 4 mice each for the vehicle PBS control group and the plasminogen group.
  • mice in the plasminogen group were given plasminogen by tail vein injection of 1mg/0.1ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS solution through the tail vein for 28 consecutive days; blank control The mice in the group were not treated with administration.
  • the start of modeling and administration was set as the first day. After the mice were sacrificed on the 29th day, the heart was taken and fixed in 4% paraformaldehyde fixative for 24 hours. The fixed heart tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the tissue section is 3 ⁇ m, the section is deparaffinized to water and then washed once, stained with 0.1% Sirius Red saturated picric acid for 30 minutes, rinsed in running water for 2 minutes, stained with hematoxylin for 1 minute, rinsed in running water, differentiated with 1% hydrochloric acid alcohol, and ammonia return Blue, rinse with running water, seal with neutral gum after drying, and observe under a 200X optical microscope.
  • mice in the blank control group had almost no collagen deposition in the heart ( Figure 10A).
  • the collagen deposition in the heart of the mice in the plasminogen group (marked by the arrow) was significantly less than that in the vehicle PBS control group ( Figure 10B). ). It shows that plasminogen can significantly reduce the fibrosis of the heart of pulmonary hypertension model mice induced by monocrotaline.
  • Example 11 Plasminogen reduces monocrotaline-induced renal fibrosis in a mouse model of pulmonary hypertension
  • mice Twelve 12-week C57 male mice were weighed and their blood pressure was measured, and they were randomly divided into 2 groups according to blood pressure, 4 mice in the blank control group, and 8 mice in the model group.
  • the mice in the blank control group were injected with 100 ⁇ l of normal saline in the tail vein, and the mice in the model group were injected with monocrotaline 60mg/kg/mouse in the tail vein for 3 consecutive days.
  • the mice had a normal diet [28,29] .
  • the blood pressure was measured after 3 days, and the model group mice were randomly divided into two groups according to the blood pressure, 4 mice each for the vehicle PBS control group and the plasminogen group.
  • mice in the plasminogen group were given plasminogen by tail vein injection of 1mg/0.1ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS solution through the tail vein for 28 consecutive days; blank control The mice in the group were not treated with administration.
  • the start of modeling and administration was set as the first day. After the mice were sacrificed on the 29th day, the kidneys were taken and fixed in 4% paraformaldehyde fixative solution for 24 hours. The fixed kidney tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the tissue section is 3 ⁇ m, the section is deparaffinized to water and then washed once, stained with 0.1% Sirius Red saturated picric acid for 30 minutes, rinsed in running water for 2 minutes, stained with hematoxylin for 1 minute, rinsed in running water, differentiated with 1% hydrochloric acid alcohol, and ammonia return Blue, rinse with running water, seal with neutral gum after drying, and observe under a 200X optical microscope.
  • Example 12 Plasminogen reduces monocrotaline-induced liver fibrosis in a mouse model of pulmonary hypertension
  • mice Twelve 12-week C57 male mice were weighed and their blood pressure was measured, and they were randomly divided into 2 groups according to blood pressure, 4 mice in the blank control group, and 8 mice in the model group.
  • the mice in the blank control group were injected with 100 ⁇ l of normal saline in the tail vein, and the mice in the model group were injected with monocrotaline 60mg/kg/mouse in the tail vein for 3 consecutive days.
  • the mice had a normal diet [28,29] .
  • the blood pressure was measured after 3 days, and the model group mice were randomly divided into two groups according to the blood pressure, 4 mice each for the vehicle PBS control group and the plasminogen group.
  • mice in the plasminogen group were given plasminogen by tail vein injection of 1mg/0.1ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS solution through the tail vein for 28 consecutive days; blank control The mice in the group were not treated with administration.
  • the start of modeling and administration was set as the first day. After the mice were sacrificed on the 29th day, the liver was taken and fixed in 4% paraformaldehyde fixative for 24 hours. The fixed liver tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the tissue section is 3 ⁇ m, the section is deparaffinized to water and then washed once, stained with 0.1% Sirius Red saturated picric acid for 30 minutes, rinsed in running water for 2 minutes, stained with hematoxylin for 1 minute, rinsed in running water, differentiated with 1% hydrochloric acid alcohol, and ammonia return Blue, rinse with running water, seal with neutral gum after drying, and observe under a 200X optical microscope.
  • Example 13 Plasminogen promotes the increase of blood pressure in mice with ischemic renal atrophy
  • mice from 6 to 7 weeks were randomly divided into two groups, 4 in the blank control group and 14 in the model group.
  • the mice in the blank control group were left untreated.
  • mice in the model group mice were injected with 3% sodium pentobarbital (50mg/kg) anesthetized in the abdominal cavity.
  • the skin, fascia, and muscle layer were cut through the midline of the abdomen by surgical methods, and the intestines, pancreas and other organs and tissues were opened and exposed.
  • mice in the model group were anesthetized by intraperitoneal injection of 3% pentobarbital sodium, opened the abdomen, loosened the left renal blood vessel ligature, and quickly sutured the abdominal cavity, disinfected the wound with medical iodophor, and placed the animal at 37 On heating pads at °C, observe and wait until the animals are awake. If the animals are not fully awake, they cannot be given water. Inject antibiotics (5000U/mouse) intramuscularly and analgesics subcutaneously (2mg/kg) for 3 consecutive days [30] . After the stitches were removed, all the mice were weighed.
  • mice in the model group were randomly divided into two groups, given to the plasminogen group and the PBS control group, 7 mice in each group, and the administration was started.
  • the start of administration was set as the first day.
  • the mice in the plasminogen group were given 1mg/0.1ml/mouse/day through the tail vein injection, and the mice in the vehicle PBS control group were given the same volume of PBS through the tail vein injection.
  • the solution was given continuously for 14 days. And on the 15th day, a non-invasive blood pressure meter (MRBP-M01, IITC Life science) was used to detect the blood pressure (systolic blood pressure and mean blood pressure) of the mice.
  • MRBP-M01 non-invasive blood pressure meter
  • mice from 8 to 9 weeks old were randomly divided into three groups: a blank control group, a vehicle group, and a drug treatment group, with 7 mice in each group.
  • the mice in the blank control group were left untreated.
  • all mice were injected subcutaneously with angiotensin II solution at a dose of 1 mg/kg/mouse 0.25 mg/ml, twice a day, with an interval of 8 hours, for continuous modeling for 6 days [31] .
  • the administration group and the vehicle group started administration, that is, the first day.
  • mice in the administration group were injected with plasmin in the tail vein at the rate of 1mg/0.1ml/mouse/day Originally, mice in the vehicle group were injected with the same volume of vehicle through the tail vein for 5 days, while the blank control group was not treated. On day 6, the blood pressure of all mice was measured. Each mouse was measured five times in a row, and the systolic blood pressure and average blood pressure of each measurement were recorded. The systolic blood pressure and the average blood pressure are the average values of the data of the systolic blood pressure and the average blood pressure obtained by five measurements, respectively. The blood pressure of mice was measured with a non-invasive blood pressure meter (MRBP-M01, IITC Life science).
  • MRBP-M01 non-invasive blood pressure meter
  • mice in the vehicle group were significantly increased.
  • the mean blood pressure and systolic blood pressure of the mice in the drug group were significantly lower than those of the mice in the vehicle group, and the statistical difference was significant (* means P ⁇ 0.05, ** means P ⁇ 0.01) ( Figure 14). It shows that plasminogen can reduce blood pressure in hypertensive model mice.
  • Example 15 Plasminogen promotes an increase in the level of angiotensin converting enzyme 2 in diabetic hypertensive model mice
  • mice Twelve db/db male mice aged 24-25 weeks were collected. The day before the administration, the basal blood pressure was measured after weighing, and the blood pressure was randomly divided into two groups, namely the vehicle group and the administration group, with 6 rats in each group. Mice in the administration group were given plasminogen by tail vein injection of 2mg/0.2ml/mouse/day, and mice in the vehicle group were given the same volume of PBS solution via tail vein injection for 28 consecutive days. On the 29th day, the eyeballs were removed and blood was collected, and the supernatant was obtained by centrifugation.
  • ACE2 detection kit manufactured by Wuhan Huamei Bioengineering Co., Ltd., article number: CSB-E17204m
  • Angiotensin-converting enzyme 2 (ACE2) is an important negative regulator of the renin-angiotensin system, and it is the main way to degrade angiotensin II (AngII) in the body. Under the action of ACE2, AngII is converted to Angl-7; in addition, ACE2 can also catalyze the degradation of AngI to Ang(1-9), which is converted to Angl-7 by ACE.
  • the affinity of ACE2 for AngII is 400 times that for Ang I, and catalyzing the conversion of AngII to Ang(1-7) is the main function of ACE2.
  • the physiological role of ACE2 is related to hypertension, heart function, heart function and diabetes, and serves as a receptor for severe acute respiratory syndrome coronavirus [32] .
  • Example 16 Plasminogen reduces the serum ACE2 level of angiotensin II-induced hypertension model mice
  • mice Twenty-one male C57 mice aged 8-9 weeks were randomly divided into three groups, 7 in the blank control group, 7 in the vehicle group and 7 in the administration group. After grouping the mice in the administration group and the vehicle group, subcutaneously inject 1 mg/kg/mouse with 0.25 mg/ml angiotensin II solution twice a day at an interval of 8 hours for continuous modeling for 7 days.
  • angiotensin II solution first take angiotensin II powder (article number: A9292-10mg, manufacturer: Beijing 7 Soleibao Biotechnology Co., Ltd.), dissolve it in 1ml of deionized water, prepare a 10mg/ml solution, and distribute it to avoid Repeated freezing and thawing, diluted 40 times when used, prepared to 0.25mg/ml, prepared for immediate use, and stored the original solution at -20°C. Two hours after the first injection, the administration group and the vehicle group started to administer, that is, the first day. The mice in the administration group were injected with plasminogen in the tail vein at the rate of 1mg/0.1ml/mouse/day.
  • the vehicle group Mice were injected with the same volume of solvent through the tail vein for 7 days, and the blank control group was not treated. On the 8th day, the eyeballs were removed and blood was collected, and the supernatant was obtained by centrifugation.
  • ACE2 detection kit manufactured by Shanghai Ximei Chemical Co., Ltd., article number: CSB-E17204m
  • Example 17 Plasminogen reduces the serum ACE level of angiotensin II-induced hypertension model mice
  • mice Twenty-one male C57 mice aged 8-9 weeks were randomly divided into three groups, 7 in the blank control group, 7 in the vehicle group and 7 in the administration group. After grouping the mice in the administration group and the vehicle group, subcutaneously inject 1 mg/kg/mouse with 0.25 mg/ml angiotensin II solution twice a day at an interval of 8 hours for continuous modeling for 7 days.
  • angiotensin II solution first take angiotensin II powder (article number: A9292-10mg, manufacturer: Beijing 7 Soleibao Biotechnology Co., Ltd.), dissolve it in 1ml of deionized water, prepare a 10mg/ml solution, and distribute it to avoid Repeated freezing and thawing, diluted 40 times when used, prepared to 0.25mg/ml, prepared for immediate use, and stored the original solution at -20°C. Two hours after the first injection, the administration group and the vehicle group started to administer, that is, the first day. The mice in the administration group were injected with plasminogen in the tail vein at the rate of 1mg/0.1ml/mouse/day.
  • the vehicle group Mice were injected with the same volume of solvent through the tail vein for 7 days, and the blank control group was not treated. On the 8th day, the eyeballs were removed and blood was collected, and the supernatant was obtained by centrifugation.
  • ACE Angiotensin Converting Enzyme
  • Angiotensin-converting enzyme is a key enzyme in the renin-angiotensin system and plays a key role in the production of AngII [32] .
  • the plasminogen used below is diluted with physiological saline at a concentration of 5 or 10 mg/ml. When spraying medicine, the plasminogen solution is sprayed with a nebulizer before use.
  • the patient was a 72-year-old female with a history of hypertension for more than 30 years.
  • Her systolic blood pressure was 160mmHg and occasionally 180mmHg. After taking antihypertensive drugs, her blood pressure was controlled at around 130/80mmHg.
  • the patient received intravenous injection of human plasminogen once a day, starting at 50 mg/time, and then gradually increasing to 135 mg/time. From the 6th day after the medication, the patient's systolic blood pressure dropped to 141mmHg, and the blood pressure fluctuated unsteadily during the 11th day (normal and abnormal alternation); after the 12th day, the blood pressure dropped and remained normal and stable within a week.
