WO2022037687A1 - 一种治疗肿瘤的方法和药物 - Google Patents

一种治疗肿瘤的方法和药物 Download PDF

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WO2022037687A1
WO2022037687A1 PCT/CN2021/113850 CN2021113850W WO2022037687A1 WO 2022037687 A1 WO2022037687 A1 WO 2022037687A1 CN 2021113850 W CN2021113850 W CN 2021113850W WO 2022037687 A1 WO2022037687 A1 WO 2022037687A1
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plasminogen
cancer
tumor
mice
group
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French (fr)
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李季男
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Talengen International Ltd
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Talengen International Ltd
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Priority to CN202180050515.0A priority Critical patent/CN115885049A/zh
Priority to US18/022,084 priority patent/US20230302102A1/en
Priority to KR1020237009007A priority patent/KR20230052929A/ko
Priority to JP2023512785A priority patent/JP7724565B2/ja
Priority to CA3190013A priority patent/CA3190013A1/en
Priority to EP21857775.7A priority patent/EP4190911A4/en
Publication of WO2022037687A1 publication Critical patent/WO2022037687A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 present invention relates to the role of components of plasminogen activation pathway, such as plasminogen, in preventing and treating tumors, thereby providing a new therapeutic strategy for preventing and/or treating tumors.
  • Malignant tumor is a disease that seriously threatens human health.
  • cancer is the leading cause of death in developed countries, accounting for 21.6% of all deaths.
  • WHO statistics in the past few years, the number of patients who died of cancer in the world has reached more than 7 million every year, which is very close to the number of cardiovascular diseases, and is expected to surpass the number of cardiovascular diseases and become the world's largest cause of death.
  • the incidence of tumors remains high and is on the rise, which has seriously threatened life and health.
  • the pathogenesis of tumors is complex, the effect of tumor treatment is poor, the recurrence and metastasis rate is high, and the side effects of tumor treatment are large. Therefore, its prevention and treatment It is also difficult to study.
  • the present invention finds that the components of the plasminogen activation pathway, such as plasminogen, have obvious inhibitory effect on the growth of tumors, and may become a new approach for the prevention and treatment of tumors.
  • the present application relates to a method of treating tumors, comprising administering to a subject an effective amount of one or more compounds selected from the group consisting of components of the plasminogen activation pathway, capable of directly activating fibrinolysis Zymogens or compounds that activate plasminogen indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, are capable of upregulating plasminogen or Antagonists of plasminogen activator-expressed compounds, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and fibrinolysis inhibitors.
  • one or more compounds selected from the group consisting of components of the plasminogen activation pathway capable of directly activating fibrinolysis Zymogens or compounds that activate plasminogen indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, are capable of upregulating plasminogen or Antagonists of plasminogen activ
  • the present application also relates to the use of one or more compounds selected from the group consisting of components of the plasminogen activation pathway, capable of directly activating plasminogen or by activating fibers in the preparation of a medicament for the treatment of tumors.
  • Compounds that indirectly activate plasminogen by activating upstream components of the protein lysinogen pathway compounds that mimic the activity of plasminogen or plasmin, and are capable of upregulating plasminogen or plasminogen activation
  • the present application also relates to the use of one or more compounds selected from the group consisting of or a pharmaceutical composition comprising one or more compounds selected from the group consisting of: a component of the plasminogen activation pathway , Compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, Compounds that up-regulate plasminogen or plasminogen activator expression, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.
  • a component of the plasminogen activation pathway Compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, Compounds that up-regulate plasminogen or plasminogen activator expression, plasminogen analogs
  • the components of the plasminogen activation pathway are selected from the group consisting of plasminogen, recombinant human plasmin, Lys-plasminogen, Glu-plasminogen, fiber Protease, plasminogen and plasmin variants and analogs containing plasminogen and one or more kringle domains and protease domains of plasmin, small plasminogen (mini-plasminogen), small plasmin (mini-plasmin), micro-plasminogen (micro-plasminogen), micro-plasmin (micro-plasmin), delta-plasminogen, delta-plasmin (delta-plasmin), plasminogen activator, tPA and uPA.
  • the antagonist of the fibrinolysis inhibitor is an inhibitor of PAI-1, complement C1 inhibitor, alpha2 antiplasmin, or alpha2 macroglobulin, eg, an antibody.
  • the compound is plasminogen.
  • the present application provides a method of treating a tumor comprising administering an effective amount of plasminogen to a tumor subject.
  • the tumor is a malignant tumor.
  • the tumor is cancer.
  • the tumor is a solid tumor.
  • the tumor is a tumor of the digestive system or a tumor of the respiratory system.
  • the tumor is selected from one or more of the following: oral cancer, esophageal cancer, gastric cancer, small bowel cancer, colon cancer, rectal cancer, lung cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, gallbladder cancer, Non-Small Cell Lung (NSCL) Cancer, Bronchoalveolar Cell Lung Cancer, Breast Cancer, Ovarian Cancer, Cervical Cancer, Fallopian Tube Cancer, Endometrial Cancer, Vaginal Cancer, Prostate Cancer, Urethral Cancer, Penile Cancer, Kidney Cancer, Ureteral Cancer, Kidney Cancer Cell carcinoma, renal pelvis, bladder, head and neck, skin, melanoma, mesothelioma, bone, thyroid, parathyroid, adrenal, soft tissue sar
  • the plasminogen described herein has one or more effects selected from the group consisting of: reducing tumor volume, improving general survival of tumor subjects, delaying tumor progression, inhibiting tumor cell proliferation growth, improve survival rate, prolong the survival of tumor subjects, reduce cancer pain, inhibit tumor angiogenesis, promote tumor cell necrosis or apoptosis, promote anti-tumor immune response, regulate the expression of tumor-associated antigens or lymphocyte surface molecules, Reduce the damage of cancer cells to tissues and organs, and promote the recovery of tumor tissue structure or function.
  • the subject's plasminogen level or plasminogen level in tumor tissue or tissue without tumor is higher, equal to or lower than healthy subject plasminogen level or plasminogen levels in corresponding tissues without tumors.
  • the subject's fibrin level or fibrin level in tumor tissue or in tumor-free tissue is greater than, equal to, or lower than a healthy subject's fibrin level or the corresponding tumor-free tissue Fibrin levels in tissues.
  • the plasminogen is administered in combination with one or more selected from the group consisting of chemotherapy, radiation therapy, surgical therapy, cell therapy, and immunotherapy.
  • the chemotherapeutic drugs include, for example, alkylating agents, antimetabolites (eg, folate analogs), pyrimidine analogs, purine analogs, antimitotic drugs, antibiotics, cytokines, platinum coordination complexes, substitutions urea, hormones, adrenal corticosteroid antagonists, anti-estrogens, anti-androgens, etc.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen, or their conserved Substitution variant.
  • the plasminogen is a plasminogen protein comprising a serine protease domain and/or a lysine binding domain.
  • the plasminogen comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 14, and plasminogen protein with protein level activity.
  • the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with sequence 2, 6, 8, 10 or 12 % sequence identity and still have plasminogen activity.
  • the plasminogen described herein is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen, or their variants that retain plasminogen activity.
  • the plasminogen is based on sequence 2, 6, 8, 10 or 12 with additions, deletions and/or substitutions of 1-100, 1-90, 1-80, 1-70 , 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1 -3, 1-2, 1 amino acid protein that still has plasminogen activity.
  • the plasminogen is natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • the plasminogen is a human plasminogen ortholog from a primate or rodent or a variant or fragment thereof that still retains plasminogen activity.
  • the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is human native plasminogen.
  • the subject is a human. In some embodiments, the subject is low, deficient or deficient in plasminogen. In some embodiments, the deficiency, deficiency or deletion is congenital, secondary and/or local.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and plasminogen for use in the aforementioned methods.
  • the kit may be a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the aforementioned methods and (ii) for delivering the plasminogen to the the subjects' means.
  • the member is a syringe or vial.
  • the kit further comprises a label or instructions for administering the plasminogen to the subject to perform any of the foregoing methods.
  • the article of manufacture comprises: a container comprising a label; and comprising (i) plasminogen or a pharmaceutical composition comprising plasminogen for use in the aforementioned methods, wherein the label indicates that the plasminogen is to be The lysinogen or composition is administered to the subject to perform any of the foregoing methods.
  • the kit or article of manufacture further comprises one or more additional components or containers that contain other medicaments.
  • the other drug is an antineoplastic drug.
  • the plasminogen can be treated by systemic or topical administration, preferably by intravenous, intramuscular, subcutaneous administration of plasminogen. In some embodiments of the foregoing methods, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer.
  • the plasminogen is administered at 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg per day /kg, 10-100mg/kg (calculated per kilogram of body weight) or 0.0001-2000mg/cm2, 0.001-800mg/cm2, 0.01-600mg/cm2, 0.1-400mg/cm2, 1-200mg/cm2, 1-100mg/ cm2, doses of 10-100 mg/cm2 (calculated per square centimeter of body surface area) are administered, preferably repeated at least once, preferably at least daily.
  • the present invention explicitly covers all combinations of technical features belonging to the embodiments of the present invention, and these combined technical solutions have been explicitly disclosed in this application, just as the above-mentioned technical solutions have been separately and explicitly disclosed.
  • the present invention also explicitly covers the combination of each embodiment and its elements, and the technical solution after the combination is explicitly disclosed herein.
  • Figure 2 Calculation results of tumor volume changes after plasminogen was administered to the tail vein of colon cancer model mice. The difference between the tumor volume on the 20th day and the tumor volume on the 10th and 13th days and the difference between the tumor volume on the 24th day and the tumor volume on the 10th day were calculated. The results showed that the increase in tumor volume in the plasminogen group was significantly smaller than that in the vehicle PBS control group, and the difference was statistically significant (* means P ⁇ 0.05).
  • FIG. 3A-B Representative images of immunohistochemical staining of CD31 in tumor masses after 29 days of plasminogen administration in the tail vein of colon cancer model mice.
  • Fig. 3A is the control group given vehicle PBS
  • Fig. 3B is the group given plasminogen.
  • the results showed that the expression of CD31 (marked by arrow) in the tumor tissue of the plasminogen group was significantly lower than that of the vehicle PBS control group. This indicates that plasminogen can inhibit the expression of vascular endothelial cell marker CD31 in tumor tissue, suggesting that plasminogen can inhibit the formation of new blood vessels in colon cancer tumor tissue.
  • Figure 4A-B Representative pictures of LYVE-1 immunohistochemical staining in tumor tissue after 29 days of plasminogen administration in the tail vein of colon cancer model mice.
  • Figure 4A is the control group given vehicle PBS
  • Figure 4B is the group given plasminogen.
  • the results showed that the expression of LYVE-1 (marked by arrow) in the tumor tissue of the plasminogen group was significantly lower than that of the vehicle PBS control group. It is suggested that plasminogen can inhibit the formation of lymphatic vessels in colon cancer and the metastasis of cancer tissue.
  • Figure 5A-D Representative images of H&E staining of tumor mass in colon cancer model mice 29 days after plasminogen was administered to the tail vein.
  • Figures 5A and C are the PBS-administered control group
  • Figures 5B and D are the plasminogen-administered groups.
  • the results showed that the colon cancer subcutaneous masses in the plasminogen group and the vehicle PBS group showed different degrees of necrosis, and the plasminogen group had more severe necrosis and wider necrosis area than the vehicle PBS group.
  • the number of tumor cells in the non-necrotic area treated with plasminogen was significantly less than that in the control group treated with vehicle PBS.
  • Figure 6A-B The results of tumor volume measurement after administration of plasminogen or vehicle to colon cancer model mice and the results of the difference in tumor volume between the two groups of mice on the 13th day and the 4th day.
  • A is the measurement result of tumor volume
  • B is the result of the difference in tumor volume between the two groups of mice on the 13th day and the 4th day.
  • the difference in tumor volume between the vehicle group and the plasminogen-administered group on the 13th day and the 4th day was compared and analyzed.
  • FIG. 7 The results of the mechanical allodynia-induced pain sensitivity test on the seventh day after plasminogen was administered to the tail vein of the lung cancer model mice.
  • the results showed that the pain threshold of the mice in the blank control group was normal, and the pain threshold of the vehicle group was increased. Compared with the vehicle group, the pain perception threshold of the mice in the plasminogen group was significantly lower, and the statistical P value of the two groups was 0.003, and The pain threshold of the administration group was close to that of the blank control group.
  • the results showed that plasminogen could significantly improve the pain sensitivity of lung cancer model mice.
  • Figure 8A-B Representative pictures were taken of lung cancer model mice on the 20th day after plasminogen was administered to the tail vein.
  • Figure A is the vehicle-administered control group
  • Figure B is the plasminogen-administered group.
  • the picture shows that on the 20th day, the mice in the PBS control group were given the vehicle to stand up, with slow movement, lethargy, severe rupture of the tumor, and obvious ulceration and bleeding of the tumor wound (marked by arrows); the mice in the plasminogen group moved freely and had a good mental state. , The tumor has no obvious ulceration, and there is a small amount of scab on the tumor epidermis.
  • the results indicate that plasminogen can improve the general physical condition and mental state of lung cancer model mice.
  • Fig. 12 Results of the boundary resting time rate in the open field experiment for 7 days after administration of plasminogen or vehicle to lung cancer model mice.
  • the results showed that the blank control mice had a certain border resting time rate; the border resting time rate of the mice in the vehicle group was significantly more than that of the blank control group; the border resting time rate of the mice in the plasminogen group was significantly less Vehicle group, and the difference was statistically significant (* means P ⁇ 0.05).
  • the results indicate that plasminogen can promote the recovery of activity and behavior in lung cancer model mice.
  • FIG. 13 Results of tumor volume measurement in lung cancer model mice after administration of plasminogen or vehicle. The results showed that the tumor volume of mice in the plasminogen group was significantly smaller than that in the vehicle group at each measurement time point. The P value on the 10th day of the drug was 0.07. It shows that plasminogen can obviously inhibit the tumor growth of lung cancer model mice.
  • FIG 14A-B Cancer pain model mice hot and cold plate pain detection results.
  • A is the statistical result of the contraction and lifting temperature of the hind foot during the detection
  • B is the statistical result of the contraction and lifting time of the hind foot during the detection.
  • the results showed that the temperature of the mice in the vehicle group was significantly higher than that of the mice in the blank control group when the hind paws were contracted and lifted.
  • the contraction and lifting time of the hind paws of the mice in the vehicle group was significantly earlier than that of the mice in the blank control group.
  • the above results indicate that plasminogen can relieve pain in cancer pain model mice.
  • FIG. 15 Results of clamp-type tenderness test in mice with cancer pain model after administration of plasminogen. The results showed that the pain threshold of the mice in the administration group was significantly higher than that in the vehicle group, and the statistical analysis P value was 0.006. It is suggested that plasminogen can relieve pain in cancer pain model mice.
  • Fig. 16 Measurement results of tumor volume in lung cancer model mice after administration of plasminogen. The results showed that the tumor volume of the mice in the administration group was no different from the vehicle group on Day 0, and the tumor volume in the administration group was significantly smaller than that in the vehicle group on Day 4, Day 7 and Day 10, and the statistical analysis P values were 0.22, 0.003 and 0.07, respectively. It shows that plasminogen can significantly inhibit the growth of lung cancer tumor.
  • Fig. 17 The results of esophageal electronic gastroscope detection in patients with esophageal cancer after treatment with plasminogen and chemotherapy. Before the first course of treatment and 4 weeks after the third course of treatment, the patients underwent electronic gastroscopic examination. The results showed that before administration, the esophagus mass (marked by arrow) was seen from 20cm-30cm away from the incisors, and the esophagus was involved for 3/4 weeks.
  • 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 plasminogen (also known as: plasminogen) , 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, which is mainly carried out on fibrin; urokinase-type plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells and can directly activate plasminogen without the need for fibrin as a cofactor.
  • Plasminogen (PLG) is synthesized by the liver. When blood coagulates, a large amount of PLG is adsorbed on the fibrin network, and under the action of t-PA or u-PA, it is activated to plasmin, which promotes fibrinolysis.
  • Plasmin (PL) is a serine protease whose functions are as follows: degrade fibrin and fibrinogen; hydrolyze various coagulation factors V, VIII, X, VII, XI, II, etc.; convert plasminogen into fibrinolytic enzymes; hydrolysis of complement, etc.
  • Fibrinolytic inhibitors including plasminogen activator inhibitor (PAI) and ⁇ 2 antiplasmin ( ⁇ 2-AP).
  • PAI mainly has two forms, PAI-1 and PAI-2, which can specifically bind to t-PA in a ratio of 1:1, thereby inactivating it and activating PLG at the same time.
  • ⁇ 2-AP is synthesized by the liver and combines with PL in a ratio of 1:1 to form a complex, which inhibits the activity of PL; FXIII makes ⁇ 2-AP covalently bound to fibrin, reducing the sensitivity of fibrin to PL.
  • Substances that inhibit the activity of the fibrinolytic system in vivo include: PAI-1, complement C1 inhibitor; ⁇ 2 antiplasmin; ⁇ 2 macroglobulin.
  • component of the plasminogen activation pathway encompasses:
  • plasmin also known as: plasmin
  • variants or analogs thereof are also known as: plasmin and variants or analogs thereof.
  • Plasminogen activators such as tPA and uPA, and tPA or uPA variants and the like comprising one or more domains of tPA or uPA, such as one or more kringle domains and serine protease domains thing.
  • 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 additions, deletions and/or 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, 1-3, 1-2, 1 amino acid protein that still has plasminogen, plasmin, tPA or uPA activity.
  • variants of plasminogen, plasmin, tPA, and uPA include, 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.
  • a "plasminogen variant” of the invention encompasses at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with sequence 2, 6, 8, 10 or 12 % sequence identity and still have plasminogen activity.
  • a "plasminogen variant” of the present invention may be based on sequence 2, 6, 8, 10 or 12 with additions, deletions and/or substitutions of 1-100, 1-90, 1-80, 1- 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid protein that still has plasminogen activity.
  • the plasminogen variants of the present invention include all naturally occurring human genetic variants as well as other mammalian forms of these proteins, as well as by 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, 1-3, 1-2, 1 amino acid obtained mutant variants of these proteins.
  • the plasminogen of the invention may be a human plasminogen ortholog from a primate or rodent or a variant thereof that still retains plasminogen activity, eg sequences 2, 6, 8, 10 Or plasminogen shown in 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 (eg, one or more kringle domains and serine protease domains) "Variants” and “analogs” of lysinogen, plasmin, tPA and uPA.
  • variants and analogs such as mini-plasminogen.
  • Variants and “analogs” of plasmin encompass “variants” of plasmin that comprise one or more plasmin domains (eg, one or more kringle domains and serine protease domains) and “analogs” such as mini-plasmin and delta-plasmin.
  • a "variant" or “analog” of the above-mentioned plasminogen, plasmin, tPA or uPA has the activity of plasminogen, plasmin, tPA or uPA, respectively, or does it provide 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, which can be measured, for example, with reference to a method selected from the literature described in: Ny, A., Leonardsson, G., Hagglund, AC, Hagglof, P.
  • the "component of the plasminogen activation pathway" of the invention is plasminogen.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen.
  • the plasminogen is a plasminogen protein comprising one or more kringle domains.
  • the plasminogen is a plasminogen protein comprising a serine protease domain.
  • the plasminogen is a plasminogen protein comprising a serine protease domain as shown in SEQ ID NO: 14.
  • the plasminogen comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 14 Plasminogen protein with a serine protease domain.
  • the plasminogen is natural or synthetic human full-length plasminogen.
  • the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is human native plasminogen.
  • the plasminogen is human native plasminogen as shown in SEQ ID NO: 2.
  • the plasminogen is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or A protein with 99% sequence identity and still having plasminogen activity. In some embodiments, the plasminogen is a conservative substitution variant of sequence 2, 6, 8, 10 or 12.
  • plasminogen activity refers to the lysine binding activity and proteolytic activity of plasminogen to its receptor or substrate.
  • Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease capable of hydrolyzing several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans [1]. In addition, plasmin can activate some metalloproteinase precursors (pro-MMPs) to form active metalloproteinases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis [2-3] .
  • ECM extracellular matrix
  • MMPs active metalloproteinases
  • Plasmin is formed by proteolysis of plasminogen by two physiological PAs: tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). Due to the relatively high levels of plasminogen in plasma and other body fluids, it has traditionally been thought 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 tightly regulated by different factors, such as hormones, growth factors and cytokines. In addition, there are specific physiological inhibitors of plasmin and PAs. The main inhibitor of plasmin is ⁇ 2-antiplasmin.
  • PAI-1 uPA plasminogen activator inhibitor-1
  • PAI-2 lysinogen activator inhibitor-2
  • uPA-specific cell surface receptors uPAR
  • Plasminogen is a single-chain glycoprotein composed of 791 amino acids with a molecular weight of about 92kDa [6-7] . Plasminogen is mainly synthesized in the liver and is abundantly present in the extracellular fluid. Plasminogen content in plasma is approximately 2 ⁇ M. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids [8-9] . Plasminogen exists in two molecular forms: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen).
  • the naturally secreted and uncleaved form of plasminogen has an amino-terminal (N-terminal) glutamate and is therefore referred to as 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, leading to the formation of the disulfide-linked double-chain protease plasmin [10] .
  • the amino-terminal part of plasminogen contains five homologous kringles, the so-called kringles domains (kringle 1, kringle 2, kringle3, kringle 4, kringle 5), 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, and are therefore also called lysine-binding domains.
  • a lysine binding domain refers to a structural region selected from any one, two, three, four or five kringle of kringle 1, kringle 2, kringle3, kringle 4 and kringle 5.
  • the main substrate of plasmin is fibrin, and the dissolution of fibrin is the key to preventing pathological thrombosis [11] .
  • Plasmin also has substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycans, and gelatin, suggesting that plasmin also plays an important role in ECM remodeling [7,12- 13] .
  • plasmin can also degrade other components of the ECM, including MMP-1, MMP-2, MMP-3 and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis [14] .
  • plasmin has the ability to activate certain latent forms of growth factors [15-17] . In vitro, plasmin also hydrolyzes components of the complement system and releases chemotactic complement fragments.
  • Pulmin is a very important enzyme present in the blood that hydrolyzes 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 according to the natural human plasminogen amino acid sequence (sequence 4) containing the signal peptide, and the molecular weight is about 90kD , is a glycoprotein that is mainly synthesized in the liver and can circulate in the blood.
  • the cDNA sequence encoding the amino acid sequence is shown in sequence 3.
  • Full-length plasminogen contains seven domains: a serine protease domain or protease domain for short at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle1-5).
  • the serine protease domain includes residues Val581-Arg804.
  • Glu-plasminogen is a natural full-length plasminogen composed of 791 amino acids (without a signal peptide of 19 amino acids), the amino acid sequence is shown in sequence 2, and the cDNA sequence encoding this sequence is shown in sequence 1. Show. In vivo, there is also a Lys-plasminogen formed by hydrolysis of amino acids 76-77 of Glu-plasminogen. The amino acid sequence is shown in sequence 6, and the cDNA sequence encoding the amino acid sequence is shown in Sequence 5 is shown. Delta-plasminogen is a fragment of full-length plasminogen that lacks the Kringle2-Kringle5 structure, and only contains Kringle1 and serine protease domains [18-19] .
  • the amino acid sequence of delta-plasminogen has been reported in the literature. (Sequence 8) [19] , the cDNA sequence encoding the amino acid sequence is as sequence 7.
  • Mini-plasminogen is composed of Kringle5 and serine protease domains, and it has been reported to include residues Val443-Asn791 (starting with the Glu residues of the Glu-plasminogen sequence without the signal peptide) amino acid) [20] , 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 contains only a serine protease domain, and it has been reported that its amino acid sequence includes residues Ala543-Asn791 (starting from the Glu residues of the Glu-plasminogen sequence without the signal peptide). starting amino acid) [21] , there are also patent documents CN102154253A reported that its sequence includes residues Lys531-Asn791 (with the Glu residues of the Glu-plasminogen sequence that does not contain the signal peptide as the starting amino acid), this patent sequence refers to the patent document CN102154253A, its amino acid sequence is shown in sequence 12, and the cDNA sequence encoding the amino acid sequence is shown in sequence 11. The amino acid sequence of the serine protease domain in this application is shown in SEQ ID NO: 14, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID 13.
  • Plasminogens comprising one or more Kringle domains selected from Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5 are encompassed by the present application.
  • a "serine protease domain”, also known as a protease domain, is a domain of plasminogen that performs proteolytic functions.
  • the technical solutions of the present invention relating to plasminogen cover all technical solutions of plasminogen comprising a serine protease domain.
  • the plasminogen fragment comprising a serine protease domain of the present invention is a protein comprising a serine protease domain of plasminogen.
  • the plasminogen fragment comprising a serine protease domain described herein is a protein comprising the amino acid sequence shown in SEQ ID NO: 14.
  • the plasminogen fragment comprising a serine protease domain described herein is at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 14
  • the plasminogen comprises conservative substitution variants of sequence 14.
  • assay methods for plasminogen and its activity include: detection of tissue plasminogen activator activity (t-PAA), detection of plasma tissue plasminogen activator antigen (t-PAA) 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 fibrinolysis Detection of zymogen activator inhibitor antigen, detection of plasma plasmin-antiplasmin complex (PAP).
  • tissue plasminogen activator activity t-PAA
  • t-PAA plasma tissue plasminogen activator antigen
  • plgA plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma tissue fibrinolysis
  • the most commonly used detection method is the chromogenic substrate method: streptokinase (SK) and chromogenic substrate are added to the test plasma, and the plasminogen in the test plasma is converted into plasmin under the action of SK. , the latter acts on a chromogenic substrate, which is subsequently measured by a spectrophotometer, and the increase in absorbance is proportional to plasminogen activity.