  • the patient did not take any antihypertensive drugs at the same time while receiving the above-mentioned intravenous injection of plasminogen.
  • the patient's blood pressure during the medication period is shown in Figure 18, which shows that plasminogen can reduce the blood pressure of hypertensive patients to close to normal levels.
  • the patient female, 79 years old, had coronary heart disease for more than 20 years and had a cerebral infarction in 2014. 23 years of diabetes history, long-term injection of insulin. He was diagnosed with hypertension for 5 years. After taking antihypertensive drugs, his blood pressure was 130-150/50-60mmHg in the morning and around 150/60mmHg in the afternoon. Human plasminogen was administered intravenously to the patient once a day at a dose of 100 mg/time.
  • the patient's blood pressure was 135/54mmHg on the 13th day after the medication, and in the later period, the blood pressure was stable at a normal level without the use of antihypertensive drugs.
  • the patient's blood pressure during medication is shown in Figure 19.
  • Human plasminogen is administered to the patient by intravenous injection, 1-2 times a day, with a daily dosage of 35-75 mg.
  • the diastolic blood pressure reached a normal value from less than 60mmHg, and the blood pressure was normal and stable during the treatment; after the treatment, the antihypertensive drug was halved, and the blood pressure was basically maintained at 130-140/64-76mmHg.
  • the patient's blood pressure during medication is shown in Figure 20.
  • Plasminogen can promote the normalization of blood pressure in patients with hypertension, and can reduce the dosage of antihypertensive drugs.
  • the patient male, 57 years old, was admitted to the hospital for review and found that the abdominal aorta was thickened and diagnosed as aortic dissection.
  • the hospital recommends surgery. He has a family history of heart disease and a history of hypertension for more than 10 years.
  • the patient is allergic to contrast agents and has eczema and urticaria.
  • the blood pressure is high in the morning and evening, and the systolic blood pressure can reach 200mmHg at the highest time, and the pressure difference is large, up to 80mmHg or more. Poor sleep, slow falling asleep, waking up 3-4 times at night, affecting sleep.
  • Human plasminogen is administered by intravenous injection, 1-2 times a day, with a dosage of 50-150 mg/day.
  • the blood pressure began to drop sooner or later after the fifth day of medication, and the pressure difference decreased.
  • the patient complained that he had not seen blood pressure drop below 140/75mmHg under the condition of taking antihypertensive drugs. This is the first time that the blood pressure has improved.
  • the blood pressure was maintained at about 136/73mmHg in the morning and evening after the sixth day of medication.
  • the patient 78 years old, was diagnosed as critically ill with new type of coronary pneumonia. During admission to the hospital, he found that his blood pressure rose to 150/78mmhg. He had no history of hypertension and had no medical examination. The patient was given human plasminogen by nebulized inhalation, 2 times a day, 10 mg each time. Blood pressure returned to normal after 1 day. The patient's blood pressure during the medication period is shown in Table 1 and Table 2.
  • the patient was diagnosed as critically ill with new coronavirus pneumonia. In addition to relevant clinical symptoms, his blood pressure was 139/94mmHg and his diastolic blood pressure was higher than normal.
  • the patient was given plasminogen by nebulized inhalation, 2 times a day, 10 mg each time. After one treatment, the diastolic blood pressure returned to normal.
  • the patient's blood pressure during the medication period is shown in Table 3.
  • Plasminogen can promote the return of hypertension to normal in critically ill patients with novel coronavirus pneumonia.
  • Patient female, 67 years old, patient is bedridden, unable to speak due to tracheotomy, no history of diabetes, heart disease, hypertension, no history of allergies, Parkinson's for 6 years, unable to walk for 4 years, bedridden for 3 years, catheterization for 1.5 years , The patient cannot turn over on his own, but can simply move his limbs by himself, and continue to inhale oxygen at a low flow rate every day. Blood pressure 80/45mmHg. Diagnosed with hypotension and Parkinson's. Plasminogen is administered to patients by intravenous injection, once a day, with a dosage of 50-250 mg/day, with an initial dosage of 50 mg/day, and then gradually increase. The medication was continued for 7 days, the medication was continued on the 9th, 11th, and 13th days, and the medication was discontinued on the 8, 10, and 12 days for observation.
  • the blood pressure showed an upward trend after medication, and normal blood pressure appeared for the first time on the third day of medication.
  • the blood pressure remained normal on the 9th day after the medication, and the blood pressure remained at 95/70mmHg after stopping the medication.
  • the results of blood pressure measurement during the patient's medication are shown in Figure 22.
  • Treatment method intravenous injection, once a day, with a dosage of 100 mg.
  • Figure 23 shows the results of blood pressure measurement during the patient's medication.

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Abstract

本发明涉及一种治疗血压异常病症的方法，包括给药受试者有效量的纤维蛋白溶酶原激活途径组分。本发明还涉及治疗血压异常病症的药物和制品及其用途。

Description

一种预防和治疗血压异常病症的方法和药物 技术领域
本发明涉及一种治疗血压异常病症及其并发症的方法和药物。
背景技术
高血压病是全球性多发病之一，其发病率在中国逐年上升。高血压导致心、脑、肾等内脏器官的并发症，导致伤残、死亡。高血压病的危害在于其能导致心、脑、肾等多个器官和系统的病变，例如最常见的并发症脑卒中。其次，高血压导致相关心脏损害，包括心肌肥厚、冠状动脉硬化、心律失常和心力衰竭等。高血压还常伴有肾脏损害和周围血管病变。高血压发展到中晚期，可发生视网膜病变。糖尿病也是高血压常见的合并症之一。防治高血压的研究越来越引起世界各国学者的重视，同时，有效防治高血压的并发症,可以显著降低患者致残率和死亡率。
本发明发现纤溶酶原能够显著降低高血压，同时改善高血压导致的组织器官损伤、纤维化和功能障碍，为高血压及其相关病症，并发症的预防和治疗开辟了新的途径。
发明概述
本申请涉及纤维蛋白溶酶原激活途径的组分，例如纤溶酶原预防和治疗血压异常病症(包括高血压病症和低血压病症)的方法，用途和药物。本申请通过研究证明，纤维蛋白溶酶原激活途径的组分，例如纤溶酶原能够促进患高血压病症或低血压病症的受试者的血压恢复至正常，同时能够减轻和缓解由于血压异常(例如高血压或低血压)导致的组织器官，例如心脏、肺脏、肾脏和肝脏的组织结构损伤和功能损伤。
具体地，本申请涉及如下各项：
1.一方面，一种预防或治疗高血压病症的方法，包括给药患高血压病症的受试者有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维 蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请还涉及选自如下的一种或多种化合物在制备预防或治疗高血压病症的药物中的用途，所述一种或多种化合物选自：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请还涉及包含选自如下的一种或多种化合物的预防或治疗高血压病症的药物，所述一种或多种化合物选自：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请还涉及选自如下的一种或多种化合物预防或治疗高血压病症的用途，所述一种或多种化合物选自：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