  • plasminogen activity eg, proteolytic activity
  • Angiostatin has a molecular weight of 38KD and is a part of plasminogen [22-23] . Its amino acid sequence has a homology of up to 98% with Kringle1-Kringle4 in the five Kringles of plasminogen.
  • Angiostatin is formed by the cleavage of plasminogen by elastase, different matrix metalloproteinases and other proteolytic enzymes in vivo [24-25] .
  • Angiostatin can bind to a variety of proteins including angiomotin, annexin II, tPA and CD26 [26] , and studies have shown that angiostatin can interact with these proteins to inhibit angiogenesis.
  • angiostatin can specifically inhibit the proliferation, migration and apoptosis of vascular endothelial cells, and its mechanism may be closely related to the lysine-binding activity of kringle in angiostatin.
  • Plasminogen Kringle5 (K5) can also significantly inhibit the growth of endothelial cells, and its activity is significantly higher than that of Angiostatin.
  • K5 also has lysine binding activity, its inhibitory effect on angiogenesis does not depend on lysine binding activity, and the structure of K5 has a unique mechanism of action [27] .
  • angiostatin has been expanded from a single protein to a class of proteins (angiostatin isoforms or angiostatin related proteins), including A series of fragments containing different Kingle structures of plasminogen and similar biological activities have been developed [28-29] .
  • This effect is likely to be that the additional increased plasminogen in the body is degraded by proteolytic enzymes such as MMPs and elastase accumulated in the tumor microenvironment, thereby promoting the production of high amounts of Angiostatin and/or its analogs, and thereby inhibiting tumor angiogenesis. generated function. That is to say, the inhibition of angiogenesis can be achieved by directly regulating the plasminogen in the body instead of directly increasing the Angiostatin and/or its analogs in the body. This opens up a new therapeutic strategy for tumor suppression.
  • proteolytic enzymes such as MMPs and elastase accumulated in the tumor microenvironment
  • plasminogen encompasses plasminogen fragments having plasminogen activity, such as plasminogen fragments comprising one or more different Kingle domains, or comprising biologically similar to one or more Kingle domains Active variant plasminogen fragment.
  • orthologs or orthologs refer to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs and vertical homologs. It specifically refers to proteins or genes that have evolved from the same ancestral gene in different species.
  • the plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs derived from different species and having plasminogen activity.
  • a “conservative substitution variant” refers to one in which a given amino acid residue is altered without altering the overall conformation and function of the protein or enzyme, including but not limited to those with similar properties (eg, 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 are interchangeable.
  • 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 of similar functions may differ.
  • Constants also include polypeptides or enzymes determined to have more than 60% amino acid identity by BLAST or FASTA algorithm, if it can reach more than 75%, it is better, preferably more than 85%, or even more than 90%. is optimal and has the same or substantially similar properties or functions as the native or parent protein or enzyme.
  • Plasmid and “plasmin” and “plasminase” can be used interchangeably and have the same meaning; “plasminogen” is used with “plasminogen” and “plasminogen” “Original” are used interchangeably and have the same meaning.
  • plasminogen is used with “plasminogen” and “plasminogen” “Original” are used interchangeably and have the same meaning.
  • Compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway refers to activating plasminogen either directly 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, anistreplase, Monteplase, lanoteplase, paamiplase, staphylokinase.
  • the "antagonist of a fibrinolysis inhibitor" of the present invention is a compound that antagonizes, weakens, blocks, or prevents the action of a fibrinolysis inhibitor.
  • fibrinolytic inhibitors are eg PAI-1, complement C1 inhibitor, alpha2 antiplasmin and alpha2 macroglobulin.
  • Such antagonists such as PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin antibodies, or block or downregulate such as PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin Antisense RNA or small RNA expressed by globulin, or occupying the binding site of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, or ⁇ 2 macroglobulin but without PAI-1, complement C1 inhibitor, ⁇ 2 antifibrinolytic A compound that functions as a lysin or alpha2 macroglobulin", or a compound that blocks the binding and/or active domains of PAI-1, complement C1 inhibitor, alpha2 antiplasmin, or alpha2 macroglobulin.
  • the "deficiency" of plasminogen means that the content or activity of plasminogen in a subject is lower than that of normal people, and is low enough to affect the normal physiological function of the subject; the The meaning of “deletion” of plasminogen is that the content or activity of plasminogen in the subject is significantly lower than that of normal people, or even the activity or expression is extremely low, and normal physiological functions can only be maintained by external supply.
  • Isolated plasminogen refers to a plasminogen protein that has been isolated and/or recovered from its natural environment.
  • the plasminogen will be purified (1) to greater than 90%, greater than 95%, or greater than 98% purity (by weight), as determined by Lowry's method, eg, 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 use of a spinning cup sequencer, or (3) to homogeneity as determined by using Determined by Coomassie blue or silver staining by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions.
  • Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.
  • polypeptide refers to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized modified amino acids, and polypeptides with modified peptide backbones.
  • the term includes fusion proteins including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without N-terminal methionine residues); and the like.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as, after gaps have been introduced as necessary to achieve maximum percent sequence identity, and no conservative substitutions are considered part of sequence identity, the Percentage of amino acid residues that are identical to amino acid residues in a reference polypeptide sequence. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes of the present invention, percent amino acid sequence identity values were generated using the sequence comparison computer program ALIGN-2.
  • % 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 comprising relative to, with, or against a given amino acid sequence A given amino acid sequence A) for a certain % amino acid sequence identity of B is calculated as follows:
  • the terms “treating” and “treating” refer to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be complete or partial prevention of the disease or symptoms thereof, and/or partial or complete cure of the disease and/or symptoms thereof, and includes: (a) preventing the occurrence of the disease in a subject, who may have Predisposing to the disease, but not yet diagnosed as having the disease; (b) inhibiting the disease, ie, blocking its development; and (c) alleviating the disease and/or its symptoms, ie, causing regression of the disease and/or its symptoms.
  • mammals including, but not limited to, murine (rat, mouse), non-human primate, human, canine, feline , hoofed animals (eg horses, cattle, sheep, pigs, goats), etc.
  • murine rat, mouse
  • non-human primate human
  • canine feline
  • hoofed animals eg horses, cattle, sheep, pigs, goats
  • a “therapeutically effective amount” or “effective amount” refers to an amount of plasminogen sufficient to effect said prevention and/or treatment of a disease when administered to a mammal or other subject to treat the disease.
  • a “therapeutically effective amount” will vary depending on the plasminogen used, the severity of the disease and/or its symptoms in the subject to be treated, as well as age, weight, and the like.
  • Plasminogen can be isolated from nature and purified for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is synthesized chemically, the synthesis can be carried out via liquid phase or solid phase.
  • Solid-phase polypeptide synthesis SPPS
  • SPPS Solid-phase polypeptide synthesis
  • Various forms of SPPS such as Fmoc and Boc, can be used to synthesize plasminogen.
  • Techniques for solid-phase synthesis are described in Barany and Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology.
  • Plasminogen of the present invention can be produced using standard recombinant methods.
  • a nucleic acid encoding plasminogen is inserted into an expression vector operably linked to regulatory sequences in the expression vector.
  • Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression control may be a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells (eg, 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 typically replicate in the host organism as episomes or as an integral part of the host chromosomal DNA.
  • the expression vector contains a selectable marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate transformation of the exogenous with the desired DNA sequence of those cells were detected.
  • a selectable marker eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone a subject protein-encoding polynucleotide.
  • 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 generated, which will typically contain expression control sequences (eg, origins of replication) that are compatible with the host cell.
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from bacteriophage lambda.
  • a promoter will typically control expression, optionally in the case of operator sequences, and have ribosome binding site sequences, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast eg, S. cerevisiae
  • Pichia are examples of suitable yeast host cells, with suitable vectors having expression control sequences (eg, promoters), origins of replication, termination sequences, etc., as desired.
  • Typical promoters contain 3-phosphoglycerate kinase and other saccharolytic enzymes.
  • Inducible yeast are initiated from promoters that include, inter alia, alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
  • mammalian cells eg, mammalian cells grown in in vitro cell culture
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas.
  • Expression vectors for use in these cells may contain expression control sequences, such as origins of replication, promoters and enhancers (Queen et al., Immunol. Rev.
  • RNA splicing sites sites for necessary processing information
  • sites for necessary processing information such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences.
  • suitable expression control sequences are promoters derived from leukoimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol. 148:1149 (1992).
  • the compounds described herein can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like Plasminogen.
  • the plasminogen is substantially pure, eg, 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, eg, free of contaminants such as cellular debris, macromolecules other than compounds of the invention such as plasminogen, and the like.
  • Lyophilized formulations can be formed by mixing plasminogen of the desired 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, stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexanediamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parahydroxybenzoic acid Esters such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues) ; proteins such as serum albumin, gelatin or immunoglobulins; 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 as required for the particular condition to be treated, preferably those which are complementary in activity and which do not have side effects with each other.
  • active compound for example, antitumor drugs, liver protective drugs, hormone drugs, etc.
  • 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 colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in macroemulsions.
  • colloidal drug delivery systems eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in macroemulsions are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
  • the plasminogen of the present invention for in vivo administration must be sterile. This can be easily achieved by filtration through sterile filters before or after lyophilization and reconstitution.
  • the plasminogen of the present invention can be prepared as a sustained-release preparation.
  • sustained release formulations include semipermeable matrices of solid hydrophobic polymers having a shape and containing glycoproteins, such as membranes or microcapsules.
  • sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981); Langer, Chem. .Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (US Pat. No.
  • Polymers such as ethylene -Vinyl acetate and lactic acid-glycolic acid can continuously release molecules for more than 100 days, while some hydrogels release proteins for shorter periods of time. Rational strategies to stabilize proteins can be designed based on the relevant mechanisms. For example, if the mechanism of aggregation is discovered Intermolecular SS bonds are formed through the exchange of thiodisulfide bonds, which can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions. Stablize.
  • compositions of the present invention can be accomplished by different means, eg, intravenously, intraperitoneally, subcutaneously, intracranally, intrathecally, intraarterally (eg, via the carotid artery), intramuscularly.
  • 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 the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, among others.
  • Dosing regimens will be determined by healthcare professionals based on various clinical factors. As is well known in the medical arts, the dosage for any patient depends on a variety of factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, number and route of administration, general health, and other concomitantly administered drugs .
  • the dosage range of the pharmaceutical composition comprising plasminogen of the invention may be, for example, about 0.0001 to 2000 mg/kg per day, or about 0.001 to 500 mg/kg (eg, 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg) per day mg/kg, 10 mg/kg, 50 mg/kg, etc.) subject body weight.
  • the dose 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. Dosages above or below this exemplary range are also contemplated, especially in view of the factors set forth above. Intermediate doses within the above ranges are also included within the scope of the present invention. Subjects may be administered such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary dosage schedule includes 1-10 mg/kg on consecutive days. Real-time evaluation of therapeutic efficacy and safety is required during the administration of the drug of the present invention.
  • the article of manufacture 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 effective to treat the disease or condition of the present invention and has a sterile access port (eg, the container may be an intravenous solution pack or vial containing a stopper penetrable by a hypodermic needle) of).
  • At least one active agent in the composition is plasminogen/plasmin.
  • the label on or attached to the container states that the composition is used to treat the tumors of the present invention.
  • the article of manufacture 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 materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution, and dextrose solution.
  • the article of manufacture comprises a package insert with instructions for use, including, for example, instructing the user of the composition to administer the plasminogen composition to the patient along with other drugs for the treatment of concomitant diseases.
  • the plasminogen used in the following examples is human plasminogen, which is obtained from the plasma of donors. Based on the purification methods described in the literature [37-39] , part of the process optimization is carried out, and it is obtained from the purification of human plasma. Among them, human Lys-plasminogen (Lys-plasminogen) and Glu-plasminogen (Glu-plasminogen)>98%.
  • CT26.WT mouse colon cancer cells are undifferentiated colon cancer cell lines induced by N-nitroso-N-methylurethane-(NNMU). Its cloned cell line was named CT26.WT (ATCC CRL-2638).
  • CT26.WT mouse colon cancer cells were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, hereinafter referred to as CT26.
  • CT26 cells were placed in PRIM-1640 (GIBCO, Cat. No. 31800022) medium containing 10% fetal bovine serum, and cultured in a 37°C, 5% carbon dioxide incubator. After the cells in the culture flask had grown to 90%, they were digested with trypsin (0.05% trypsin with 0.02% EDTA) (Sigma, 74799), then washed three times with normal saline, and resuspended in normal saline at a concentration of 10 7 pcs/ml.
  • trypsin 0.05% trypsin with 0.02% EDTA
  • mice Fourteen male Balb/c mice aged 7-9 weeks were anesthetized by intraperitoneal injection of sodium pentobarbital at 50 mg/kg body weight. After anesthesia, mice were subcutaneously inoculated with 10 6/100 ⁇ l CT26 resuspension on the back with a 27-gauge needle, and then the volume of the mouse subcutaneous tumor (length x width 2 x 0.52) was measured with a vernier caliper every day for seven consecutive days [40-41] . According to the tumor volume on the seventh day, the mice were randomly divided into two groups, 7 mice each in the plasminogen group and the PBS control group. The administration of CT26 started on the eighth day of inoculation, and was designated as the first day.
  • Human plasminogen 1 mg/0.1 mL/mice/day was injected into the tail vein of the mice in the plasminogen group, and the vehicle PBS (20 mM citric acid-lemon sodium, 2% arginine hydrochloride, 3% mannitol, pH 7.4, the same below) the control group was injected with the same volume of PBS through the tail vein for 23 consecutive days. On the 1st, 4th, 7th, 10th, 13th, 17th, 20th, and 24th days, the length and width of the tumor were measured, and the tumor volume was calculated.
  • mice 14 male Balb/c mice aged 7-9 weeks.
  • the mice were anesthetized by intraperitoneal injection of sodium pentobarbital at 50 mg/kg body weight. After anesthesia, the mice were subcutaneously inoculated on the back with a 27-gauge needle. Suspension, and then the volume of the mouse subcutaneous tumor (length x width 2 x 0.52) was measured with a vernier caliper every day [40-41] for seven consecutive days. According to the volume of the tumor on the seventh day, the mice in the model group were randomly divided into two groups, the plasminogen group and the PBS control group, with 7 mice in each group. The administration of CT26 began on the eighth day of inoculation and was designated as the first day.
  • Human plasminogen 1 mg/0.1 mL/mice/day was injected into the tail vein of the plasminogen group, and the same as the tail vein injection of the vehicle PBS control group. volume of PBS for 29 consecutive days.
  • the mice were sacrificed on the 30th day, and the tumor tissues were fixed in 10% neutral formaldehyde for 24-48 hours, and then dehydrated and embedded.
  • the fixed tissue samples were dehydrated in an alcohol gradient and cleared with xylene before paraffin-embedding.
  • the thickness of tumor tissue sections was 4 ⁇ m, and the sections were deparaffinized and rehydrated, and washed once with water. Repair with citric acid for 30 minutes, cool at room temperature for 10 minutes and rinse gently with water.
  • the color was developed according to the DAB kit (Vector laboratories, Inc., USA), washed with water for 3 times, counterstained with hematoxylin for 30 seconds, returned to blue under running water for 5 minutes, and then washed once with PBS. Gradient dehydration was made transparent and mounted, and the sections were observed under a 200-fold optical microscope.
  • CD31 is usually located in vascular endothelial cells, platelets, macrophages and kuffer cells, granulocytes, T/NK cells, lymphocytes, megakaryocytes, osteoclasts, and neutrophils. In immunohistochemistry, CD31 is a marker of vascular endothelial cells and can be used to assess tumor angiogenesis [42-43] .
  • mice 14 male Balb/c mice aged 7-9 weeks.
  • the mice were anesthetized by intraperitoneal injection of sodium pentobarbital at 50 mg/kg body weight. After anesthesia, the mice were subcutaneously inoculated on the back with a 27-gauge needle. Suspension, and then the volume of the mouse subcutaneous tumor (length x width 2 x 0.52) was measured with a vernier caliper every day [40-41] for seven consecutive days. According to the volume of the tumor on the seventh day, the mice in the model group were randomly divided into two groups, the plasminogen group and the PBS control group, with 7 mice in each group. The administration of CT26 began on the eighth day of inoculation and was designated as the first day.
  • Human plasminogen 1 mg/0.1 mL/mice/day was injected into the tail vein of the plasminogen group, and the same as the tail vein injection of the vehicle PBS control group. volume of PBS for 29 consecutive days.
  • the mice were sacrificed on the 30th day, and the tumor tissues were collected and fixed in 10% neutral formaldehyde for 24-48 hours, then dehydrated and embedded.
  • the fixed tissue samples were dehydrated in an alcohol gradient and cleared with xylene before paraffin-embedding.
  • the thickness of tumor tissue sections was 4 ⁇ m, and the sections were deparaffinized and rehydrated, and washed once with water. Repair with citric acid for 30 minutes, cool at room temperature for 10 minutes and rinse gently with water.
  • the color was developed according to the DAB kit (Vector laboratories, Inc., USA), washed with water for 3 times, counterstained with hematoxylin for 30 seconds, returned to blue under running water for 5 minutes, and then washed once with PBS. Gradient dehydration was made transparent and mounted, and the sections were observed under a 400-fold optical microscope.
  • Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is a receptor on the surface of lymphatic endothelial cells and can be used as a marker of lymphatic endothelial cells [44-45] .
  • LYVE-1 (marked by arrow) in the plasminogen group (Fig. 4B) was significantly lower than that in the vehicle PBS control group (Fig. 4A).
  • plasminogen could inhibit the expression of LYVE-1, a marker of intratumoral lymphatic endothelial cells, suggesting that plasminogen inhibits the formation of lymphatic vessels in colon cancer tissue.
  • the mice were anesthetized by intraperitoneal injection of sodium pentobarbital at 50 mg/kg body weight. After anesthesia, the mice were subcutaneously inoculated with 10 6/100 ⁇ l CT26 on the back with a 27-gauge needle. After resuspension, the volume of mouse subcutaneous tumors (length x width 2 x 0.52) was measured with a vernier caliper every day [40-41] for seven consecutive days. According to the volume of the tumor on the seventh day, the mice in the model group were randomly divided into two groups, 7 mice in the plasminogen group and 7 mice in the PBS control group, with 7 mice in each group.
  • CT26 The administration of CT26 began on the eighth day of inoculation and was designated as the first day.
  • Human plasminogen 1 mg/0.1 mL/mice/day was injected into the tail vein of the plasminogen group, and the same as the tail vein injection of the vehicle PBS control group. volume of PBS for 29 consecutive days.
  • the mice were sacrificed on the 30th day, and the tumor tissues were fixed in 10% neutral formaldehyde for 24-48 hours.
  • the fixed tumor tissue was dehydrated in alcohol gradient and cleared with xylene before being embedded in paraffin.
  • the thickness of the tissue section was 3 ⁇ m.
  • FIG. 5C and FIG. 5D are enlarged pictures of the block area in FIG. 5A and FIG. 5B , respectively.
  • mice Twenty-seven 9-week-old Balb/c female mice were randomly divided into 2 groups after weighing, 9 mice in the blank control group and 18 mice in the model group. After the grouping was completed, all mice in the model group were anesthetized with 2% isoflurane, sterilized one armpit and injected subcutaneously with 1 ⁇ 10 6 cells of CT-26 cell suspension, and observed for 7 days. On the 8th day of tumor cell inoculation, all mice were tested for tumor volume. When the tumor volume of the mice reached more than 100 mm 3 , all mice were weighed and randomly divided into groups according to the results of the detected body weight and tumor volume. There were 9 mice in the vehicle group. Mice, 9 mice in the administration group.
  • mice in the administration group were injected with human plasminogen in the tail vein of 1 mg/0.1ml/mouse/day, and the mice in the vehicle group and the mice in the blank control group were injected with the same volume of vehicle PBS in the tail vein, on the first day of administration. Defined as day 1, dosing for 13 consecutive days. The volume of mouse subcutaneous tumors (length x width 2 x 0.52) was measured with vernier calipers on days 0 (pre-dose), 4, 7, 10, and 13 of dosing [40-41] .
  • Mouse Lewis lung cancer cells (Lewis lung cancer cells, LLC) were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, hereinafter referred to as LLC.
  • LLC was placed in DMEM (GIBCO, Cat. No. 15140122) medium containing 10% fetal bovine serum, and cultured in a 37°C, 5% carbon dioxide incubator. After the cells in the culture flask had grown to 90%, they were digested with trypsin (0.02% EDTA added to 0.05% trypsin) (Sigma, 74799), and then washed three times with normal saline. Cells were resuspended in physiological saline and counted under a microscope to confirm that the cells were in single suspension. Finally, the cells were resuspended with physiological saline, the cell concentration was 10 7 cells/ml, and the cells were kept on ice for use.
  • DMEM fetal bovine serum
  • mice in the model group were selected and randomly divided into 2 groups after weighing, 6 mice in the blank control group and 24 mice in the model group. After the grouping was completed, the mice were anesthetized with 2% isoflurane using a respiratory anesthesia machine; the left leg was depilated, and the skin of the distal condyle of the femur was disinfected with iodine solution, and a 0.5 cm shallow incision was made; then a blunt ligament was made on the patella Sexual dissection to expose the distal condyle of the femur with minimal damage; mice in the model group were slowly injected with 10ul LLC cell suspension (2 ⁇ 10 5 cells/ul) into the distal medullary cavity of the left femur with a 50ul sterile microsyringe.
  • mice in the control group were injected with 10 ul of sterile saline into the bone cavity; after the injection was completed, the syringe was placed for 90 s to allow cells to fill the bone cavity, and the injection hole was sealed with sterile bone wax to prevent cell leakage after removing the syringe, and then injected with 5.0
  • the wound was sutured with silk thread; the mice after surgery were placed on a heating pad to keep warm, and returned to the cage after the mice recovered.
  • all mice were weighed and electronic pain detection was performed.
  • the mice in the model group were divided into groups according to the results of pain and body weight, with 12 mice in the vehicle group and 12 mice in the administration group.
  • mice in the administration group were injected with human plasminogen in the tail vein of 50 mg/kg/mouse/day, and the mice in the vehicle group and the mice in the blank control group were injected with the vehicle in the tail vein of 5ml/kg/mouse/day.
  • the first day of the drug was defined as the first day of dosing.
  • Von-Frey filaments (Stoelting, USA) were used to detect the sensitivity of animals to mechanical damage. With 2.0g force as the starting force, test its left foot first. If 4 times out of 5 stimulations, the paw withdrawal response is positive, which is recorded as the threshold of the animal to mechanical damage.
  • mice Eleven female C57 mice aged 6-7 weeks were randomly divided into two groups according to body weight, the plasminogen group (5 mice) and the vehicle control group (6 mice). Two groups of mice were injected intraperitoneally with sodium pentobarbital at 50 mg/kg body weight, and after anesthesia, mice were subcutaneously inoculated with 2 x 10 6 mice/200 ⁇ l LLC resuspension [46,47] . The mice were given cells on the day of inoculation, which was recorded as the first day. Human plasminogen 1 mg/0.1 mL/mice/day was injected into the tail vein of the mice in the plasminogen group, and the same volume was injected into the tail vein of the vehicle control group every day. The vehicle PBS was observed and photographed every day to record the tumor situation and the mental state of the mice.
  • mice in the control group who were given the vehicle PBS had erect hair, slow movement, lethargy, severe rupture of the tumor, and obvious ulceration and bleeding of the tumor wound (marked by arrows);
  • the mice in the plasminogen group (Fig. 8B) The mice move freely and have good mental state.
  • the tumor has no obvious rupture phenomenon, and the tumor epidermis has a small amount of scab. This observation suggests that plasminogen can improve general physical and mental status in lung cancer model mice.
  • mice Eleven female C57 mice aged 6-7 weeks were randomly divided into two groups according to body weight, the plasminogen group (5 mice) and the vehicle control group (6 mice). Two groups of mice were injected intraperitoneally with sodium pentobarbital at 50 mg/kg body weight, and after anesthesia, mice were subcutaneously inoculated with 2 x 10 6 mice/200 ⁇ l LLC resuspension [46,47] . The day after the mice were inoculated with cells, the administration started, which was recorded as the first day.
  • Human plasminogen 1 mg/0.1 mL/mice/day was injected into the tail vein of the mice in the plasminogen (PLG) group, and 1 mg/0.1 mL/mice/day was injected into the tail vein of the mice in the vehicle control group. The same volume of vehicle PBS was injected. The survival of the mice was observed and recorded every day for 24 consecutive days. During the observation period, the animals were administered daily according to the protocol.
  • mice Thirty male C57 mice aged 13-14 weeks were randomly divided into two groups according to body weight, 6 in the blank control group and 24 in the model group. Two groups of mice were anesthetized with 2% isoflurane. After the mice were anesthetized, the back skin of 4 to 6 lumbar vertebrae was sterilized and 1 x 10 6 LLC resuspension was subcutaneously inoculated [46,47] . After the cell injection was completed, the tumor growth status of the mice was observed. Seven days after tumor cell inoculation, all mice were tested for body weight, tumor volume and open field. The mice in the model group were randomly divided into two groups according to the test results, the vehicle control group and the fibrinolytic group.
  • mice in the zymogen group there were 12 mice in each group, and the administration was started, which was recorded as the first day.
  • the mice in the plasminogen group were injected with human plasminogen 1 mg/0.1 mL/mice/day through the tail vein, and the mice in the vehicle control group were given 1 mg/0.1 mL/mouse/day.
  • mice Thirty male C57 mice aged 13-14 weeks were randomly divided into two groups according to body weight, 6 in the blank control group and 24 in the model group. Two groups of mice were anesthetized with 2% isoflurane. After the mice were anesthetized, the back skin of 4 to 6 lumbar vertebrae was sterilized and 1 x 10 6 LLC resuspension was subcutaneously inoculated [46,47] . After the cell injection was completed, the tumor growth status of the mice was observed. Seven days after tumor cell inoculation, all mice were tested for body weight, tumor volume and open field. The mice in the model group were randomly divided into two groups according to the test results, the vehicle control group and the fibrinolytic group.