2.项1所述的方法、用途或药物，其中所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro- plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。
3.项1的方法、用途或药物，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抑制剂，例如抗体。
4.项1的方法、用途或药物，其中所述高血压病症包括高血压导致的心、脑、肺、肝、肾或血管的损伤或并发症。
5.项4的方法、用途或药物，其中所述并发症包括心律失常、心力衰竭、脑出血、脑血栓、脑梗塞、高血压肾病、肾衰、尿毒症、肝硬化、肺动脉高压、肺纤维化、微血栓。
6.项1-5任一项的方法、用途或药物，其中所述化合物具有选自如下的一项或多项作用：降低患高血压病症的受试者的血压、降低患高血压病症的受试者的血清血管紧张素II水平、调节受试者ACE或ACE2的水平、减轻高血压导致的组织器官损伤、促进损伤的组织器官的修复、减轻组织器官纤维化和促进自由基的清除。
7.项6的方法、用途或药物，其中所述促进损伤的组织器官的修复为促进损伤的心脏组织、脑组织、肺组织、肾脏组织或肝组织的结构或功能的恢复。
8.项6的方法、用途或药物，其中所述减轻组织器官纤维化包括减轻心脏组织、肺组织、肾脏组织或肝组织的纤维化。
9.项6的方法、用途或药物，其中所述化合物通过促进SOD的产生消除自由基。
10.项1-9任一项的方法、用途或药物，其中所述化合物为纤溶酶原。
11.项1-9任一项的方法、用途或药物，其中所述纤溶酶原与序列2具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且具有纤溶酶原的蛋白水解活性或赖氨酸结合活性。
12.项1-11任一项的方法、用途或药物，所述纤溶酶原包含序列14所示的纤溶酶原活性片段、具有纤溶酶原的蛋白水解活性。
13.项1-9任一项的方法、用途或药物，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原蛋白水解活性的变体。
14.项1-9任一项的方法、用途或药物，所述纤溶酶原为天然或合成的人纤溶酶原、或其保留纤溶酶原的蛋白水解活性或赖氨酸结合活性的变体或片段。
15.项1-14任一项的方法、用途或药物，其中所述化合物与一种或多种其他治疗方法或药物联合使用。
16.项15的方法、用途或药物，其中所述其他药物包括降压药、激素、免疫抑制剂、抗菌素或抗病毒药物。
17.项1-16任一项的方法、用途或药物，其中所述化合物通过鼻腔吸入、雾化吸入、滴鼻液、滴耳液或滴眼液形式给药，静脉内、腹膜内、皮下、颅内、鞘内、动脉内、直肠内和/或肌肉内给药。
本发明还涉及如下各项：
1.一方面，本申请涉及一种预防或治疗血压异常或血压异常病症的方法，包括给药患血压异常病症的受试者有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请还涉及选自如下的一种或多种化合物在制备预防或治疗血压异常或血压异常病症的药物中的用途，所述一种或多种化合物选自：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请还涉及包含选自如下的一种或多种化合物的预防或治疗血压异常或血压异常病症的药物，所述一种或多种化合物选自：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟 纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请还涉及选自如下的一种或多种化合物预防或治疗血压异常或血压异常病症的用途，所述一种或多种化合物选自：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
本申请所述血压异常或血压异常病症包括血压高于正常水平的高血压或高血压病症和血压低于正常水平的低血压或低血压病症。因此，本申请涉及使用上述一种或多种化合物使患高血压病症受试者的高血压或患低血压病症受试者的低血压恢复至正常水平的方法，用途和药物。
2.项1所述的方法、用途或药物，其中所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro-plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。
3.项1的方法、用途或药物，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抑制剂，例如抗体。
4.项1-3任一项的方法、用途或药物，其中所述血压异常病症包括血压异常病症导致的组织器官损伤或并发症。在一些实施方案中，所述组织器官损伤或并发症为心、脑、肺、肝、肾或血管的损伤或并发症。在一些实施方案中，所述组织器官损伤为组织器官结构损伤(例如正常组织结构改变)或组织器官功能损伤(例如组织器官功能下降)。
在上述实施方案中，所述血压异常病症包括高血压病症或低血压病症。
5.项4的方法、用途或药物，其中所述血压异常病症导致的并发症为高血压病症导致的并发症，包括心律失常、心力衰竭、冠心病、脑出血、脑血栓、脑梗塞、高血压肾病、肾衰、尿毒症、肝硬化、肺动脉高压、肺纤维化或微血栓。
项4的方法、用途或药物，其中所述血压异常病症导致的并发症为低血压病症导致的并发症，包括低血压病症导致的组织器官供血不足，例如心脏供血不足、心绞痛、休克、脑供血不足、晕厥、脑梗死、肾脏供血不足、少尿、蛋白尿、肾功能不全。
6.项1-5任一项的方法、用途或药物，其中所述化合物具有选自如下的一项或多项作用：降低患高血压病症的受试者的血压、降低患高血压病症的受试者的血清血管紧张素II水平、调节受试者ACE或ACE2的水平、升高患低血压病症的受试者的血压、促进患高血压病症的受试者或患低血压病症的受试者的血压恢复至正常水平、减轻高血压或低血压导致的组织器官损伤、促进损伤的组织器官的修复、减轻组织器官纤维化和促进自由基的清除。
7.项1-6任一项的方法、用途或药物，其中所述促进损伤的组织器官的修复为促进损伤的心脏组织、脑组织、肺组织、肾脏组织或肝组织的结构或功能的恢复。
8.项1-7任一项的方法、用途或药物，其中所述减轻组织器官纤维化包括减轻心脏组织、肺组织、肾脏组织或肝组织的纤维化。
9.项1-8任一项的方法、用途或药物，其中所述化合物通过促进SOD的产生消除自由基。
10.项1-9任一项的方法、用途或药物，其中所述化合物为纤溶酶原。
11.项1-10任一项的方法、用途或药物，其中所述纤溶酶原与序列2具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且具有纤溶酶原的蛋白水解活性或赖氨酸结合活性。
12.项1-11任一项的方法、用途或药物，所述纤溶酶原包含序列14所示的纤溶酶原活性片段、具有纤溶酶原的蛋白水解活性。
13.项1-12任一项的方法、用途或药物，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原蛋白水解活性的变体。
14.项1-13任一项的方法、用途或药物，所述纤溶酶原为天然或合成的人纤溶酶原、或其保留纤溶酶原的蛋白水解活性或赖氨酸结合活性的变体或片段。
15.项1-14任一项的方法、用途或药物，其中所述化合物与一种或多种其他治疗方法或药物联合使用。
16.项15的方法、用途或药物，其中所述其他药物为治疗血压异常病症受试者血压异常病症外的其他疾病的药物。
17.项1-16任一项的方法、用途或药物，其中所述化合物通过鼻腔吸入、雾化吸入、滴鼻液、滴耳液或滴眼液形式给药，静脉内、腹膜内、皮下、颅内、鞘内、动脉内、直肠内和/或肌肉内给药。
本发明一方面还涉及用于预防或治疗受试者血压异常病症(例如高血压病症或低血压病症)及其相关损伤或并发症的纤溶酶原、包含纤溶酶原的药物、药物组合物、试剂盒、制品。
本发明一方面还涉及纤溶酶原、包含纤溶酶原的药物、药物组合物、试剂盒、制品。的以上方法中的药物、药物组合物、试剂盒、制品用于预防或治疗患血压异常病症的受试者血压异常病症(例如高血压病症或低血压病症)及其相关损伤或并发症的用途。
在本发明的上述任一实施方案中，所述纤溶酶原可与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有蛋白水解活性或赖氨酸结合活性的蛋白质。
在一些实施方案中，所述纤溶酶原是包含蛋白水解活性或赖氨酸结合活性片段、并且仍然具有蛋白水解活性或赖氨酸结合活性的蛋白质。在一些实施方案中，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶 酶原、微纤溶酶原、delta-纤溶酶原或它们的保留蛋白水解活性或赖氨酸结合活性的变体。在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留蛋白水解活性或赖氨酸结合活性的变体或片段。在一些实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。
在一些实施方案中，所述受试者是人。在一些实施方案中，所述受试者缺乏或缺失纤溶酶原。在一些实施方案中，所述缺乏或缺失是先天的、继发的和/或局部的。
在一些实施方案中，所述药物组合物包含药学上可接受的载剂和用于前述方法的纤溶酶原。在一些实施方案中，所述试剂盒可以是预防性或治疗性试剂盒，其包含：(i)用于前述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。在一些实施方案中，所述构件为注射器或小瓶。在一些实施方案中，所述试剂盒还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施前述任一方法。
在一些实施方案中，所述制品包含：含有标签的容器；和包含(i)用于前述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施前述任一方法。
在一些实施方案中，所述试剂盒或制品还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。
在前述方法的一些实施方案中，所述纤溶酶原通过全身或局部给药，优选通过以下途径施用：鼻腔吸入、雾化吸入、滴鼻液或滴眼液形式给药，静脉内、腹膜内、皮下、颅内、鞘内、动脉内、直肠内和/或肌肉内给药纤溶酶原来进行治疗。在前述方法的一些实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在前述方法的一些实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400 mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。
本发明明确涵盖了属于本发明实施方案之间的技术特征的所有组合，并且这些组合后的技术方案在本申请中已经明确公开，就像上述技术方案已经单独且明确公开一样。另外，本发明还明确涵盖各个实施方案及其要素的之间的组合，该组合后的技术方案在本文中明确公开。
附图简述
图1A-1B显示15-16周龄糖尿病高血压模型小鼠给予纤溶酶原21天血压测定结果。图1A为收缩压，图1B为平均压。结果显示，给药21天后PBS对照组小鼠血压与给药前相比并没有明显改变，而给纤溶酶原组小鼠的收缩压和平均压明显降低，均明显低于给溶媒PBS对照组，且统计差异显著(*表示P<0.05,**表示P<0.01)。说明纤溶酶原能够显著降低15-16周龄糖尿病小鼠的高血压。
图2A-2B显示25-26周龄糖尿病高血压模型小鼠给予纤溶酶原21天血压测定结果。图2A为收缩压，图2B为平均压。结果显示，给药21天后PBS对照组小鼠收缩压与给药前相比并没有明显改变，而给纤溶酶原组小鼠收缩压在给药7天已经开始显著降低，明显低于给溶媒PBS对照组，且统计差异显著(P＝0.019)；在给药的14、21天后，两组小鼠同样差异显著。在给药21天后，纤溶酶原组小鼠平均压低于给溶媒PBS对照组，差异接近显著(P＝0.09)。说明纤溶酶原能够显著降低25-26周龄糖尿病小鼠的高收缩压。
图3显示15-16周龄糖尿病高血压模型小鼠给予纤溶酶原28天血清血管紧张素II检测结果。结果显示，给予纤溶酶原28天后，给纤溶酶原组血清血管紧张素II水平明显低于给溶媒PBS对照组。说明纤溶酶原能够降低糖尿病高血压模型小鼠血清血管紧张素II水平，从而改善高血压。
图4A-4B显示25-26周龄糖尿病高血压模型小鼠给予纤溶酶原28天心脏H&E染色代表性图片。图4A给溶媒PBS对照组，图4B为给纤溶酶原组。结果显示，相比于给溶媒PBS对照组(图4A)，给纤溶酶原组(图4B)小鼠心肌细胞间更加紧密，排列更加规则。图5A-5B显示25-26周龄糖尿病高血压模型小鼠给予纤溶酶原28天肾脏SR染色代表性图片。图5A 给溶媒PBS对照组，图5B为给纤溶酶原组。结果显示，给予纤溶酶原28天后，给纤溶酶原组肾脏胶原纤维沉积(箭头标识)明显少于给溶媒PBS对照组。说明纤溶酶原能够明显降低糖尿病高血压模型小鼠肾脏纤维化。
图6血管紧张素II诱导的高血压模型小鼠给予纤溶酶原14天后血清SOD水平检测结果。结果显示，空白对照组血清中具有一定水平的SOD，给溶媒PBS对照组血清SOD水平明显降低，给纤溶酶原组血清SOD水平明显高于给溶媒PBS对照组，且统计差异显著(*表示P<0.05)。说明纤溶酶原能够增强机体清除自由基的能力。
图7A-7C血管紧张素II诱导的高血压模型小鼠给予纤溶酶原14天后肾脏天狼星红染色代表性图片。图7A为空白对照组，图7B给溶媒PBS对照组，图7C为给纤溶酶原组。结果显示，空白对照组小鼠肾脏无明显的胶原纤维沉积，给纤溶酶原组胶原纤维的沉积(箭头标识)明显少于给溶媒PBS对照组。说明纤溶酶原能够明显减少血管紧张素II诱导的高血压模型小鼠肾脏纤维化，改善高血压所致的肾脏病变。
图8A-8C血管紧张素II诱导的高血压模型小鼠给予纤溶酶原14天后心脏天狼星红染色代表性图片。图8A为空白对照组，图8B给溶媒PBS对照组，图8C为给纤溶酶原组。结果显示，空白对照组小鼠心脏无明显的胶原纤维沉积，给纤溶酶原组胶原纤维的沉积(箭头标识)明显少于给溶媒PBS对照组。说明纤溶酶原能够明显减少血管紧张素II诱导的高血压模型小鼠心脏纤维化，改善高血压所致的心脏病变。
图9A-9C野百合碱诱导的肺动脉高压模型小鼠给予纤溶酶原28天后肺天狼星红染色代表性图片。图9A为空白对照组，图9B给溶媒PBS对照组，图9C为给纤溶酶原组。结果显示，空白对照组小鼠肺基本无胶原蛋白沉积，给纤溶酶原组小鼠肺组织胶原蛋白沉积(箭头标识)要明显少于给溶媒PBS对照组。说明纤溶酶原能够明显减轻野百合碱诱导的肺动脉高压模型小鼠肺的纤维化。
图10A-10C百合碱诱导的肺动脉高压模型小鼠给予纤溶酶原28天后心脏天狼星红染色代表性图片。图10A为空白对照组，图10B给溶媒PBS对照组，图10C为给纤溶酶原组。结果显示，空白对照组小鼠心脏基本无胶原蛋白沉积，给纤溶酶原组小鼠心脏胶原蛋白沉积(箭头标识)要 明显少于给溶媒PBS对照组。说明纤溶酶原能够明显减轻野百合碱诱导的肺动脉高压模型小鼠心脏的纤维化。
图11A-11C野百合碱诱导的肺动脉高压模型小鼠给予纤溶酶原28天后肾脏天狼星红染色代表性图片。图11A为空白对照组，图11B给溶媒PBS对照组，图11C为给纤溶酶原组。结果显示，空白对照组小鼠肾脏基本无胶原蛋白沉积，给纤溶酶原组小鼠肾脏胶原蛋白沉积(箭头标识)要明显少于给溶媒PBS对照组。说明纤溶酶原能够明显减轻野百合碱诱导的肺动脉高压模型小鼠肾脏的纤维化。
图12A-12C野百合碱诱导的肺动脉高压模型小鼠给予纤溶酶原28天后肝脏天狼星红染色代表性图片。图12A为空白对照组，图12B给溶媒PBS对照组，图12C为给纤溶酶原组。结果显示，空白对照组小鼠肝脏基本无胶原蛋白沉积，给纤溶酶原组小鼠肝脏胶原蛋白沉积(箭头标识)要明显少于给溶媒PBS对照组。说明纤溶酶原能够明显减轻野百合碱诱导的肺动脉高压模型小鼠肝脏的纤维化。
图13肾萎缩模型小鼠给予纤溶酶原14天血压测定结果。结果显示，给纤溶酶原组和空白对照收缩压和平均压明显高于PBS对照组血压，并且纤溶酶原组和PBS对照组之间有统计差异显著(*表示P<0.05)。说明纤溶酶原能够促进肾萎缩所致低血压恢复正常水平。
图14血管紧张素II诱导的高血压模型小鼠给予纤溶酶原5天平均压和收缩压测定结果。结果显示，空白对照组小鼠具有一定水平的平均压和收缩压，溶媒组小鼠平均压和收缩压明显升高，给药组小鼠的平均压和收缩压明显低于溶媒组小鼠，且统计差异显著(*表示P<0.05，**表P<0.01)。表明纤溶酶原能够降低高血压模型小鼠血压。
图15 24-25周龄糖尿病小鼠给予纤溶酶原28天血清ACE2水平检测结果。结果显示，给药组小鼠血清ACE2水平明显高于溶媒组，且统计差异接近显著(P＝0.057)。提示纤溶酶原能够促进糖尿病高血压模型小鼠血清ACE2水平增加。
图16血管紧张素II诱导的高血压模型小鼠给予纤溶酶原7天血清ACE2水平检测结果。结果显示，空白对照组小鼠血液中具有一定水平的ACE2，溶媒组小鼠血液中ACE2水平明显高于空白对照组小鼠，给药组小 鼠血液中ACE2水平明显低于溶媒组小鼠，且统计差异极为显著(**表示P<0.01)。提示纤溶酶原能够促进AngII诱导的高血压模型小鼠血清ACE2水平降低。
图17血管紧张素II诱导的高血压模型小鼠给予纤溶酶原7天血清ACE水平检测结果。结果显示，空白对照组小鼠血液中具有一定水平的ACE，溶媒组小鼠血液中ACE水平明显高于空白对照组小鼠，给药组小鼠血液中ACE水平明显低于溶媒组小鼠，且统计差异接近显著(P＝0.051)。提示纤溶酶原能够促进AngII诱导的高血压模型小鼠血清ACE水平降低。
图18患者用药期间收缩压(高压)和舒张压(低压)检测结果。结果显示，用药后第6天开始，患者收缩压下降至141mmHg，至11天期间血压波动不稳定(正常异常交替)；第12天后，血压下降，且一周内保持正常稳定。表明纤溶酶原能够降低高血压患者的血压。
图19患者用药期间收缩压(高压)和舒张压(低压)检测结果。结果显示，用药后第13天患者血压135/54mmHg，后期正常稳定，基本不用降压药。表明纤溶酶原能够治疗高血压。
图20患者用药期间收缩压(高压)和舒张压(低压)检测结果。结果显示，用药后舒张压达到正常值，原来曾低于60mmHg，治疗期间血压正常稳定；治疗结束后，降压药减半，血压基本控制在130-140/64-76mmHg。表明纤溶酶原能够促进高血压患者血压恢复正常，可减少降压药的使用剂量。
图21A-21B患者用药期间早间(21A)和晚间(221B)收缩压(高压)和舒张压(低压)检测结果。第五天用药后早晚血压开始下降，压差减少。患者自诉，先前在吃降压药的情况下，也没有出现血压下降至140/75mmHg以下的情况，这是第一次出现好转。第六天用药后早晚血压维持在136/73mmHg左右。第七天用药后早晚血压维持平稳，血压在130/70mmHg左右。表明纤溶酶原能够改善患者高血压症状。
图22患者用药期间收缩压(高压)和舒张压(低压)检测结果。结果显示，用药后血压呈上升趋势，用药第3天第一次出现正常血压。用药第9天后血压开始正常，且一直血压保持正常，停药后血压保持在95/70mmHg。表明纤溶酶原能够促进低血压患者血压恢复。
图23患者用药期间收缩压(高压)和舒张压(低压)检测结果。结果显示，用药后患者精神状态好转，血压在用药后第二天即达到正常，一周后达到110/70mmHg左右。表明给药纤溶酶原能够促进低血压患者血压恢复。
发明详述
纤维蛋白溶解系统(Fibrinolytic system)也称纤溶系统，为参与纤维蛋白溶解(纤溶)过程的一系列化学物质组成的系统，主要包括纤维蛋白溶解酶原(纤溶酶原)、纤溶酶、纤溶酶原激活物、纤溶抑制剂。纤溶酶原激活物包括组织型纤溶酶原激活物(t-PA)和尿激酶型纤溶酶原激活物(u-PA)。t-PA是一种丝氨酸蛋白酶，由血管内皮细胞合成。t-PA激活纤溶酶原，此过程主要在纤维蛋白上进行；尿激酶型纤溶酶原激活物(u-PA)由肾小管上皮细胞和血管内皮细胞产生，可以直接激活纤溶酶原而不需要纤维蛋白作为辅因子。纤溶酶原(PLG)由肝脏合成，当血液凝固时，PLG大量吸附在纤维蛋白网上，在t-PA或u-PA的作用下，被激活为纤溶酶，促使纤维蛋白溶解。纤溶酶(PL)是一种丝氨酸蛋白酶，作用如下：降解纤维蛋白和纤维蛋白原；水解多种凝血因子Ⅴ、Ⅷ、Ⅹ、Ⅶ、Ⅺ、Ⅱ等；使纤溶酶原转变为纤溶酶；水解补体等。纤溶抑制物：包括纤溶酶原激活物抑制剂(PAI)和α2抗纤溶酶(α2-AP)。PAI主要有PAI-1和PAI-2两种形式，能特异性与t-PA以1：1比例结合，从而使其失活，同时激活PLG。α2-AP由肝脏合成，与PL以1：1比例结合形成复合物，抑制PL活性；FⅩⅢ使α2-AP以共价键与纤维蛋白结合，减弱了纤维蛋白对PL作用的敏感性。体内抑制纤溶系统活性的物质：PAI-1，补体C1抑制物；α2抗纤溶酶；α2巨球蛋白。
本发明的术语“纤维蛋白溶酶原激活途径的组分”涵盖：
1.纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、微纤溶酶原(micro-plasminogen)、delta-纤溶酶原；它们的变体或类似物；
2.纤维蛋白溶酶以及它们的变体或类似物；和
3.纤维蛋白溶酶原激活剂，例如tPA和uPA以及包含一个或多个tPA或uPA的结构域(如一个或多个kringle结构域和蛋白水解结构域)的tPA或uPA变体和类似物。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式，以及通过添加、删除和/或取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸、仍然具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA活性的蛋白质。例如，纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”包括通过例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个保守性氨基酸取代获得的这些蛋白质的突变变体。
本发明的“纤溶酶原变体”涵盖与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有蛋白水解活性或赖氨酸结合活性的蛋白质。例如本发明的“纤溶酶原变体”可以是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有蛋白水解活性或赖氨酸结合活性的蛋白质。具体地，本发明纤溶酶原变体包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式，以及通过保守性氨基酸取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸获得的这些蛋白质的突变变体。
本发明的纤溶酶原可以为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留蛋白水解活性或赖氨酸结合活性的变体，例如序列2、6、8、10或12所示的纤溶酶原，例如序列2所示的人天然纤溶酶原。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“类似物”包括分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用的化合物。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和“类似物”涵盖包含一个或多个结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和 “类似物”。例如，纤维蛋白溶酶原的“变体”和“类似物”涵盖包含一个或多个纤溶酶原结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶原变体和类似物，例如小纤维蛋白溶酶原(mini-plasminogen)。纤维蛋白溶酶的“变体”和“类似物”涵盖包含一个或多个纤维蛋白溶酶结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶“变体”和“类似物”，例如小纤维蛋白溶酶(mini-plasmin)和δ-纤维蛋白溶酶(delta-plasmin)。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的“变体”或“类似物”是否分别具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的活性，或者是否分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用可以通过本领域已知方法进行检测，例如，通过基于酶谱法(enzymography)、ELISA(酶联免疫吸附测定)和FACS(荧光激活细胞分选方法)通过激活的纤维蛋白溶酶活性水平来衡量，例如可以参照选自如下文献中记载的方法测量：Ny，A.，Leonardsson，G.，Hagglund，A.C，Hagglof，P.，Ploplis，V.A.，Carmeliet，P.and Ny，T.(1999).Ovulation inplasminogen-deficient mice.Endocrinology 140，5030-5035；Silverstein RL,Leung LL,Harpel PC,Nachman RL(November 1984)."Complex formation of platelet thrombospondin with plasminogen.Modulation of activation by tissue activator".J.Clin.Invest.74(5):1625–33；Gravanis I,Tsirka SE(February2008)."Tissue-type plasminogen activator as a therapeutic target in stroke".Expert Opinion on Therapeutic Targets.12(2):159–70；Geiger M,Huber K,Wojta J,Stingl L,Espana F,Griffin JH,Binder BR(Aug 1989)."Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo".Blood.74(2):722–8.
在本发明的一些实施方案中，本发明的“纤维蛋白溶酶原激活途径的组分”为纤溶酶原，选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留蛋白水解活性或赖氨酸结合活性的变体。在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留蛋白水解活性或赖氨酸结合活性的保守突变变体或其片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶 酶原直向同系物或其仍然保留蛋白水解活性或赖氨酸结合活性的保守突变变体或其片段。在一些实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。在一些实施方案中，所述纤溶酶原是如序列2所示的人天然纤溶酶原。
“能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物”指能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的任何化合物，例如tPA、uPA、链激酶、沙芦普酶、阿替普酶、瑞替普酶、替奈普酶、阿尼普酶、孟替普酶、拉诺替普酶、帕米普酶、葡激酶。
本发明“纤溶抑制剂的拮抗剂”为拮抗、减弱、封闭、阻止纤溶抑制剂作用的化合物。所述纤溶抑制剂例如PAI-1、补体C1抑制物、α2抗纤溶酶和α2巨球蛋白。所述拮抗剂例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抗体，或阻断或下调例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白表达的反义RNA或小RNA，或占据PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的结合位点但无PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白功能的化合物”，或封闭PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的结合结构域和/或活性结构域的化合物。
纤溶酶是纤溶酶原激活系统(PA系统)的关键组分。它是一种广谱的蛋白酶，能够水解细胞外基质(ECM)的几个组分，包括纤维蛋白、明胶、纤连蛋白、层粘连蛋白和蛋白聚糖。此外，纤溶酶能将一些金属蛋白酶前体(pro-MMPs)激活形成具有活性的金属蛋白酶(MMPs)。因此纤溶酶被认为是胞外蛋白水解作用的一个重要的上游调节物。纤溶酶是由纤溶酶原通过两种生理性的PAs：组织型纤溶酶原激活剂(tPA)或尿激酶型纤溶酶原激活剂(uPA)蛋白水解形成的。由于纤溶酶原在血浆和其他体液中相对水平较高，传统上认为PA系统的调节主要通过PAs的合成和活性水平实现。PA系统组分的合成受不同因素严格调节，如激素、生长因子和细胞因子。此外，还存在纤溶酶和PAs的特定生理抑制剂。纤溶酶的主要抑制剂是α2-抗纤溶酶(α2-antiplasmin)。PAs的活性同时被uPA和tPA的纤溶酶原激活剂抑制剂-1(PAI-1)抑制以及主要抑制uPA的溶酶原激活剂抑 制剂-2(PAI-2)调节。某些细胞表面具有直接水解活性的uPA特异性细胞表面受体(uPAR)。