  • mice in the zymogen group there were 12 mice in each group, and the administration was started, which was recorded as the first day.
  • the mice in the plasminogen group were injected with human plasminogen 1 mg/0.1 mL/mice/day through the tail vein, and the mice in the vehicle control group were given 1 mg/0.1 mL/mouse/day.
  • the same volume of vehicle PBS was injected into the tail vein and administered continuously for 7 days. Open field experiments were performed on the 8th day of dosing.
  • the Smart System is a complete and easy-to-use video tracking system for evaluating the behavior of laboratory animals. It allows to record trajectories, activities, specific behaviors (such as rotation, stretching and feeding) and events, and to perform calculations of various analytical parameters.
  • the Smart3.0 system was used to record and analyze the movement of the mice, and the boundary resting time rate of the mice was recorded. The box was wiped with 70% alcohol to prevent olfactory preference in each experiment.
  • mice severe combined immunodeficiency mice (NOD.CB17-Prkdcscid/NcrCrl, referred to as NOD SCID, purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd., strain code 40624) were taken from 7 to 10 weeks old, and were called The mice were randomly divided into 2 groups, 8 mice in the blank control group and 16 mice in the model group. After grouping, all mice in the model group were anesthetized with 2% isoflurane, sterilized their backs and inoculated subcutaneously with 1 ⁇ 10 6 Lewis lung carcinoma cells-GFP cells (mouse lung cancer cells stably transduced with green fluorescent protein GFP, LLC- for short).
  • NOD SCID severe combined immunodeficiency mice
  • mice in the model group was randomly divided into 2 groups, 8 mice in the vehicle group, 8 mice in the administration group, and blank control. The mice in the group were not treated. After the mice were grouped, the mice in the administration group were injected with human plasminogen in the tail vein of 1 mg/0.1ml/mouse/day, the mice in the vehicle group were injected with the same volume of PBS in the tail vein, and the mice in the blank control group were not given administration. Treatment, continuous administration for 10 days. The volume of mouse subcutaneous tumors (length x width 2 x 0.52) was measured with a caliper on day 0 (pre-dose), day 4, day 7 and day 10 of administration [40-41] .
  • mice 18 female and 18 male Balb/c mice aged 8-9 weeks were selected and randomly divided into 2 groups after weighing, 12 mice in the blank control group and 24 mice in the model group. After the grouping was completed, the mice were anesthetized with 2% isoflurane using a respiratory anesthesia machine; the left leg was depilated, and the skin of the distal condyle of the femur was disinfected with iodine solution, and a 0.5 cm shallow incision was made; then a blunt ligament was made on the patella Sexual dissection to expose the distal condyle of the femur with minimal damage; mice in the model group were slowly injected with 5 ⁇ l of CT-26 cell suspension (2 ⁇ 10 4 cells/ ⁇ l) into the medullary cavity of the left distal femur with a 50 ⁇ l sterile microsyringe .
  • mice in the control group were injected with 5 ⁇ l of sterile normal saline into the bone cavity; after the injection was completed, the syringe was placed for 90 s to allow cells to fill the bone cavity, and the injection hole was sealed with sterile bone wax to prevent cell leakage after removing the syringe, and then with 5.0
  • the wound was sutured with silk thread; the mice after surgery were placed on a heating pad to keep warm, and returned to the cage when the mice were awake. All mice were weighed on the 8th day of surgical modeling. Mice in model group were randomly divided into vehicle group and administration group according to the results of body weight, with 12 mice in each group.
  • mice in the administration group were injected with human plasminogen in the tail vein of 50 mg/kg/mouse/day, and the mice in the vehicle group and the blank control group were injected with the vehicle PBS in the tail vein of 5ml/kg/mouse/day.
  • the first day of dosing was defined as day 1, and the dosing was continued for 7 days.
  • hot and cold disk pain detection was performed, and the mice began to show pain response.
  • the temperature of the equipment was set to a constant temperature of 20 °C, and the mice were put in one by one to adapt for 5 min.
  • the hot and cold plate RAMP mode select the hot and cold plate RAMP mode, set the initial temperature to 20°C and the final temperature to 4°C, and set the time from initial temperature to final temperature of 5 minutes. After the device temperature reaches the initial temperature, place the mouse to be tested on the cold plate and click Start. Start timing and cooling, observe the mouse's hind foot contraction and lift and stop, record the time and display temperature of the mouse's hind foot contraction and lift, the longest detection time is 6 minutes, and the measurement is performed 3 times in a row. Note that after each mouse experiment, the hot and cold plates need to be cleaned and wiped with disinfectant before starting the next mouse to avoid mutual interference.
  • mice in the vehicle group was significantly higher than that of the mice in the blank control group when the hind paws were contracted and lifted.
  • mice in the model group were selected and randomly divided into 2 groups after weighing, 6 mice in the blank control group and 24 mice in the model group. After the grouping was completed, the mice were anesthetized with 2% isoflurane using a respiratory anesthesia machine; the left leg was depilated, and the skin of the distal condyle of the femur was disinfected with iodine solution, and a 0.5 cm shallow incision was made; then a blunt ligament was made on the patella Sexual dissection to expose the distal condyle of the femur with minimal damage; mice in the model group were slowly injected with 10ul LLC cell suspension (2 ⁇ 10 5 cells/ul) into the distal medullary cavity of the left femur with a 50ul sterile microsyringe.
  • mice in the control group were injected with 10 ul of sterile saline into the bone cavity; after the injection was completed, the syringe was placed for 90 s to allow cells to fill the bone cavity, and the injection hole was sealed with sterile bone wax to prevent cell leakage after removing the syringe, and then injected with 5.0
  • the wound was sutured with silk thread; the mice after surgery were placed on a heating pad to keep warm, and returned to the cage when the mice were awake.
  • all mice were weighed and electronic pain detection was performed.
  • the mice in the model group were divided into groups according to the results of pain and body weight. There were 12 mice in the vehicle group and 12 mice in the administration group.
  • mice in the administration group were injected with human plasminogen in the tail vein of 50 mg/kg/mouse/day, and the mice in the vehicle group and the mice in the blank control group were injected with the vehicle in the tail vein of 5ml/kg/mouse/day.
  • the first day of the drug was defined as the first day of dosing, and the drug was administered for 20 consecutive days.
  • a clamp-type tenderness test was performed. The specific steps are as follows: the animal to be tested is placed on the fixing frame, and the left and right paws are exposed. Use forceps to clamp the mouse's hind paw and measure the maximum pressure. Record the desired measured pressure value. The measurements were repeated 4 times on the left and right hind paws of each mouse.
  • mice Twenty-one 7-10-week-old SCID (Severe Combined Immunodeficiency) mice (NOD.CB17-Prkdcscid/NcrCrl, referred to as NOD SCID, purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd., strain code 40624) were selected. The mice were randomly divided into two groups, 7 mice in the blank control group and 14 mice in the model group. After the grouping was completed, all mice in the model group were anesthetized with 2% isoflurane (purchased from Lunan Beite Pharmaceutical Co., Ltd.), sterilized their backs and injected subcutaneously with 1 ⁇ 10 6 Lewis lung carcinoma cells-GFP cells (stable to green fluorescence).
  • NOD SCID severe Combined Immunodeficiency mice
  • mice were suspended and observed for 7 days.
  • All mice were tested for body weight, and mice in the model group were tested for tumor volume.
  • the model group was randomly divided into 2 groups, 7 mice in the vehicle group, 7 mice in the administration group, and blank control. The mice in the group were not treated.
  • mice in the administration group were injected with human plasminogen in the tail vein of 1 mg/0.1ml/mouse/day, the mice in the vehicle group were injected with the same volume of PBS in the tail vein, and the mice in the blank control group were not given administration. Dosing for 10 consecutive days. The first day of dosing was designated as day 0, and tumor volume was measured on day 0, day 4, day 7 and day 10.
  • Example 15 Therapeutic effect of plasminogen on patients with esophageal cancer
  • the patient was a 72-year-old male with clear consciousness, no history of diabetes and heart disease, and a history of hypertension. Diagnosed with esophageal cancer six months ago and started chemotherapy.
  • the patient signed an informed consent form and voluntarily received human plasminogen therapy, which was approved by the hospital ethics committee. Human plasminogen was started after the fourth chemotherapy, and human plasminogen therapy was also used after the fifth, sixth, and seventh chemotherapy.
  • Human plasminogen dosing regimen The mode of administration is intravenous bolus. Dosing for 21 days is a course of treatment, and the frequency of dosing is twice a day for the first 5 days and once a day thereafter. Daily dose: 40 mg on day 1, then gradually increased to 190 mg on day 21.
  • the curative effect on patients after administration was recorded on a scale of 1-10, and the condition without administration on the first day was recorded as 10. If the symptom aggravation score increases, the symptom relief score decreases, and 1 is the lightest.
  • the patient's condition improved, the overall impression score was 7 points, the mental state score was 8 points, the appetite score was 7 points, the nausea subsidence score was 0 points, the lower lip ulceration was scabbed, and the hair basically did not fall out .
  • the patient's condition was further improved.
  • the overall impression score was 4 points, the mental state score was 5 points, the appetite score was 6 points, and the color scores of the left and right palms were both 8 points.
  • the lower lip ulceration was basically healed, and the hair basically did not fall out.
  • the overall impression score was 1, the mental state score was 0, the appetite score was 0, and the left and right palm color scores were 3 and 4, respectively.
  • Dosing regimen for the second course of human plasminogen The mode of administration is intravenous bolus injection. The dosing period is 21 days, the frequency of dosing is once a day, and the dose is 100-120 mg.
  • Efficacy The patient's mental state is good, his appetite is good, his sleep is good, his body softens and improves, and he has no other discomfort.
  • the sixth chemotherapy was performed 5 days after the end of the second course of administration. After the chemotherapy, the patient reported no discomfort.
  • Other drugs taken at the same time are: thymosin enteric-coated tablets.
  • the dosing regimen of the third course of human plasminogen is intravenous bolus.
  • the dosing period is 7 days, the frequency of dosing is once a day, and the dose is 100-115 mg.
  • Angiostatin a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell. 1994 Oct 21;79(2):315-28.
  • Angiostatin a circulating endothelial cell inhibitor that suppresses angiogenesis and tumor growth. Cold Spring Harb Symp Quant Biol. 1994;59:471-82.