纤溶酶原是一个单链糖蛋白，由791个氨基酸组成，分子量约为92kDa。纤溶酶原主要在肝脏合成，大量存在于胞外液中。血浆中纤溶酶原含量约为2μM。因此纤溶酶原是组织和体液中蛋白质水解活性的一个巨大的潜在来源。纤溶酶原存在两种分子形式：谷氨酸-纤溶酶原(Glu-plasminogen)和赖氨酸-纤溶酶原(Lys-plasminogen)。天然分泌和未裂解形式的纤溶酶原具有一个氨基末端(N-末端)谷氨酸，因此被称为谷氨酸-纤溶酶原。然而，在纤溶酶存在时，谷氨酸-纤溶酶原在Lys76-Lys77处水解成为赖氨酸-纤溶酶原。与谷氨酸-纤溶酶原相比，赖氨酸-纤溶酶原与纤维蛋白具有更高的亲和力，并可以更高的速率被PAs激活。这两种形式的纤溶酶原的Arg560-Val561肽键可被uPA或tPA切割，导致二硫键连接的双链蛋白酶纤溶酶的形成。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringles，羧基末端部分包含蛋白酶结构域。一些kringles含有介导纤溶酶原与纤维蛋白及其抑制剂α2-AP特异性相互作用的赖氨酸结合位点。最新发现一个纤溶酶原为38kDa的片段，其中包括kringles1-4，是血管生成的有效抑制剂。这个片段被命名为血管抑素，可通过几个蛋白酶水解纤溶酶原产生。
纤溶酶的主要底物是纤维蛋白，纤维蛋白的溶解是预防病理性血栓形成的关键。纤溶酶还具有对ECM几个组分的底物特异性，包括层粘连蛋白、纤连蛋白、蛋白聚糖和明胶，表明纤溶酶在ECM重建中也起着重要作用。间接地，纤溶酶还可以通过转化某些蛋白酶前体为活性蛋白酶来降解ECM的其他组分，包括MMP-1，MMP-2，MMP-3和MMP-9。因此，有人提出，纤溶酶可能是细胞外蛋白水解的一个重要的上游调节器。此外，纤溶酶具有激活某些潜在形式的生长因子的能力。在体外，纤溶酶还能水解补体系统的组分并释放趋化补体片段。
“纤溶酶”是存在于血液中的一种非常重要的酶，能将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体。
“纤溶酶原”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源纤溶酶原氨基酸序列(序列4)计算由810个氨基酸组成， 分子量约为90kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的纤溶酶原包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基Cys103-Cys181，Kringle2包括残基Glu184-Cys262，Kringle3包括残基Cys275-Cys352，Kringle4包括残基Cys377-Cys454，Kringle5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是人天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。Delta-纤溶酶原(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶域(也称蛋白酶结构域(protease domain，PD))，有文献报道了delta-纤溶酶原的氨基酸序列(序列8)，编码该氨基酸序列的cDNA序列如序列7所示。小纤溶酶原(Mini-plasminogen)由Kringle5和丝氨酸蛋白酶域组成，有文献报道其包括残基Val443-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，其氨基酸序列如序列10所示，编码该氨基酸序列的cDNA序列如序列9所示。而微纤溶酶原(Micro-plasminogen)仅含有丝氨酸蛋白酶结构域，有文献报道其氨基酸序列包括残基Ala543-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，也有专利文献CN102154253A报道其序列包括残基Lys531-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，在本专利申请中微纤溶酶原序列参考专利文献CN102154253A，其氨基酸序列如序列12所示，编码该氨基酸序列的cDNA序列如序列11所示。
全长纤溶酶原的结构也描述在Aisina等(Aisina R B,Mukhametova L I.Structure and function of plasminogen/plasmin system[J].Russian Journal of  Bioorganic Chemistry,2014,40(6):590-605)的文章中。在该文章中，Aisina等描述纤溶酶原包括Kringle1、2、3、4、5结构域和丝氨酸蛋白酶结构域(也称蛋白酶结构域(protease domain，PD))，其中，Kringles负责纤溶酶原与低分子量和高分子量的配体结合(即赖氨酸结合活性)，导致纤溶酶原转变成一个更加开放的构型，从而更容易被活化；蛋白酶结构域(PD)为残基Val562-Asn791，tPA和UPA特异性切割纤溶酶原的Arg561-Val562位活化键，从而使纤溶酶原形成纤溶酶，因此，蛋白酶结构域(PD)是赋予纤溶酶原蛋白水解活性的区域。
本发明的“纤溶酶”与“纤维蛋白溶酶”、“纤维蛋白溶解酶”可互换使用，含义相同；“纤溶酶原”与“纤溶酶原”、“纤维蛋白溶解酶原”可互换使用，含义相同。
在本申请中，所述纤溶酶原“缺乏”的含义或活性为受试者体内纤溶酶原的含量比正常人低，低至足以影响所述受试者的正常生理功能；所述纤溶酶原“缺失”的含义或活性为受试者体内纤溶酶原的含量显著低于正常人，甚至活性或表达极微，只有通过外源提供才能维持正常生理功能。
本领域技术人员可以理解，本发明纤溶酶原的所有技术方案适用于纤溶酶，因此，本发明描述的技术方案涵盖了纤溶酶原和纤溶酶。在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时，在纤溶酶原激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性纤溶酶。具有活性的纤溶酶可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中纤溶酶原的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为纤溶酶原激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”
是指具有与底物靶序列中赖氨酸结合的活性(赖氨酸结合活性)、或发挥蛋白水解功能的活性(蛋白水解活性)、或蛋白水解活性和赖氨酸结合活性的片段。本发明涉及纤溶酶原的技术方案涵盖了用纤溶酶原活性片段代替纤溶酶原的技术方案。在一些实施方案中，本发明所述的纤溶酶原 活性片段包含纤溶酶原的丝氨酸蛋白酶结构域或由纤溶酶原的丝氨酸蛋白酶结构域组成，优选，本发明所述的纤溶酶原活性片段包含序列14、或与序列14具有至少80％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列或由序列14、或与序列14具有至少80％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列组成。在一些实施方案中，本发明所述的纤溶酶原活性片段包含选自Kringle 1、Kringle 2、Kringle 3、Kringle 4、Kringle 5中一个或多个的区域，或由选自Kringle 1、Kringle 2、Kringle 3、Kringle 4、Kringle 5中一个或多个的区域组成。在一些实施方案中，本发明所述的纤溶酶原包括含有上述纤溶酶原活性片段的蛋白质。
目前，对于血液中纤溶酶原及其活性测定方法包括：对组织纤溶酶原激活剂活性的检测(t-PAA)、血浆组织纤溶酶原激活剂抗原的检测(t-PAAg)、对血浆组织纤溶酶原活性的检测(plgA)、血浆组织纤溶酶原抗原的检测(plgAg)、血浆组织纤溶酶原激活剂抑制物活性的检测、血浆组织纤溶酶原激活剂抑制物抗原的检测、血浆纤维蛋白溶酶-抗纤维蛋白溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤溶酶原活性成正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤溶酶原包括人的天然纤溶酶原，还包括来源于不同物种的、具有纤溶酶原活性的纤溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性，碱性，疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸，蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性 可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤溶酶原是指从其天然环境分离和/或回收的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原会纯化(1)至大于90％、大于95％、或大于98％的纯度(按重量计)，如通过Lowry法所确定的，例如超过99％(按重量计)，(2)至足以通过使用旋转杯序列分析仪获得N端或内部氨基酸序列的至少15个残基的程度，或(3)至同质性，该同质性是通过使用考马斯蓝或银染在还原性或非还原性条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定的。分离的纤溶酶原也包括通过生物工程技术从重组细胞制备，并通过至少一个纯化步骤分离的纤溶酶原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指任何长度的氨基酸的聚合形式，其可以包括遗传编码的和非遗传编码的氨基酸，化学或生物化学修饰的或衍生化的氨基酸，和具有经修饰的肽主链的多肽。该术语包括融合蛋白，包括但不限于具有异源氨基酸序列的融合蛋白，具有异源和同源前导序列(具有或没有N端甲硫氨酸残基)的融合物；等等。
关于参照多肽序列的“氨基酸序列同一性百分数(％)”定义为在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式实现，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员能决定用于比对序列的适宜参数，包括对所比较序列全长实现最大对比需要的任何算法。然而，为了本发明的目的，氨基酸序列同一性百分数值是使用序列比较计算机程序ALIGN-2产生的。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相 对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：
分数X/Y乘100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基的数目，且其中Y是B中的氨基酸残基的总数。应当领会，在氨基酸序列A的长度与氨基酸序列B的长度不相等的情况下，A相对于B的％氨基酸序列同一性会不等于B相对于A的％氨基酸序列同一性。除非另有明确说明，本文中使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
如本文中使用的，术语“治疗”指获得期望的药理和/或生理效果。所述效果可以是完全或部分预防疾病或其症状的发生、发作，部分或完全减轻疾病和/或其症状，和/或部分或完全治愈疾病和/或其症状，包括：(a)预防疾病在受试者体内发生或发作，所述受试者可以具有疾病的素因，但是尚未诊断为具有疾病；(b)抑制疾病，即阻滞其形成；和(c)减轻疾病和/或其症状，即引起疾病和/或其症状消退或消失。
术语“个体”、“受试者”和“患者”在本文中可互换使用，指哺乳动物，包括但不限于鼠(大鼠、小鼠)、非人灵长类、人、犬、猫、有蹄动物(例如马、牛、绵羊、猪、山羊)等。
“治疗有效量”或“有效量”指在对哺乳动物或其它受试者施用以治疗疾病时足以实现对疾病的所述预防和/或治疗的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)的量。“治疗有效量”会根据所使用的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)、要治疗的受试者的疾病和/或其症状的严重程度以及年龄、体重等而变化。
本申请“促进损伤的组织器官修复”指促进损伤的组织器官的结构和功能的修复，使损伤的组织器官解剖结构的完整性和功能尽量恢复至正常。
本申请“高血压”或“高血压病症”是指体循环动脉压力高于正常的状态。根据1999年世界卫生组织/国际高血压学会治疗指南，高血压诊断标准是收缩压≥18.7Kpa(140mmHg)或舒张压≥12.0Kpa(90mmHg)。“低血压”或“低血压病症”是指体循环动脉压力低于正常的状态。低血压的诊 断尚无统一标准。一般认为成年人上肢动脉收缩压/舒张压分别低于12/8kPa(90/60mmHg)即为低血压。“高血压”或“高血压病症”和“低血压”或“低血压病症”的血压指标诊断标准也可参照各国疾病诊断手册中的具体规定。
在本申请中，治疗“高血压”或“高血压病症”受试者包括使患“高血压”或“高血压病症”的受试者的高血压“下降”或“降低”，所述“下降”或“降低”是指使受试者的血压与没有给药的对照相比，或与给药之前受试者的血压相比，血压下降，例如使受试者血压趋于正常、接近正常或恢复至正常水平。同样，在本申请中，治疗“低血压”或“低血压病症”受试者包括使患“低血压”或“低血压病症”的受试者的低血压“升高”。所述“升高”是指使受试者的血压与没有给药的对照相比，或与给药之前的受试者的血压相比，血压升高，例如使受试者的血压趋于正常、接近正常或恢复至正常水平。
“并发症”是指一种疾病在发展过程中引起另一种疾病或症状的发生，后者即为前者的并发症，即后—种疾病的发生是由前一种疾病所引起的，或在疾病诊疗过程中，患者合并发生了与这种疾病有关的另一种或几种疾病。
“高血压并发症”，是指由高血压所引起的并发症，包括高血压导致的心、脑、肺、肝、肾或血管损伤。高血压导致的心脏损伤并发症例如左心室肥厚、心绞痛、心肌梗死和心力衰竭；高血压导致的脑组织损伤并发症例如脑出血、脑血栓、脑梗塞，出血性脑卒中、缺血性脑卒中、高血压脑病；高血压导致的肾损害例如进展缓慢的小动脉性肾硬化症、恶性小动脉性肾硬化症、慢性肾功能衰竭、高血压肾病、肾衰、尿毒症。高血压病最常见的并发症是脑血管意外，其次是高血压性心脏病心力衰竭，再是肾功能衰竭。较少见但严重的并发症为主动脉夹层动脉瘤。
“低血压并发症”是指由低血压所引起的并发症。低血压常见的并发症多是由重要脏器的灌注不足所致。如出现在大脑的供血不足，多表现为耳鸣、头晕眼花等，严重者可并发脑梗死；若心脏出现供血不足，多表现为心慌气短、胸闷等；若肾脏出现供血不足，多表现为少尿、蛋白尿，严重者出现无尿等肾功能不全现象。
发明纤溶酶原的制备
纤溶酶原可以从自然界分离并纯化用于进一步的治疗用途，也可以通过标准的化学肽合成技术来合成。当通过化学合成多肽时，可以经液相或固相进行合成。固相多肽合成(SPPS)(其中将序列的C末端氨基酸附接于不溶性支持物，接着序贯添加序列中剩余的氨基酸)是适合纤溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤溶酶原。用于固相合成的技术描述于Barany和Solid-Phase Peptide Synthesis；第3-284页于The Peptides:Analysis,Synthesis,Biology.第2卷：Special Methods in Peptide Synthesis,Part A.,Merrifield,等J.Am.Chem.Soc.,85:2149-2156(1963)；Stewart等,Solid Phase Peptide Synthesis,2nd ed.Pierce Chem.Co.,Rockford,Ill.(1984)；和Ganesan A.2006 Mini Rev.Med Chem.6:3-10和Camarero JA等2005 Protein Pept Lett.12:723-8中。简言之，用其上构建有肽链的功能性单元处理小的不溶性多孔珠。在偶联/去保护的重复循环后，将附接的固相游离N末端胺与单个受N保护的氨基酸单元偶联。然后，将此单元去保护，露出可以与别的氨基酸附接的新的N末端胺。肽保持固定在固相上，之后将其切掉。
可以使用标准重组方法来生产本发明的纤溶酶原。例如，将编码纤溶酶原的核酸插入表达载体中，使其与表达载体中的调控序列可操作连接。表达调控序列包括但不限于启动子(例如天然关联的或异源的启动子)、信号序列、增强子元件、和转录终止序列。表达调控可以是载体中的真核启动子系统，所述载体能够转化或转染真核宿主细胞(例如COS或CHO细胞)。一旦将载体掺入合适的宿主中，在适合于核苷酸序列的高水平表达及纤溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆主题化合物编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(enterobacteriaceae)，诸如沙门氏 菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物种。在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子系统，色氨酸(trp)启动子系统，beta-内酰胺酶启动子系统，或来自噬菌体λ的启动子系统。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
在微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达并生成本发明的纤溶酶原(例如编码纤溶酶原的多核苷酸)。参见Winnacker,From Genes to Clones,VCH Publishers,N.Y.,N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起点，启动子和增强子(Queen等,Immunol.Rev.89:49(1986))，以及必需的加工信息位点，诸如核糖体结合位点，RNA剪接位点，多聚腺苷酸化位点，和转录终止子序列。合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛乳头瘤病毒、巨细胞病毒等衍生的启动子。参见Co等,J.Immunol.148:1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀，亲和柱，柱层析，高效液相层析(HPLC)，凝胶电泳等来纯化本发明所述的纤溶酶原。该纤溶酶原是基本上纯的，例如至少约80％至85％纯的，至少约85％至90％纯的，至少约90％至95％纯的，或98％至99％纯的或更纯的，例如不含污染物，所述污染物如细胞碎片，除纤溶酶原以外的大分子，等等。
药物配制剂
可以通过将具有所需纯度的纤溶酶原与可选的药用载体，赋形剂，或稳定剂(Remington's Pharmaceutical Sciences,16版，Osol,A.ed.(1980))混合形成冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。例如，抗高血压的药物，抗心律失常的药物，治疗糖尿病的药物等。
本发明的纤溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送系统(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980)。
用于体内给药的本发明的纤溶酶原必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤溶酶原可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J.Biomed.Mater.Res.,15:167-277(1981)；Langer,Chem.Tech.,12:98-105(1982))或聚(乙烯醇)，聚交酯(美国专利3773919,EP 58,481)，L-谷氨酸与γ乙基-L-谷氨酸的共聚物(Sidman，等，Biopolymers 22:547(1983))，不 可降解的乙烯－乙烯乙酸酯(ethylene-vinyl acetate)(Langer，等，出处同上)，或可降解的乳酸-羟基乙酸共聚物如Lupron DepotTM(由乳酸-羟基乙酸共聚物和亮氨酰脯氨酸(leuprolide)乙酸酯组成的可注射的微球体)，以及聚D-(-)-3-羟丁酸。聚合物如乙烯-乙酸乙烯酯和乳酸-羟基乙酸能持续释放分子100天以上，而一些水凝胶释放蛋白的时间却较短。可以根据相关机理来设计使蛋白稳定的合理策略。例如，如果发现凝聚的机理是通过硫代二硫键互换而形成分子间S-S键，则可通过修饰巯基残基、从酸性溶液中冻干、控制湿度、采用合适的添加剂、和开发特定的聚合物基质组合物来实现稳定。
给药和剂量
可以通过不同方式，例如通过鼻腔吸入、雾化吸入、滴鼻液或滴眼液，静脉内，腹膜内、皮下、颅内、鞘内、动脉内(例如经由颈动脉)、直肠内、肌肉内、直肠给药来实现本发明药物组合物的施用。
用于胃肠外施用的制备物包括无菌水性或非水性溶液、悬浮液和乳剂。非水性溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油，和可注射有机酯，如油酸乙酯。水性载体包括水、醇性/水性溶液、乳剂或悬浮液，包括盐水和缓冲介质。胃肠外媒介物包含氯化钠溶液、林格氏右旋糖、右旋糖和氯化钠、或固定油。静脉内媒介物包含液体和营养补充物、电解质补充物，等等。也可以存在防腐剂和其他添加剂，诸如例如，抗微生物剂、抗氧化剂、螯合剂、和惰性气体，等等。
医务人员会基于各种临床因素确定剂量方案。如医学领域中公知的，任一患者的剂量取决于多种因素，包括患者的体型、体表面积、年龄、要施用的具体化合物、性别、施用次数和路径、总体健康、和同时施用的其它药物。本发明包含纤溶酶原的药物组合物的剂量范围可以例如为例如每天约0.0001至2000mg/kg，或约0.001至500mg/kg(例如0.02mg/kg，0.25mg/kg，0.5mg/kg，0.75mg/kg，10mg/kg，50mg/kg等等)受试者体重。例如，剂量可以是1mg/kg体重或50mg/kg体重或在1-50mg/kg的范围，或至少1mg/kg。高于或低于此例示性范围的剂量也涵盖在内，特别是考虑到上述的因素。上述范围中的中间剂量也包含在本发明的范围内。受试者可以每天、隔天、每周或根据通过经验分析确定的任何其它日程表施用此类 剂量。例示性的剂量日程表包括连续几天0.01-100mg/kg。在本发明的药物施用过程中需要实时评估治疗效果和安全性。
制品或药盒
本发明的一个实施方案涉及一种制品或药盒，其包含可用于治疗高血压及其相关病症的本发明纤溶酶原或纤溶酶。所述制品优选包括一个容器，标签或包装插页。适当的容器有瓶子，小瓶，注射器等。容器可由各种材料如玻璃或塑料制成。所述容器含有组合物，所述组合物可有效治疗本发明的疾病或病症并具有无菌入口(例如所述容器可为静脉内溶液包或小瓶，其含有可被皮下注射针穿透的塞子的)。所述组合物中至少一种活性剂为纤溶酶原/纤溶酶。所述容器上或所附的标签说明所述组合物用于治疗本发明所述高血压及其相关病症。所述制品可进一步包含含有可药用缓冲液的第二容器，诸如磷酸盐缓冲的盐水，林格氏溶液以及葡萄糖溶液。其可进一步包含从商业和使用者角度来看所需的其它物质，包括其它缓冲液，稀释剂，过滤物，针和注射器。此外，所述制品包含带有使用说明的包装插页，包括例如指示所述组合物的使用者将纤溶酶原组合物以及治疗伴随的疾病的其它药物给药患者。
实施例
以下所有实施例中使用的纤溶酶原均为人纤溶酶原，来自捐赠者血浆，基于如下文献描述的方法：KennethC Robbins,Louis Summaria,David Elwyn et al.Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin.Journal of Biological Chemistry,1965,240(1):541-550；Summaria L,Spitz F,Arzadon L et al.Isolation and characterization of the affinity chromatography forms of human Glu-and Lys-plasminogens and plasmins.J Biol Chem.1976 Jun 25；251(12):3693-9；HAGAN JJ,ABLONDI FB,DE RENZO EC.Purification and biochemical properties of human plasminogen.J Biol Chem.1960 Apr；235:1005-10，并进行工艺优化，从人血浆中纯化所得，其中人Lys-纤维蛋白溶酶原(Lys-纤溶酶原)和Glu-纤维蛋白溶酶原(Glu-纤溶酶原)>98％。
以下实施例1-5中以db/db小鼠(购自南京生物医药研究院，品系名称BKS.Cg-Dock7m+/-Leprdb/JNju)作为高血压模型，研究纤溶酶原对高血压 的作用。有多篇文献报道db/db小鼠在11-14周自发形成高血压，且随着周龄的增长，血压有持续升高的趋势 [24,25]。
实施例1纤溶酶原降低15-16周龄糖尿病小鼠高血压
取15-16周龄db/db雄性小鼠12只。在给药前一天，称重后开始检测基础血压，根据血压随机分为两组，分别为给溶媒PBS对照组和给纤溶酶原组，每组各6只。给纤溶酶原组小鼠按照2mg/0.2ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予21天。开始给药定为第1天，在第0、8、15、22天测量血压，每只小鼠连续测量五次，记录每次测量的收缩压和平均压。收缩压和平均压分别为五次测量所获得的收缩压和平均压数据的平均值。平均血压的计算方法是1/3的收缩压+2/3的舒张压。小鼠的血压使用无创血压仪(MRBP-M01,IITC Life science)进行检测。
结果显示，给药21天后PBS对照组小鼠血压与给药前相比并没有明显改变，而给纤溶酶原组小鼠的收缩压(图1A)和平均压(图1B)明显降低，均明显低于给溶媒PBS对照组，且统计差异显著(*表示P<0.05,**表示P<0.01)。说明纤溶酶原能够显著降低15-16周龄糖尿病小鼠的高血压。
实施例2纤溶酶原降低25-26周龄糖尿病小鼠高血压
取25-26周龄db/db雄性小鼠13只。在给药前一天，称重后开始检测基础血压，根据血压随机分为两组，给溶媒PBS对照组6只和给纤溶酶原组7只。给纤溶酶原组小鼠按照2mg/0.2ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予21天。开始给药定为第1天，在第0、8、15、22天测量血压，每只小鼠连续测量五次，记录每次测量的收缩压和平均压。收缩压和平均压分别为五次测量所获得的收缩压和平均压数据的平均值。小鼠的血压使用无创血压仪(MRBP-M01,IITC Life science)进行检测。
结果显示，给药21天后PBS对照组小鼠收缩压与给药前相比并没有明显改变，而给纤溶酶原组小鼠收缩压在给药7天已经开始显著降低，明显低于给溶媒PBS对照组，且统计差异显著(P＝0.019)；在给药的14、21天，两组小鼠收缩压同样差异显著(图2A)。在给药21天后，纤溶酶原 组小鼠平均压低于给溶媒PBS对照组，差异接近显著(P＝0.09)(图2B)。说明纤溶酶原能够显著降低25-26周龄糖尿病小鼠的高收缩压。
实施例3纤溶酶原降低糖尿病高血压模型小鼠血清血管紧张素II水平
取15-16周龄db/db雄性小鼠12只。在给药前一天，称重后开始检测基础血压，根据血压随机分为两组，分别为给溶媒PBS对照组和给纤溶酶原组，每组各6只。给纤溶酶原组小鼠按照2mg/0.2ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予28天。第29天摘除眼球取血，离心获得上清。按照血管紧张素II检测试剂盒(厂家：武汉华美生物工程有限公司，货号：CSB-E04495m)说明书操作检测血清血管紧张素II水平。
结果显示，给予纤溶酶原28天后，给纤溶酶原组血清血管紧张素II水平明显低于给溶媒PBS对照组(图3)。说明纤溶酶原能够降低糖尿病高血压模型小鼠血清血管紧张素II水平，从而纠正高血压。
实施例4纤溶酶原促进糖尿病高血压模型小鼠心脏损伤修复
取25-26周龄db/db雄性小鼠13只。在给药前一天，称重后开始检测基础血压，根据血压随机分为两组，给溶媒PBS对照组6只和给纤溶酶原组7只。给纤溶酶原组小鼠按照2mg/0.2ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予28天。第29天处死小鼠后取材心脏在4％多聚甲醛固定液中固定24小时。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为4μm，切片脱蜡复水并用苏木素和伊红染色(H&E染色)，1％盐酸酒精分化后氨水返蓝并酒精梯度脱水封片，切片在200倍光学显微镜下观察。
结果显示，相比于给溶媒PBS对照组(图4A)，给纤溶酶原组(图4B)小鼠心肌细胞间更加紧密，排列更加规则的。说明纤溶酶原能够明显改善糖尿病高血压模型小鼠心脏损伤。
实施例5纤溶酶原减轻糖尿病高血压模型小鼠肾脏纤维化