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Abstract

本发明提供一种用于治疗受试者肿瘤以及维持或改善在肿瘤条件下机体器官功能并延长受试者生存期的方法，包括给药受试者预防和/或治疗有效量的纤维蛋白溶酶原激活途径的组分例如纤溶酶原。本发明还提供用于治疗受试者肿瘤的包含纤溶酶原的药物、药物组合物、制品、试剂盒。

Description

一种治疗肿瘤的方法和药物 技术领域
本发明涉及纤维蛋白溶酶原激活途径的组分例如纤溶酶原在预防和治疗肿瘤方面的作用，进而为预防和/或治疗肿瘤提供全新的治疗策略。
背景技术
恶性肿瘤是一种严重威胁人类健康的疾病。据美国癌症协会统计，癌症在发达国家为第一大死因，占其死亡人数的21.6％。据WHO统计，过去几年全球死于癌症的患者人数每年都高达700万以上，已经与心血管病的人数非常接近，预计将超过心血管疾病人数跃居全球第一大死因。肿瘤的发病率居高不下，还有上升的趋势，已经严重地威胁了生命健康，但肿瘤发病机制复杂、肿瘤治疗效果差、复发转移率高且肿瘤治疗副作用大，因此对其的预防和治疗也是研究的难点。本发明发现纤维蛋白溶酶原激活途径的组分例如纤溶酶原对肿瘤的生长有明显的抑制作用，可能成为肿瘤的预防和治疗的新途径。
发明概述
一方面，本申请涉及一种治疗肿瘤的方法，包括给药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治疗肿瘤的药物中的用途：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或包含选自如下的一种或多种化合物的药物组合物用于治疗肿瘤的用途：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。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，所述化合物为纤溶酶原。
在一些实施方案中，本申请提供了一种治疗肿瘤的方法，包括给药肿瘤受试者有效量的纤溶酶原。
在一些实施方案中，所述肿瘤为恶性肿瘤。在一些实施方案中，所述肿瘤为癌症。在一些实施方案中，所述肿瘤为实体瘤。在一些实施方案中，所述肿瘤为消化系统肿瘤或呼吸系统肿瘤。在一些实施方案中，所述肿瘤选自如下的一种或多种：口腔癌、食管癌、胃癌、小 肠癌、结肠癌、直肠癌、肺癌、肝癌、肝细胞癌、胰腺癌、胆囊癌、非小细胞肺(NSCL)癌、支气管肺泡细胞肺癌、乳腺癌、卵巢癌、宫颈癌，输卵管癌、子宫内膜癌、阴道癌、前列腺癌、尿道癌、阴茎癌、肾癌、输尿管癌、肾细胞癌、肾盂癌、膀胱癌、头颈部癌、皮肤癌、黑素瘤、间皮瘤、骨癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、胶质瘤、多形性成胶质细胞瘤、星形细胞瘤、施旺细胞瘤、室管膜瘤、髓母细胞瘤、脑脊膜瘤、鳞状细胞癌、垂体腺瘤。
在一些实施方案中，本申请所述纤溶酶原具有选自如下的一种或多种作用：减小肿瘤体积、改善肿瘤受试者的一般生存状况、延迟肿瘤的进展、抑制肿瘤细胞的生长、提高存活率、延长肿瘤受试者生存期、减轻癌性疼痛、抑制肿瘤血管形成、促进肿瘤细胞坏死或凋亡、促进抗肿瘤免疫应答、调节肿瘤相关抗原或淋巴细胞表面分子的表达、减轻癌细胞对组织器官的损伤、促进肿瘤损伤组织结构或功能恢复。
在一些实施方案中，所述受试者血纤溶酶原水平或肿瘤组织中或未患肿瘤的组织中纤溶酶原水平高于、等于或低于健康受试者血纤溶酶原水平或未患肿瘤的相应组织中的纤溶酶原水平。在一些实施方案中，所述受试者血纤维蛋白水平或肿瘤组织中或未患肿瘤的组织中纤维蛋白水平高于、等于或低于健康受试者血纤维蛋白水平或未患肿瘤的相应组织中的纤维蛋白水平。
在一些实施方案中，所述纤溶酶原与选自如下的一项或多项联合施用：化疗、放疗、手术疗法、细胞疗法和免疫疗法。在一些实施方案中，所述化疗药物包括例如烷化剂、抗代谢物(例如叶酸类似物)、嘧啶类似物、嘌呤类似物、抗有丝分裂药物、抗生素、细胞因子、铂配位复合物、取代的脲、激素、肾上腺皮质类固醇拮抗剂、抗雌激素、抗雄激素等。
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守取代变体。在一些实施方案中，所述纤溶酶原是包含丝氨酸蛋白酶结构域和/或赖氨酸结合结构域的纤溶酶原蛋白。在一些实施方案中，所述 纤溶酶原是包含与序列14具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并具有蛋白水平活性的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原与序列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，本申请所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。在一些实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。
在一些实施方案中，所述受试者是人。在一些实施方案中，所述受试者低下、缺乏或缺失纤溶酶原。在一些实施方案中，所述低下、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。在一些实施方案中，所述其他药物为抗肿瘤药物。
在前述方法的一些实施方案中，所述纤溶酶原可通过全身或局部给药，优选通过以下途径施用：静脉内、肌内、皮下给予纤溶酶原来进行治疗。在前述方法的一些实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在前述方法的一些实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。
本发明明确涵盖了属于本发明实施方案之间的技术特征的所有组合，并且这些组合后的技术方案在本申请中已经明确公开，就像上述技术方案已经单独且明确公开一样。另外，本发明还明确涵盖各个实施方案及其要素的之间的组合，该组合后的技术方案在本文中明确公开。
附图说明
图1结肠癌模型小鼠尾静脉给予纤溶酶原23天肿瘤体积测量结果。结果显示，给药期间给纤溶酶原组结肠肿瘤生长速度明显小于给溶媒PBS对照组，并且在第20和24天，两组小鼠肿瘤大小的统计学差异接近显著(P＝0.06)；在第17天纤溶酶原组肿瘤体积相较于给PBS对照组减小了40％，第20天减小了26％，第24天减小了32％。
图2结肠癌模型小鼠尾静脉给予纤溶酶原后肿瘤体积变化计算结果。计算第20天肿瘤体积与第10、13天肿瘤体积差值和第24天肿瘤体积与第10天肿瘤体积的差值。结果显示，给纤溶酶原组肿瘤 体积增加值明显小于给溶媒PBS对照组，且统计差异显著(*表示P<0.05)。
图3A-B结肠癌模型小鼠尾静脉给予纤溶酶原29天后肿瘤肿块CD31免疫组化染色代表性图片。图3A为给溶媒PBS对照组，图3B为给纤溶酶原组。结果显示，给纤溶酶原组肿瘤组织中CD31的表达(箭头标识)明显少于给溶媒PBS对照组。说明纤溶酶原能够抑制肿瘤组织内部血管内皮细胞标志物CD31的表达，提示纤溶酶原能抑制结肠癌肿瘤组织内部新生血管的形成。
图4A-B结肠癌模型小鼠尾静脉给予纤溶酶原29天后肿瘤组织LYVE-1免疫组化染色代表性图片。图4A为给溶媒PBS对照组，图4B为给纤溶酶原组。结果显示，给纤溶酶原组肿瘤组织LYVE-1的表达(箭头标识)明显少于给溶媒PBS对照组。提示纤溶酶原能够抑制结肠癌内部淋巴管的形成和癌组织的转移。
图5A-D结肠癌模型小鼠尾静脉给予纤溶酶原29天后肿瘤肿块H&E染色代表性图片。图5A、C为给溶媒PBS对照组，图5B、D为给纤溶酶原组。结果显示，给纤溶酶原组和给溶媒PBS对照组结肠癌皮下肿块均呈现有不同程度的坏死，给纤溶酶原组较之给溶媒PBS对照组坏死严重，坏死面积广。此外，非坏死区域给纤溶酶原组肿瘤细胞数明显少于给溶媒PBS对照组。
图6A-B结肠癌模型小鼠给予纤溶酶原或溶媒后肿瘤体积测量结果及第13天与第4天两组小鼠肿瘤体积差值结果。A为肿瘤体积测量结果，B为第13天与第4天两组小鼠肿瘤体积差值结果。结果显示，第7、10和13天溶媒组小鼠肿瘤体积大于给纤溶酶原组小鼠，且第13天统计差异接近显著(P＝0.05)。比较分析了第13天与第4天溶媒组和给纤溶酶原组肿瘤体积的差值，结果显示，溶媒组小鼠增加的肿瘤体积明显大于给纤溶酶原组小鼠，且统计差异显著(P＝0.01)。以上表明纤溶酶原能够抑制结肠癌模型小鼠肿瘤生长。
图7肺癌模型小鼠尾静脉给予纤溶酶原第7天机械触诱发痛感应能力检测结果。结果显示，空白对照组小鼠痛觉阈值正常，溶媒组痛觉阈值升高，与溶媒组相比，给纤溶酶原组小鼠的痛觉感应阈值明 显降低，两组比较统计P值为0.003，且给药组痛觉阈值接近于空白对照组小鼠。结果表明，纤溶酶原能够明显改善肺癌模型小鼠痛觉敏感性。
图8A-B尾静脉给予纤溶酶原第20天肺癌模型小鼠拍摄代表性图片。图A为给溶媒对照组，图B为给纤溶酶原组。图片显示，第20天，给溶媒PBS对照组小鼠立毛，行动迟缓，精神萎靡，肿瘤严重破裂，肿瘤伤口明显溃烂出血(箭头标识)；给纤溶酶原组小鼠行动自如，精神状态良好，肿瘤无明显破溃现象，肿瘤表皮有少量结痂。该结果表明纤溶酶原能够改善肺癌模型小鼠一般身体状况和精神状态。
图9肺癌模型小鼠尾静脉给予纤溶酶原24天生存曲线。由生存曲线可知，观察期间给溶媒对照组小鼠的生存率明显低于给纤溶酶原组，且统计差异显著(P＝0.03)。结果表明，纤溶酶原能提高肺癌模型小鼠生存率，延长小鼠生存时间。
图10肺癌模型小鼠给予纤溶酶原或溶媒14天肿瘤系数结果。结果显示，给纤溶酶原组肿瘤系数明显小于溶媒组，且统计差异接近显著(P＝0.09)。
图11肺癌模型小鼠给予纤溶酶原或溶媒14天肿瘤体积结果。结果显示，给纤溶酶原组小鼠肿瘤体积明显小于溶媒组，且在第4天统计差异极为显著(**表示P<0.01)。
图12肺癌模型小鼠给予纤溶酶原或溶媒7天旷场实验边界静息时间率结果。结果显示，空白对照小鼠有一定的边界静息时间率；溶媒组小鼠边界静息时间率明显多于空白对照组小鼠；给纤溶酶原组小鼠边界静息时间率明显少于溶媒组，且统计差异显著(*表示P<0.05)。该结果表明纤溶酶原能促进肺癌模型小鼠活动行为恢复。
图13肺癌模型小鼠给予纤溶酶原或溶媒后肿瘤体积测量结果。结果显示，每个测量时间点给纤溶酶原组小鼠肿瘤体积明显小于溶媒组小鼠肿瘤体积，给药第4天统计P值为0.22，给药第7天统计P值为0.003，给药第10天P值为0.07。说明纤溶酶原能够明显抑制肺癌模型小鼠肿瘤生长。
图14A-B癌性疼痛模型小鼠冷热盘痛觉检测结果。A为检测中后脚收缩抬起温度统计结果，B为检测中后脚收缩抬起时间统计结果。结果显示，溶媒组小鼠后脚收缩抬起时温度明显高于空白对照组小鼠，给药组小鼠后脚收缩抬起时温度明显低于溶媒组小鼠且接近于空白对照组小鼠。统计学分析结果显示，雌性给药组和溶媒组小鼠之间比较P＝0.007，雄性给药组和溶媒组之间P＝0.043。溶媒组小鼠后脚收缩抬起时间明显早于空白对照组小鼠，给药组小鼠后脚收缩抬起时时间明显晚于溶媒组小鼠且接近于空白对照组小鼠。统计学分析结果显示，雌性给药组和溶媒组小鼠之间比较P＝0.004，雄性给药组和溶媒组之间P＝0.023。以上结果说明，纤溶酶原能够缓解癌性疼痛模型小鼠痛觉。
图15给药纤溶酶原后癌性疼痛模型小鼠钳式压痛仪检测结果。结果显示，给药组小鼠疼痛阈值明显高于溶媒组，且统计分析P值为0.006。提示纤溶酶原能够缓解癌性疼痛模型小鼠痛觉。
图16给药纤溶酶原后肺癌模型小鼠肿瘤体积测量结果。结果显示，给药组小鼠肿瘤体积在Day0时于溶媒组无差别，在Day4、Day7和Day10时给药组肿瘤体积明显小于溶媒组，且统计分析P值分别为0.22、0.003和0.07。说明纤溶酶原能够明显抑制肺癌肿瘤生长。
图17食道癌患者给予纤溶酶原和化疗治疗后食道电子胃镜检测结果。第1疗程用药前和第3疗程结束4周后，患者进行电子胃镜检测。结果显示，给药前患者自距门齿20cm-30cm可见食道肿物(箭头标识)，累及食道3/4周，肿物脆易出血，食道狭窄，镜身勉强通过。第3个疗程结束4周后，电子胃镜检测结果显示，距离门齿21cm见一0.8cm大小的隆起型病变(箭头标识)，黏膜较光滑，22-24cm处可见一2cm大小的溃疡隆起型病变，占2/5周；27cm处可见一约0.3cm息肉，粘膜光滑，三个病灶互不相连。患者经纤溶酶原和化疗治疗后食道癌肿物体积明显缩小。
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(也称为：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包括：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(也称为：纤溶酶)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结构域和丝氨酸蛋白酶结构域)的tPA或uPA变体和类似物。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式， 以及通过添加、删除和/或取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸、仍然具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA活性的蛋白质。例如，纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”包括通过例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个保守性氨基酸取代获得的这些蛋白质的突变变体。
本发明的“纤溶酶原变体”涵盖与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性的蛋白质。例如本发明的“纤溶酶原变体”可以是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。具体地，本发明纤溶酶原变体包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式，以及通过保守性氨基酸取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸获得的这些蛋白质的突变变体。
本发明的纤溶酶原可以为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体，例如序列2、6、8、10或12所示的纤溶酶原，例如序列2所示的人天然纤溶酶原。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“类似物”包括分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用的化合物。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和“类似物”涵盖包含一个或多个结构域(例如一个或多个kringle结构域和丝氨酸蛋白酶结构域)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结构域和丝氨酸蛋白酶结构域)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结构域和丝氨酸蛋白酶结构域)的纤维蛋白溶酶“变体”和“类似物”，例如小纤维蛋白溶酶(mini-plasmin)和δ-纤维蛋白溶酶(delta-plasmin)。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的“变体”或“类似物”是否分别具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的活性，或者是否分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用可以通过本领域已知方法进行检测，例如，通过基于酶谱法(enzymography)、ELISA(酶联免疫吸附测定)和FACS(荧光激活细胞分选方法)通过激活的纤维蛋白溶酶活性水平来衡量，例如可以参照选自如下文献中记载的方法测量：Ny，A.，Leonardsson，G.，Hagglund，A.C，Hagglof，P.，Ploplis，V.A.，Carmeliet，P.and Ny，T.(1999).Ovulation inplasminogen-deficient mice.Endocrinology 140，5030-5035；Silverstein RL,Leung LL,Harpel PC,Nachman RL(November 1984)."Complex formation of platelet thrombospondin with plasminogen.Modulation of activation by tissue activator".J.Clin.Invest.74(5):1625–33；Gravanis I,Tsirka SE(February 2008)."Tissue-type plasminogen activator as a therapeutic target in stroke".Expert Opinion on Therapeutic Targets.12(2):159–70；Geiger M,Huber K,Wojta J,Stingl L,Espana F,Griffin JH,Binder BR(Aug 1989)."Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo".Blood.74(2):722–8.