取25-26周龄db/db雄性小鼠13只。在给药前一天，称重后开始检测基础血压，根据血压随机分为两组，给溶媒PBS对照组6只和给纤溶酶原组7只。给纤溶酶原组小鼠按照2mg/0.2ml/只/天尾静脉注射给予纤溶酶 原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予28天。第29天处死小鼠后取材肾脏在4％多聚甲醛固定液中固定24小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2分钟，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
天狼星红染色可使胶原持久染色，作为病理切片特殊染色方法，天狼星红染色可以特异显示胶原组织。
结果显示，给予纤溶酶原28天后，给纤溶酶原组(图5B)肾脏胶原纤维沉积(箭头标识)明显少于给溶媒PBS对照组(图5A)。说明纤溶酶原能够明显降低糖尿病高血压模型小鼠肾脏纤维化。
实施例6纤溶酶原升高血管紧张素II诱导高血压模型小鼠血清SOD水平
取21-22周Plg+/+雄性小鼠14只测量血压、体重，根据血压、体重将小鼠随机分为3组，空白对照组4只，给溶媒PBS对照组和给纤溶酶原组各5只。空白对照组小鼠皮下注射生理盐水0.1ml，给溶媒PBS对照组和给纤溶酶原组小鼠皮下注射血管紧张素II 1mg/kg/只/天，连续注射造模14天 [26]。造模开始的同时开始给药纤溶酶原或溶媒，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予14天；空白对照组小鼠不做给药处理。开始造模给药定为第1天，第15天摘除眼球取血，离心获得上清，用以检测血清超氧化物歧化酶(Super Oxide Dismutase,SOD)水平。SOD检测采用SOD检测试剂盒(南京建成生物工程研究所，货号A001-1)，按照说明书所示方法进行检测。
SOD是机体清除自由基的重要酶系统，能清除超氧阴离子(o2-)，而o2-则是氧自由基的启动自由基，研究显示SOD对高血压具有保护效应，高血压患者SOD水平降低 [27]。
结果显示，空白对照组血清中具有一定水平的SOD，给溶媒PBS对照组血清SOD水平明显降低，给纤溶酶原组血清SOD水平明显高于给溶媒 PBS对照组，且统计差异显著(*表示P<0.05)(图6)。说明纤溶酶原能够增强机体清除自由基的能力。
实施例7纤溶酶原减轻血管紧张素II诱导高血压模型小鼠肾脏纤维化
取21-22周Plg+/+雄性小鼠14只测量血压、体重，根据血压、体重将小鼠随机分为3组，空白对照组4只，给溶媒PBS对照组和给纤溶酶原组各5只。空白对照组小鼠皮下注射生理盐水0.1ml，给溶媒PBS对照组和给纤溶酶原组小鼠皮下注射血管紧张素II 1mg/kg/只/天，连续皮下注射造模14天 [26]。造模开始的同时开始给药纤溶酶原或溶媒，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予14天；空白对照组小鼠不做给药处理。开始造模给药定为第1天，在第15天处死小鼠后取材肾脏在4％多聚甲醛固定液中固定24小时。固定后的肾脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2分钟，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，空白对照组(图7A)小鼠肾脏无明显的胶原纤维沉积，给纤溶酶原组(图7C)胶原纤维的沉积(箭头标识)明显少于给溶媒PBS对照组(图7B)。说明纤溶酶原能够明显减少血管紧张素II诱导的高血压模型小鼠肾脏纤维化，改善高血压所致的肾脏病变。
实施例8纤溶酶原减轻血管紧张素II诱导高血压模型小鼠心脏纤维化
取21-22周Plg +/+雄性小鼠14只测量血压、体重，根据血压、体重将小鼠随机分为3组，空白对照组4只，给溶媒PBS对照组和给纤溶酶原组各5只。空白对照组小鼠皮下注射生理盐水0.1ml，给溶媒PBS对照组和给纤溶酶原组小鼠皮下注射血管紧张素II 1mg/kg/只/天，连续皮下注射造模14天 [26]。造模开始的同时开始给药纤溶酶原或溶媒，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予14天；空白对照组小鼠不做给药处理。开始造模给药定为第1天，在第15天处死小鼠后取材心脏在4％多聚甲醛固定液中固定24小时。固定后的心脏组织经酒精梯度脱水和二甲苯 透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2分钟，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，空白对照组(图8A)小鼠心脏无明显的胶原纤维沉积，给纤溶酶原组(图8C)胶原纤维的沉积(箭头标识)明显少于给溶媒PBS对照组(图8B)。说明纤溶酶原能够明显减少血管紧张素II诱导的高血压模型小鼠心脏纤维化，改善高血压所致的心脏病变。
实施例9纤溶酶原减轻野百合碱诱导肺动脉高压模型小鼠肺纤维化
取12周C57雄性小鼠12只称重、测量血压，根据血压随机分为2组，空白对照组4只小鼠，模型组8只小鼠。空白对照组小鼠尾静脉注射生理盐水100μl，模型组小鼠尾静脉单次注射野百合碱60mg/kg/只，连续注射3天，小鼠正常饮食 [28,29]。3天后测量血压，并根据血压将模型组小鼠随机分为两组，给溶媒PBS对照组和给纤溶酶原组各4只。给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予28天；空白对照组小鼠不做给药处理。开始造模给药定为第1天，在第29天处死小鼠后取材肺在4％多聚甲醛固定液中固定24小时。固定后的肺组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2分钟，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
野百合碱是双吡咯类生物碱，在肝脏内经P450单氧化酶转化后，经由血液循环到达肺脏，可引起肺血管不可逆性损伤。肺血管内皮细胞被认为是野百合碱的靶细胞，内皮细胞损伤在肺血管重建过程中起着关键作用 [28,29]。
结果显示，空白对照组小鼠肺基本无胶原蛋白沉积(图9A)，给纤溶酶原组(图9C)小鼠肺组织胶原蛋白沉积(箭头标识)明显少于给溶媒PBS对照组(图9B)。说明纤溶酶原能够明显减轻野百合碱诱导的肺动脉高压模型小鼠肺的纤维化。
实施例10纤溶酶原减轻野百合碱诱导肺动脉高压模型小鼠心脏纤维化
取12周C57雄性小鼠12只称重、测量血压，根据血压随机分为2组，空白对照组4只小鼠，模型组8只小鼠。空白对照组小鼠尾静脉注射生理盐水100μl，模型组小鼠尾静脉单次注射野百合碱60mg/kg/只，连续注射3天，小鼠正常饮食 [28,29]。3天后测量血压，并根据血压将模型组小鼠随机分为两组，给溶媒PBS对照组和给纤溶酶原组各4只。给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予28天；空白对照组小鼠不做给药处理。开始造模给药定为第1天，在第29天处死小鼠后取材心脏在4％多聚甲醛固定液中固定24小时。固定后的心脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2分钟，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，空白对照组小鼠心脏基本无胶原蛋白沉积(图10A)，给纤溶酶原组(图10C)小鼠心脏胶原蛋白沉积(箭头标识)明显少于给溶媒PBS对照组(图10B)。说明纤溶酶原能够明显减轻野百合碱诱导的肺动脉高压模型小鼠心脏的纤维化。
实施例11纤溶酶原减轻野百合碱诱导肺动脉高压模型小鼠肾脏纤维化
取12周C57雄性小鼠12只称重、测量血压，根据血压随机分为2组，空白对照组4只小鼠，模型组8只小鼠。空白对照组小鼠尾静脉注射生理盐水100μl，模型组小鼠尾静脉单次注射野百合碱60mg/kg/只，连续注射3天，小鼠正常饮食 [28,29]。3天后测量血压，并根据血压将模型组小鼠随机分为两组，给溶媒PBS对照组和给纤溶酶原组各4只。给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予28天；空白对照组小鼠不做给药处理。开始造模给药定为第1天，在第29天处死小鼠后取材肾脏在4％多聚甲醛固定液中固定24小时。固定后的肾脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2分钟，苏木素 染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，空白对照组小鼠肾脏基本无胶原蛋白沉积(图11A)，给纤溶酶原组(图11C)小鼠肾脏胶原蛋白沉积(箭头标识)明显少于给溶媒PBS对照组(图11B)。说明纤溶酶原能够明显减轻野百合碱诱导的肺动脉高压模型小鼠肾脏的纤维化。
实施例12纤溶酶原减轻野百合碱诱导肺动脉高压模型小鼠肝脏纤维化
取12周C57雄性小鼠12只称重、测量血压，根据血压随机分为2组，空白对照组4只小鼠，模型组8只小鼠。空白对照组小鼠尾静脉注射生理盐水100μl，模型组小鼠尾静脉单次注射野百合碱60mg/kg/只，连续注射3天，小鼠正常饮食 [28,29]。3天后测量血压，并根据血压将模型组小鼠随机分为两组，给溶媒PBS对照组和给纤溶酶原组各4只。给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予28天；空白对照组小鼠不做给药处理。开始造模给药定为第1天，在第29天处死小鼠后取材肝脏在4％多聚甲醛固定液中固定24小时。固定后的肝脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2分钟，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，空白对照组小鼠肝脏基本无胶原蛋白沉积(图12A)，给纤溶酶原组(图12C)小鼠肝脏胶原蛋白沉积(箭头标识)明显少于给溶媒PBS对照组(图12B)。说明纤溶酶原能够明显减轻野百合碱诱导的肺动脉高压模型小鼠肝脏的纤维化。
实施例13纤溶酶原促进缺血肾萎缩小鼠血压升高
取6～7周C57雄性小鼠18只，随机分为两组，空白对照组4只，模型组14只。空白对照组小鼠，不做任何处理。模型组小鼠腹腔注射3％戊巴比妥钠(50mg/kg)麻醉，通过外科手术方法在腹中线切开皮肤、筋膜、及肌肉层，翻开肠道、胰腺等脏器组织，暴露术野，钝性分离左侧肾脏的肾盂部位的主动脉与主静脉，用5号丝线对分离的血管进行结扎，确保结扎完 全后，把肠道、胰腺等脏器组织放回对应生理位置上，通过外科手术方法连续缝合肌肉层，结节缝合皮肤，缝合后，用医用碘伏对创口进行消毒，把动物置于37℃的加热垫，观察等候其恢复清醒，在动物没完全恢复清醒不能给予饮水。肌肉注射抗生素(5000U/只)，皮下注射止痛药(2mg/kg)，连续注射3天。手术结扎14天后，模型组小鼠腹腔注射3％戊巴比妥钠麻醉，开腹，松开左侧肾血管结扎线，迅速缝合腹腔，用医用碘伏对创口进行消毒，把动物置于37℃的加热垫，观察等候其清醒，在动物没完全清醒不能给予饮水，肌肉注射抗生素(5000U/只)，皮下注射止痛药(2mg/kg)，连续注射3天 [30]。拆线后全部小鼠称重，模型组小鼠随机分为两组，给纤溶酶原组和PBS对照组，每组各7只，并开始给药。开始给药定为第1天，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射给予纤溶酶原，给溶媒PBS对照组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予14天。并在第15天使用无创血压仪(MRBP-M01,IITC Life science)检测小鼠血压(收缩压和平均压)。
结果显示，给纤溶酶原组和空白对照收缩压和平均压明显高于PBS对照组血压，并且纤溶酶原组和PBS对照组之间有统计差异显著(*表示P<0.05)(图13)。说明纤溶酶原能够促进肾萎缩所致低血压恢复正常水平。
实施例14纤溶酶原降低血管紧张素II诱导的高血压模型小鼠血压
取8～9周C57雄性小鼠21只，随机分为三组，空白对照组、溶媒组和给药组，每组7只。空白对照组小鼠，不做任何处理。给药组和溶媒组，所有小鼠按1mg/kg/只小鼠0.25mg/ml的剂量皮下注射血管紧张素II溶液，每天2次，中间间隔8小时，连续造模6天 [31]。第一次注射血管紧张素II造模2小时后，给药组及溶媒组开始给药，即为第1天，给药组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原，溶媒组小鼠尾静脉注射相同体积溶媒，持续给药5天，空白对照组不做给药处理。第6天，测量所有小鼠血压。每只小鼠连续测量五次，记录每次测量的收缩压和平均压。收缩压和平均压分别为五次测量所获得的收缩压和平均压数据的平均值。小鼠的血压使用无创血压仪(MRBP-M01,IITC Life science)进行检测。
结果显示，溶媒组小鼠平均压和收缩压明显升高，给药组小鼠的平均压和收缩压明显低于溶媒组小鼠，且统计差异显著(*表示P<0.05，**表示P<0.01)(图14)。表明纤溶酶原能够降低高血压模型小鼠血压。
实施例15纤溶酶原促进糖尿病高血压模型小鼠血管紧张素转化酶2水平增加
取24-25周龄db/db雄性小鼠12只。在给药前一天，称重后开始检测基础血压，根据血压随机分为两组，分别为溶媒组和给药组，每组各6只。给药组小鼠按照2mg/0.2ml/只/天尾静脉注射给予纤溶酶原，溶媒组小鼠尾静脉注射给予相同体积的PBS溶液，连续给予28天。第29天摘除眼球取血，离心获得上清。按照血管紧张素转化酶2(ACE2)检测试剂盒(厂家：武汉华美生物工程有限公司，货号：CSB-E17204m)说明书操作检测血清ACE2水平。
血管紧张素转化酶2(ACE2)是肾素血管紧张素系统重要的负向调节酶，是体内降解血管紧张素II(AngII)的主要途径。在ACE2作用下AngII转变成Angl-7；此外，ACE2也可催化AngI降解成Ang(1-9)，后者经ACE转化为Angl-7。ACE2对AngII的亲和力是对Ang I的400倍，催化AngII转化为Ang(1-7)是ACE2的主要功能。ACE2的生理作用与高血压，心脏功能，心脏功能和糖尿病有关，并作为严重急性呼吸系统综合症冠状病毒的受体 [32]。
结果显示，给药组小鼠血清ACE2水平明显高于溶媒组，且统计差异接近显著(P＝0.057)(图15)。提示纤溶酶原能够促进糖尿病高血压模型小鼠血清ACE2水平增加。
实施例16纤溶酶原降低血管紧张素II诱导的高血压模型小鼠血清ACE2水平
取8-9周龄的雄性C57小鼠21只，随机分为三组，空白对照组7只，溶媒组7只和给药组7只。给药组和溶媒组小鼠分组完成后，皮下注射1mg/kg/只小鼠0.25mg/ml血管紧张素II溶液，每天2次，间隔8小时，连续造模7天。血管紧张素II溶液配制：先取血管紧张素II粉末(货号：A9292-10mg，厂商：北京7索莱宝生物科技有限公司)溶于1ml去离子水中，配制成10mg/ml溶液，分装，避免反复冻融，使用时稀释40倍，配制 成0.25mg/ml，现配现用，原液于-20℃保存。第一次注射造模2小时后，给药组及溶媒组开始给药，即为第1天，给药组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原，溶媒组小鼠尾静脉注射相同体积溶媒，持续给药7天，空白对照组不做给药处理。第8天摘除眼球取血，离心获得上清。按照血管紧张素转化酶2(ACE2)检测试剂盒(厂家：上海希美化学有限公司，货号：CSB-E17204m)说明书操作检测血清ACE2水平。