在本发明的一些实施方案中，本发明的“纤维蛋白溶酶原激活途径的组分”为纤溶酶原。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。在一些实施方案中，所述纤溶酶原为包含一个或多个kringle结构域的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原为包含丝氨酸蛋白酶结构域的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原为包含如序列14所示的丝氨酸蛋白酶结构域的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原为包含与序列14所示的氨基酸序列有至少 80％、85％、90％、95％、96％、97％、98％、99％同一性的丝氨酸蛋白酶结构域的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原为天然或合成的人全长纤溶酶原。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。
在一些实施方案中，所述纤溶酶原为与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性并且仍然具有纤溶酶原活性的蛋白质。在一些实施方案中，纤溶酶原是序列2、6、8、10或12的保守取代变体。
本申请的“纤溶酶原活性”指纤溶酶原对其受体或底物的赖氨酸结合活性和蛋白水解活性。
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[1]。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 [2-3]。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。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) [4-5]。
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 [6-7]。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 [8-9]。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 [10]。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringles结构域(kringle 1，kringle 2，kringle3，kringle 4，kringle 5)，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，因此也称为赖氨酸结合结构域。在本申请中，赖氨酸结合结构域指选自kringle 1，kringle 2，kringle3，kringle 4和kringle 5中任一个、二个、三个、四个或五个kringle的结构区域。多个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 [11]。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 [7,12-13]。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 [14]。此外，纤溶酶具有激活某些潜在形式的生长因子的能力 [15-17]。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，是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或简称蛋白酶结构域、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，Kringle 1包括残基Cys103-Cys181，Kringle 2包括残基Glu184-Cys262，Kringle 3包括残基Cys275-Cys352，Kringle 4包括残基Cys377-Cys454，Kringle 5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶结构域包括残基Val581-Arg804。
Glu-纤溶酶原是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)，氨基酸序列如序列2所示，编码该序列的cDNA序列如序列1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，氨基酸序列如序列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，是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶结构域 [18-19]，有文献报道了delta-纤溶酶原的氨基酸序列(序列8) [19]，编码该氨基酸序列的cDNA序列如序列7。小纤溶酶原(Mini-plasminogen)由Kringle5和丝氨酸蛋白酶结构域组成，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) [20]，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) [21]，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)，本专利序列参考专利文献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。本申请中丝氨酸蛋白酶结构域氨基酸序列如序列14所示，编码该氨基酸序列的cDNA序列如序列13所示。
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Kringle结构域则能够与存在于受体和底物上的赖氨酸残基结合。本申请纤溶酶原涵盖了包含选自Kringle 1、Kringle 2、Kringle 3、Kringle 4和Kringle 5中的一个或多个Kringle结构域的纤溶酶原。
“丝氨酸蛋白酶结构域”也称蛋白酶结构域，是纤溶酶原发挥蛋白水解功能的结构域。本发明涉及纤溶酶原的技术方案涵盖了包含丝氨酸蛋白酶结构域的纤溶酶原的所有技术方案。本发明所述的包含丝氨酸蛋白酶结构域的纤溶酶原片段为包含纤溶酶原的丝氨酸蛋白酶结构域的蛋白质。在一些实施方案中，本申请所述的包含丝氨酸蛋白酶结构域的纤溶酶原片段为包含序列14所示氨基酸序列的蛋白质。在一些实施方案中，本申请所述的包含丝氨酸蛋白酶结构域的纤溶酶原片段为包含与序列14具有至少80％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列的蛋白质。在一些实施方案中，纤溶酶原包含序列14的保守取代变体。
目前，对于纤溶酶原及其活性(例如蛋白水解活性)的测定方法包括：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，受检血浆中的纤溶酶原在SK的作用下，转变成纤溶酶，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纤溶酶原活性(例如蛋白水解活性)进行测定。
血管抑素(Angiostatin)分子量为38KD，是纤溶酶原的一部分 [22-23]，其氨基酸序列与纤溶酶原5个Kringle中的Kringle1-Kringle4部分同源性高达98﹪。血管抑素在体内是由纤溶酶原在弹性蛋白酶、不同基质金属蛋白酶以及其它蛋白水解酶等作用下切割而形成的 [24-25]。血管抑素能够结合包括angiomotin,annexin II,tPA以及CD26等在内的多种蛋白质 [26]，并且研究显示血管抑素可以与上述几种蛋白相互作用抑制血管形成。
研究发现，血管抑素能特异性抑制血管内皮细胞增殖、迁移以及诱导细胞凋亡，其机理可能与血管抑素中kringle的赖氨酸结合(lysine-binding)活性紧密相关。纤溶酶原Kringle5(K5)亦能显著地抑制内皮细胞的生长，且活性明显高于Angiostatin。但研究表明，K5虽同样具有赖氨酸结合活性，其对血管形成的抑制作用却并不依赖于赖氨酸结合活性，K5结构具有独特的作用机制 [27]。随着后续大量研究表明，不同形式的纤溶酶原的Kringle结构已经展示了不同抑制血管活性，因此，Angiostatin的概念已由单一的蛋白扩展为一类蛋白(angiostatin isoforms or angiostatin related protein)，包含了一系列含有纤溶酶原不同Kingle结构和相似生物活性片段 [28-29]。
1971年，哈佛大学Judah Folkman教授首次提出切断肿瘤血管、饿死癌细胞理论 [30]，并在1994年发现了血管抑素这一由内源的纤溶酶原通过MMPs酶解而产生的具有抑制血管形成作用的分子 [23]。后来多个研究数据显示Angiostatin能够通过抑制血管形成来抑制肿瘤的生长和转移。然而，在Folkma教授等人的研究中，明确指出这些作用只有血管抑素或kringle5具备，完整的纤溶酶原并不具有抑制血管和抑制肿瘤的作用 [23,31,32]。因此，佛克曼教授等人提出了在临床上可以通过使用Angiostatin来抑制肿瘤血管形成，从而抑制癌症。然而，经过20年左右的努力，Angiostatin及其类似物并未如人们想象的那样成为一类有效的抑制肿瘤血管生成的药物 [33-34]。反而是VEGF 抗体等抑制物从2000年左右开始逐渐成为一类主流的抑制肿瘤血管生成的药物开发策略，并在结肠癌等临床上得到广泛使用 [35]。由上可知，抑制血管形成抑制肿瘤的思路是正确的，但直接使用Angiostain及其类似物抑制肿瘤的方法可能存在缺陷。
然而，在我们的研究中，我们明确看到了完整的纤溶酶原具有抑制血管生成、抑制肿瘤生长和转移的作用。特别是当我们基本上重复了佛克曼教授等1994年发表在《CELL》和1996年发表在《NATURE MEDICINE》的实验 [23,36]，只是将纤溶酶原的注射量提升后，我们清楚地得到了与佛克曼教授等人不同的结果，注射人纤溶酶原后不但能够明显地减少血管生成，而且能抑制肠癌肿瘤体积增大，并能显著地延长小鼠在LLC肿瘤条件下的生存期和生存率。值得指出的是，在这些实验中我们提高了纤溶酶原的注射量，这是因为我们认为在《CELL》等文章中，Angiostatin的对照用的是同样质量的纤溶酶原是值得商榷的——既然Angiostatin是纤溶酶原蛋白酶水解的产物，那么Angiostatin的对照应当用至少是同样摩尔量的纤溶酶原而非同样质量的纤溶酶原。不管怎样，这些结果说明直接使用纤溶酶原本身就可以抑制血管生成。这种作用很有可能是体内额外增加的纤溶酶原被肿瘤微环境内聚集的MMPs和elastase等蛋白水解酶降解从而促进产生高量的Angiostatin及/或其类似物，并进而产生抑制肿瘤血管生成的功能。也就是说，抑制血管生成可以不通过直接增加机体内的Angiostatin及/或其类似物，而直接通过调节机体内的纤溶酶原而实现。这为抑制肿瘤开辟了一条全新的治疗策略。
本申请纤溶酶原涵盖具有纤溶酶原活性的纤溶酶原片段，例如包含一个或多个不同Kingle结构域的纤溶酶原片段，或包含与一个或多个Kingle结构域有相似生物活性的变体的纤溶酶原片段。
“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。
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“纤维蛋白溶酶原”、“纤维蛋白溶解酶原”可互换使用，含义相同。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。
“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。
在本申请中，所述纤溶酶原“缺乏”的含义为受试者体内纤溶酶原的含量或活性比正常人低，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”的含义为受试者体内纤溶酶原的含量或活性显著低于正常人，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。
“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”和“处理”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，和/或部分或完全治愈疾病和/或其症状，并且包括：(a)预防疾病在受试者体内发生，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。
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/或治疗的纤溶酶原的量。“治疗有效量”会根据所使用的纤溶酶原、要治疗的受试者的疾病和/或其症状的严重程度以及年龄、体重等而变化。
本发明纤溶酶原的制备
纤溶酶原可以从自然界分离并纯化用于进一步的治疗用途，也可以通过标准的化学肽合成技术来合成。当通过化学合成多肽时，可以经液相或固相进行合成。固相多肽合成(SPPS)(其中将序列的C末端氨基酸附接于不溶性支持物，接着序贯添加序列中剩余的氨基酸)是适合纤溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤溶酶原。用于固相合成的技术描述于Barany和Solid-Phase Peptide Synthesis；第3-284页于The Peptides:Analysis,Synthesis,Biology.第2卷：Special Methods in Peptide Synthesis,Part A.,Merrifield,等J.Am.Chem.Soc.,85:2149-2156(1963)；Stewart等,Solid Phase Peptide Synthesis,2nd ed.Pierce Chem.Co.,Rockford,Ill.(1984)；和Ganesan A.2006 Mini Rev.Med Chem.6:3-10和Camarero JA等2005 Protein Pept Lett.12:723-8中。简言之，用其上构建有肽链的功能性单元处理小的不溶性多孔珠。在偶联/去保护的重复循环后，将附接的固相游离N末端胺与单个受N保护的氨基酸单元偶联。然后，将此单元去保护，露出可以与别的氨基酸附接的新的N末端胺。肽保持固定在固相上，之后将其切掉。