结果显示，空白对照组小鼠血液中具有一定水平的ACE2，溶媒组小鼠血液中ACE2水平明显高于空白对照组小鼠，给药组小鼠血液中ACE2水平明显低于溶媒组小鼠，且统计差异极为显著(**表示P<0.01)(图16)。提示纤溶酶原能够促进血管紧张素II诱导的高血压模型小鼠血清ACE2水平降低。
实施例17纤溶酶原降低血管紧张素II诱导的高血压模型小鼠血清ACE水平
取8-9周龄的雄性C57小鼠21只，随机分为三组，空白对照组7只，溶媒组7只和给药组7只。给药组和溶媒组小鼠分组完成后，皮下注射1mg/kg/只小鼠0.25mg/ml血管紧张素II溶液，每天2次，间隔8小时，连续造模7天。血管紧张素II溶液配制：先取血管紧张素II粉末(货号：A9292-10mg，厂商：北京7索莱宝生物科技有限公司)溶于1ml去离子水中，配制成10mg/ml溶液，分装，避免反复冻融，使用时稀释40倍，配制成0.25mg/ml，现配现用，原液于-20℃保存。第一次注射造模2小时后，给药组及溶媒组开始给药，即为第1天，给药组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原，溶媒组小鼠尾静脉注射相同体积溶媒，持续给药7天，空白对照组不做给药处理。第8天摘除眼球取血，离心获得上清。按照血管紧张素转化酶(ACE)检测试剂盒(厂家：武汉华美生物工程有限公司，货号：CSB-E04492m)说明书操作检测血清ACE水平。
血管紧张素转化酶(ACE)是肾素血管紧张素系统中的关键酶，在AngII产生过程中起着关键作用 [32]。
结果显示，空白对照组小鼠血液中具有一定水平的ACE，溶媒组小鼠血液中ACE水平明显高于空白对照组小鼠，给药组小鼠血液中ACE水平 明显低于溶媒组小鼠，且统计差异接近显著(P＝0.051)(图17)。提示纤溶酶原能够促进AngII诱导的高血压模型小鼠血清ACE水平降低。
实施例18纤溶酶原对临床自愿者的治疗效果
以下是临床自愿者数据，所有患者均签署知情同意书自愿用药，并获得医院伦理委员会批准。以下使用的纤溶酶原用生理盐水稀释，浓度为5或10mg/ml。雾化用药时，纤溶酶原溶液用雾化器雾化后使用。
案例1
患者，女，72岁，有30多年高血压病史，收缩压160mmHg、偶尔180mmHg，服用降压药后，血压控制在130/80mmHg左右。患者每天静脉注射人纤溶酶原，每日一次，起始剂量为50mg/次，之后逐渐增加至135mg/次。用药后第6天开始，患者收缩压下降至141mmHg，至11天期间血压波动不稳定(正常异常交替)；第12天后，血压下降，且一周内保持正常稳定。患者在接受上述纤溶酶原静脉注射治疗期间，没有同时服用任何降压药。用药期间患者血压情况见图18，说明纤溶酶原能够降低高血压患者的血压，使其接近正常水平。
案例2
患者，女，79岁，冠心病20余年，2014年发生脑梗。糖尿病史23年，长期注射胰岛素。诊断为高血压5年，服用降压药后，晨起血压130-150/50-60mmHg，下午150/60mmHg左右。静脉注射给予患者人纤溶酶原，每日一次，剂量为100mg/次。
用药后第13天患者血压135/54mmHg，后期在不用降压药的情况下血压也稳定在正常水平。患者用药期间血压情况见图19。
案例3
患者，女，76岁，糖尿病史20年，高血压史10年，血压最高时168/70mmHg，口服硝苯地平缓释片，血压控制在114/55mmHg左右，舒张压低于正常值。通过静脉注射对患者施予人纤溶酶原，每日1-2次，每日用药量35-75mg。
用药后舒张压从低于60mmHg达到正常值，治疗期间血压正常稳定；治疗结束后，降压药减半，血压基本维持在130-140/64-76mmHg。用药期间患者血压情况见图20。
纤溶酶原能够促进高血压患者血压恢复正常，可减少降压药的使用剂量。
案例4
患者，男，57岁，入院复查，发现腹主动脉增粗，诊断为主动脉夹层。医院建议手术治疗。有心脏病家族史，高血压病史10余年。患者对造影剂过敏，身上有湿疹和荨麻疹。血压早晚偏高，收缩压最高时可达200mmHg，压差较大，可达80mmHg以上。睡眠状况不佳，入睡慢，夜间起夜3-4次，影响睡眠。
通过静脉注射给予人纤溶酶原，每天1-2次，用药量50-150mg/天。
第五天用药后早晚血压开始下降，压差减少。患者自诉，先前在吃降压药的情况下，也没有出现血压下降至140/75mmHg以下的情况，这是第一次出现好转。第六天用药后早晚血压维持在136/73mmHg左右。
第七天用药后早晚血压维持平稳，血压在130/70mmHg左右。用药期间患者血压情况见图21。
此外，用药第五天，患者的睡眠明显好转，夜间无起夜。湿疹和荨麻疹症状也明显好转。此后，患者症状持续好转。
案例5
患者，78岁，诊断为新型冠状肺炎危重型，入院治疗期间发现血压升至150/78mmhg，原无高血压病史，也没有就诊检查过。通过雾化吸入给予患者人纤溶酶原，每日2次，每次10mg。1天后血压恢复正常。用药期间患者血压情况见表1和表2。
表明人纤溶酶原能够促进新型冠状病毒肺炎危重型患者高血压恢复正常。
表1新冠状病毒肺炎危重型患者用药第一天血压检测结果
项目 首次雾化前 二次雾化前 雾化后
血压(mmHg)   150/78 150/78
表2新冠状病毒肺炎危重型患者用药第二天血压检测结果
项目 首次雾化前后 二次雾化前 雾化后
血压(mmHg) 129/61 130/64 130/64
案例6
患者诊断为新冠状病毒肺炎危重型，除相关临床症状外，血压139/94mmHg，舒张压高于正常值。通过雾化吸入给予患者纤溶酶原，每日2次，每次10mg。治疗1次后舒张压即恢复正常。用药期间患者血压情况见表3。
纤溶酶原能够促进新型冠状病毒肺炎危重型患者高血压恢复正常。
表3新冠状病毒肺炎危重型患者用药期间血压检测结果
项目 首次治疗前 二次治疗前 治疗后1h
血压(mmHg) 139/94 130/87 120/83
案例7
患者，女，67岁，患者卧床，因气管切开不能讲话，无糖尿病、心脏病、高血压病史，无过敏史，帕金森6年，不能行走4年，卧床3年，导尿留置1.5年，患者不能自主翻身，可以自己简单活动肢体，每天低流量持续吸氧。血压80/45mmHg。诊断为低血压和帕金森。通过静脉注射给予患者纤溶酶原，每日一次，用药剂量50-250mg/天，起始剂量50mg/天，后逐渐增加。连续用药7天，第9天、第11天、第13天继续用药，第8、10、12天停药观察。
治疗效果：
用药后血压呈上升趋势，用药第3天第一次出现正常血压。用药第9天后血压保持正常，停药后血压保持在95/70mmHg。患者用药期间血压测量结果见图22。
表明给药纤溶酶原能够促进低血压患者血压恢复。
案例8
患者，女，50岁，低血压持续很多年，平日血压偏低，80/60mmHg，早上起床后有时会头晕。诊断为低血压病。治疗方法：静脉注射，每日一次，用药剂量100mg。
治疗结果：
用药后患者血压在用药后第二天即达到正常，一周后达到110/70mmHg左右。患者用药期间血压测量结果见图23。
表明给药纤溶酶原能够促进低血压患者血压恢复。
参考文献
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序列表：
序列1：
Figure PCTCN2021078200-appb-000001
Figure PCTCN2021078200-appb-000002
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Figure PCTCN2021078200-appb-000003
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Figure PCTCN2021078200-appb-000004
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Figure PCTCN2021078200-appb-000005
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Figure PCTCN2021078200-appb-000006
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Figure PCTCN2021078200-appb-000007
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Figure PCTCN2021078200-appb-000008
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Figure PCTCN2021078200-appb-000009
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Figure PCTCN2021078200-appb-000010
Figure PCTCN2021078200-appb-000011
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Figure PCTCN2021078200-appb-000012
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Figure PCTCN2021078200-appb-000013
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Figure PCTCN2021078200-appb-000014
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Figure PCTCN2021078200-appb-000015
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Figure PCTCN2021078200-appb-000016

Claims (18)

  1. 一种双向治疗血压异常的方法，包括给药患血压异常的受试者有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂，其中，所述血压异常为高血压或低血压，并且所述化合物使患高血压或低血压的受试者的血压趋于正常、接近正常或恢复至正常。
  2. 权利要求1所述的方法，其中所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro-plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。
  3. 权利要求1的方法，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抑制剂，例如抗体。
  4. 权利要求1-3任一项的方法，其中所述化合物进一步缓解血压异常导致的组织器官损伤或并发症。
  5. 权利要求4的方法，其中所述血压异常导致的并发症为高血压导致的并发症，该并发症选自如下的一种或多种：心律失常、心力衰竭、冠心病、脑出血、脑血栓、脑梗塞、高血压肾病、肾衰、尿毒症、肝硬化、肺动脉高压、肺纤维化和微血栓。
  6. 权利要求4的方法，其中所述血压异常导致的并发症为低血压导致的并发症，该并发症选自如下的一种或多种：心脏供血不足、心绞痛、休克、脑供血不足、晕厥、脑梗死、肾脏供血不足、少尿、蛋白尿和肾功能不全。
  7. 权利要求1-6任一项的方法，其中所述化合物具有选自如下的一项或多项作用：降低患高血压的受试者的血压、降低患高血压的受试者的血清血管紧张素II水平、调节受试者ACE或ACE2的水平、促进患高血压的受试者的血压恢复至正常水平、升高患低血压的受试者的血压、促进患低血压的受试者的血压恢复至正常水平、减轻血压异常导致的组织器官损伤、促进损伤的组织器官的修复、减轻组织器官纤维化和促进自由基的清除。
  8. 权利要求7的方法，其中所述促进损伤的组织器官的修复为促进损伤的心脏组织、脑组织、肺组织、肾脏组织或肝组织的结构或功能的恢复。
  9. 权利要求7或8方法，其中所述减轻组织器官纤维化为减轻心脏组织、肺组织、肾脏组织或肝组织的纤维化。
  10. 权利要求1-9任一项的方法，其中所述化合物通过促进SOD的产生消除自由基。
  11. 权利要求1-10任一项的方法，其中所述化合物为纤溶酶原。
  12. 权利要求1-11任一项的方法，其中所述纤溶酶原与序列2具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且具有纤溶酶原的蛋白水解活性或赖氨酸结合活性。
  13. 权利要求1-12任一项的方法，所述纤溶酶原包含序列14所示的纤溶酶原活性片段、具有纤溶酶原的蛋白水解活性。
  14. 权利要求1-13任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原蛋白水解活性的变体。
  15. 权利要求1-14任一项的方法，所述纤溶酶原为天然或合成的人纤溶酶原、或其保留纤溶酶原的蛋白水解活性或赖氨酸结合活性的变体或片段。
  16. 权利要求1-15任一项的方法，其中所述化合物与一种或多种其他治疗方法或药物联合使用。
  17. 权利要求16的方法，其中所述其他药物为治疗血压异常病症受试者血压异常病症外的其他疾病的药物。
  18. 权利要求1-17任一项的方法，其中所述化合物通过鼻腔吸入、雾化 吸入、滴鼻液、滴耳液或滴眼液形式给药，静脉内、腹膜内、皮下、颅内、鞘内、动脉内、直肠内和/或肌肉内给药。
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CN108210917A (zh) * 2016-12-15 2018-06-29 深圳瑞健生命科学研究院有限公司 一种预防和治疗肥胖症的方法和药物
CN108210895A (zh) * 2016-12-15 2018-06-29 深圳瑞健生命科学研究院有限公司 预防动脉粥样硬化及其并发症的药物及其用途
CN108210915A (zh) * 2016-12-15 2018-06-29 深圳瑞健生命科学研究院有限公司 改善心脏病变的药物及其用途
CN108210897A (zh) * 2016-12-15 2018-06-29 深圳瑞健生命科学研究院有限公司 预防和治疗肺纤维化的药物及其用途
CN109125715A (zh) * 2017-06-19 2019-01-04 深圳瑞健生命科学研究院有限公司 一种调控glp-1/glp-1r的方法和药物

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