可以使用标准重组方法来生产本发明的纤溶酶原。例如，将编码纤溶酶原的核酸插入表达载体中，使其与表达载体中的调控序列可操作连接。表达调控序列包括但不限于启动子(例如天然关联的或异源的启动子)、信号序列、增强子元件、和转录终止序列。表达调控可以是载体中的真核启动子系统，所述载体能够转化或转染真核宿主细胞(例如COS或CHO细胞)。一旦将载体掺入合适的宿主中，在适合于核苷酸序列的高水平表达及纤溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆主题蛋白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化合物例如纤溶酶原。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，除本发明的化合物例如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，抗肿瘤药物、肝脏保护药物、激素类药物等。
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,EP58,481)，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，例如通过静脉内，腹膜内，皮下，颅内，鞘内，动脉内(例如经由颈动脉)，肌内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1-10mg/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，其包含可用于治疗肿瘤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肿瘤。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
以下实施例中使用的纤溶酶原为人纤溶酶原，来自捐赠者血浆，基于文献所描述的纯化方法 [37-39]，进行部分工艺优化，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％。
实施例1纤溶酶原抑制结肠癌生长
CT26.WT小鼠结肠癌细胞是N-nitroso-N-methylurethane-(NNMU)诱导形成的未分化结肠癌细胞株。它克隆形成的细胞株命名为CT26.WT(ATCC CRL-2638)。
CT26.WT(ATCC CRL-2638)小鼠结肠癌细胞购自中国科学院上海生命科学研究院，以下简称CT26。将CT26细胞置入含10％胎牛血清的PRIM-1640(GIBCO,货号31800022)培养液中，37摄氏度，5％二氧化碳培养箱培养。待培养瓶中细胞生长达到90％后，用胰酶(0.05％胰酶中加入0.02％EDTA)(Sigma,74799)消化处理，然后用生理盐水洗3次，生理盐水重悬，浓度为10 7个/ml。
7-9周龄雄性Balb/c小鼠14只，小鼠按照50mg/kg体重腹腔注射戊巴比妥钠进行麻醉。麻醉后小鼠用27-gauge针背部皮下接种10 6个/100μl CT26重悬液，然后每天用游标卡尺测量小鼠皮下肿瘤的体积(长x宽 2x 0.52) [40-41]，连续测量七天。根据第七天肿瘤的体积将小鼠随机分为两组，给纤溶酶原组和给PBS对照组每组各7只。CT26接种第八天开始给药，并定为第1天，给纤溶酶原组小鼠尾静脉注射人纤溶酶原1mg/0.1mL/只/天，给溶媒PBS(20mM柠檬酸-柠檬酸钠，2％盐酸精氨酸，3％甘露醇，PH7.4，以下相同)对照组尾静脉注射同体积的PBS，连续给药23天。第1、4、7、10、13、17、20、24天测量肿瘤的长和宽，计算肿瘤体积。
肿瘤体积
结果显示，给药期间给纤溶酶原组结肠肿瘤生长速度明显小于给溶媒PBS对照组，并且在第20和24天两组小鼠肿瘤大小的统计学差异接近显著(P＝0.06)；在第17天给纤溶酶原组肿瘤体积相较于给溶媒PBS对照组减小了40％，第20天减小26％，第24天减小了32％(图1)。
肿瘤体积差
计算第20天肿瘤体积与第10、13天肿瘤体积差值和第24天肿瘤体积与第10天肿瘤体积的差值。结果显示，给纤溶酶原组肿瘤体积增加值明显小于给溶媒PBS对照组，且统计差异显著(*表示P<0.05)(图2)。
上述结果显示，纤溶酶原可以抑制结肠癌细胞的生长。
实施例2纤溶酶原抑制结肠癌组织内微血管的形成
7-9周龄雄性Balb/c小鼠14只，小鼠按照50mg/kg体重腹腔注射戊巴比妥钠进行麻醉，麻醉后小鼠用27-gauge针背部皮下接种10 6个/100μl CT26重悬液，然后每天用游标卡尺测量小鼠皮下肿瘤的体积(长x宽 2x 0.52) [40-41]，连续测量七天。根据第七天肿瘤的体积模型组小鼠随机分为两组，给纤溶酶原组和给PBS对照组，每组各7只。CT26接种第八天开始给药，并定为第1天，给纤溶酶原组小鼠尾静脉注射人纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药29天。第30天处死小鼠，取材的肿瘤组织于10％中性甲醛中固定24-48小时，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。肿瘤组织切片厚度为4μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.，USA)封闭1小时；之后，弃除羊血清液。兔源抗CD31抗体(Abcam，ab28364)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水返蓝5分钟，然后PBS洗1次。梯度脱水透明并封片，切片在200倍光学显微镜下观察。
CD31通常位于血管内皮细胞、血小板、巨噬细胞和kuffer细胞、粒细胞、T/NK细胞、淋巴细胞、巨核细胞、破骨细胞、嗜中性粒细胞。在免疫组化中，CD31是血管内皮细胞的标志物，可用于评估肿瘤血管生成 [42-43]。
结果显示，给纤溶酶原组(图3B)肿块CD31的表达(箭头标识)明显少于给溶媒PBS对照组(图3A)。说明纤溶酶原能够抑制肿瘤组织血管内皮细胞标志物CD31的表达，提示纤溶酶原能抑制结肠癌组织内部新生血管的形成。
实施例3纤溶酶原抑制结肠癌淋巴管形成
7-9周龄雄性Balb/c小鼠14只，小鼠按照50mg/kg体重腹腔注射戊巴比妥钠进行麻醉，麻醉后小鼠用27-gauge针背部皮下接种10 6个/100μl CT26重悬液，然后每天用游标卡尺测量小鼠皮下肿瘤的体积(长x宽 2x 0.52) [40-41]，连续测量七天。根据第七天肿瘤的体积模型组小鼠随机分为两组，给纤溶酶原组和给PBS对照组，每组各7只。CT26接种第八天开始给药，并定为第1天，给纤溶酶原组小鼠尾静脉注射人纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药29天。第30天处死小鼠，取材肿瘤组织于10％中性甲醛中固定24-48小时，脱水包埋。固定后的组织样本经酒精梯度脱水和二甲苯透明后进行石蜡包埋。肿瘤组织切片厚度为4μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.,USA)封闭1小时；之后，弃除羊血清液。用兔源抗LYVE-1抗体(Abcam，ab14917)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.,USA)显色，水洗3次后苏木素复染30秒，流水返蓝5分钟，然后PBS洗1次。梯度脱水透明并封片，切片在400倍光学显微镜下观察。
淋巴管内皮透明质酸受体1(Lymphatic vessel endothelial hyaluronan receptor 1，LYVE-1)是淋巴管内皮细胞表面受体，可以作为淋巴管内皮细胞标志物 [44-45]。
结果显示，给纤溶酶原组(图4B)肿块LYVE-1的表达(箭头标识)明显少于给溶媒PBS对照组(图4A)。实验结果表明纤溶酶原可以抑 制肿瘤内淋巴管内皮细胞标志物LYVE-1的表达，提示纤溶酶原有抑制结肠癌组织内部淋巴管形成的作用。
实施例4纤溶酶原促进结肠癌细胞的坏死
7-9周龄雄性Balb/c小鼠14只，小鼠按照50mg/kg体重腹腔注射戊巴比妥钠进行麻醉，麻醉后小鼠用27-gauge针后背部皮下接种10 6个/100μl CT26重悬液，然后每天用游标卡尺测量小鼠皮下肿瘤的体积(长x宽 2x 0.52) [40-41]，连续测量七天。根据第七天肿瘤的体积模型组小鼠随机分为两组，给纤溶酶原组和给PBS对照组7只，每组各7只。CT26接种第八天开始给药，并定为第1天，给纤溶酶原组小鼠尾静脉注射人纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药29天。第30天处死小鼠，取材的肿瘤组织于10％中性甲醛中固定24-48小时。固定后的肿瘤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，切片脱蜡复水并用苏木素和伊红染色(H&E染色)，1％盐酸酒精分化，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。图5C、图5D分别为图5A、图5B方框区域放大图片。
结果显示，给纤溶酶原组(图5B)和给溶媒PBS对照组(图5A)结肠癌皮下肿块均呈现有不同程度的坏死(箭头标识)，给纤溶酶原组较之给溶媒PBS对照组坏死严重，坏死的面积广。此外，给纤溶酶原组(图5D)非坏死区域肿瘤细胞数明显少于给溶媒PBS对照组(图5C)。说明纤溶酶原可促进结肠癌细胞坏死。
实施例5纤溶酶原抑制结肠癌模型小鼠肿瘤生长
取9周龄的Balb/c雌性小鼠27只，称重后随机分为2组，空白对照组小鼠9只，模型组小鼠18只。分组完成后，模型组所有小鼠使用2％异氟烷麻醉，消毒一侧腋下并皮下注射1×10 6个细胞的CT-26细胞悬液，观察7天。肿瘤细胞接种第8天所有小鼠开始进行肿瘤体积检测，待小鼠肿瘤体积达到100mm 3以上后，对所有小鼠进行称重，并根据检测体重及肿瘤体积结果进行随机分组，溶媒组9只小鼠，给药组9只小鼠。分组完成后，给药组小鼠按照1mg/0.1ml/只/天尾静脉注射人纤溶酶原，溶媒组小鼠及空白对照组小鼠尾静脉 注射相同体积溶媒PBS，给药第一天定义为第1天，连续给药13天。给药第0天(给药前)、第4天、第7天、第10天和第13天用游标卡尺测量小鼠皮下肿瘤的体积(长x宽 2x 0.52) [40-41]。
结果显示，第7、10和13天溶媒组小鼠肿瘤体积大于给纤溶酶原组小鼠，且第13天统计差异接近显著(P＝0.05)(图6A)。比较分析了第13天与第4天溶媒组和给纤溶酶原组肿瘤体积的差值，结果显示，溶媒组小鼠增加的肿瘤体积明显大于给纤溶酶原组小鼠，且统计差异显著(P＝0.01)(图6B)。以上结果提示纤溶酶原能够抑制结肠癌肿瘤生长。
实施例6纤溶酶原改善肺癌模型小鼠痛觉敏感性
小鼠Lewis肺癌细胞(Lewis lung cancer cells，LLC)购自中国科学院上海生命科学研究院，以下简称LLC。将LLC置入含10％胎牛血清的DMEM(GIBCO,货号15140122)培养液中，37摄氏度，5％二氧化碳培养箱培养。待培养瓶中细胞生长达到90％后，用胰酶(0.05％胰酶中加入0.02％EDTA)(Sigma,74799)消化处理，然后用生理盐水洗3次。细胞用生理盐水重悬，显微镜下计数并确定细胞是单个悬浮的。最后细胞用生理盐水重悬，细胞浓度为10 7个/ml，放在冰上待用。
取8-9周龄的C57小鼠30只，称重后随机分为2组，空白对照组小鼠6只，模型组小鼠24只。分组完成后，小鼠使用2％异氟烷用呼吸麻醉机进行麻醉；左腿脱毛，在股骨远端髁突皮肤上加碘溶液消毒后，作0.5cm浅切口；然后在髌骨韧带上进行钝性解剖，暴露股骨远端髁部，损伤最小；模型组小鼠将10ul LLC细胞悬液(2×10 5细胞/ul)用50ul无菌显微注射器缓慢注射至左股骨远端髓腔。对照组小鼠骨腔注射10ul无菌生理盐水；注射完闭后，注射器放置90s以允许细胞填充骨腔，注射孔用无菌骨蜡密封，以防止移除注射器后细胞渗漏，然后用5.0丝线缝合伤口；手术完成的小鼠放置与加热垫上保温，待小鼠苏醒后放回笼盒。手术造模第8天所有小鼠称量体重及电子疼痛检测，模型组小鼠根据疼痛及体重结果进行分组，溶媒组12只，给药组12只。分组完成后，给药组小鼠按照50mg/kg/只/天尾 静脉注射人纤溶酶原，溶媒组小鼠及空白对照组小鼠按照5ml/kg/只/天尾静脉注射溶媒，给药第一天定义为给药第1天。第7天用Von-Frey纤维丝(Stoelting，USA)检测动物对机械性损伤的敏感程度。以2.0g力为起始力，先检测其左脚。若5次刺激有4次有缩爪反应即为阳性，记为该动物对机械损伤的阈值。若2.0g力刺激反应为阴性则以大一级的力刺激其右脚，若为阳性则计为其阈值，若为阴性则继续以大一级的力刺激其左脚，如此交替刺激小鼠右脚直到出现阳性反应 [48]。Von-Frey纤维丝电子测痛实验是临床前研究和临床研究常用癌性疼痛常用评价方法。
结果显示，空白对照组小鼠痛觉阈值正常，溶媒组痛觉阈值升高，与溶媒组相比，给纤溶酶原组小鼠的痛觉感应阈值明显降低，两组比较统计P值为0.003，且给药组痛觉阈值接近于空白对照组小鼠(图7)。结果表明，纤溶酶原能够明显改善肺癌模型小鼠痛觉敏感性。
实施例7纤溶酶原改善肺癌模型小鼠一般身体状况和精神状态
6-7周龄的雌性C57小鼠11只，根据体重随机分为两组，给纤溶酶原组(5只)和给溶媒对照组(6只)。两组小鼠按照50mg/kg体重腹腔注射戊巴比妥钠，小鼠麻醉后背部皮下接种2x 10 6个/200μl LLC重悬液 [46,47]。小鼠接种细胞当天开始给药，记为第1天，给纤溶酶原组小鼠尾静脉注射人纤溶酶原1mg/0.1mL/只/天，给溶媒对照组每天尾静脉注射同体积的溶媒PBS，每天观察拍摄记录肿瘤情况和小鼠精神状态。
结果显示，第20天，给溶媒PBS对照组(图8A)小鼠立毛，行动迟缓，精神萎靡，肿瘤严重破裂，肿瘤伤口明显溃烂出血(箭头标识)；给纤溶酶原组(图8B)小鼠行动自如，精神状态良好，肿瘤无明显破溃现象，肿瘤表皮有少量结痂。该观察结果表明纤溶酶原能够改善肺癌模型小鼠一般身体状况和精神状态。
实施例8纤溶酶原提高肺癌模型小鼠生存率
6-7周龄的雌性C57小鼠11只，根据体重随机分为两组，给纤溶酶原组(5只)和给溶媒对照组(6只)。两组小鼠按照50mg/kg体重腹腔注射戊巴比妥钠，小鼠麻醉后背部皮下接种2x 10 6个/200μl  LLC重悬液 [46,47]。小鼠接种细胞当天开始给药，记为第1天，给纤溶酶原(PLG)组小鼠尾静脉注射人纤溶酶原1mg/0.1mL/只/天，给溶媒对照组每天尾静脉注射同体积的溶媒PBS。每天观察记录小鼠生存情况，连续观察24天，观察期间动物每天按照方案给药。
由生存曲线可知，在观察的24天期间给溶媒对照组小鼠的生存率明显低于给纤溶酶原组，统计差异显著(图9)。结果表明，纤溶酶原能提高肺癌模型小鼠生存率，延长生存时间。
实施例9纤溶酶原抑制肺癌生长
13-14周龄的雄性C57小鼠30只，根据体重随机分为两组，空白对照组6只，模型组24只。两组小鼠按照使用2％异氟烷麻醉，小鼠麻醉后消毒腰椎4～6处背部皮肤并皮下接种1 x 10 6个LLC重悬液 [46,47]。细胞注射完成后注意观察小鼠肿瘤生长状态，肿瘤细胞接种七天后所有小鼠进行体重、肿瘤体积及旷场检测，模型组小鼠根据检测结果随机分为两组，溶媒对照组和给纤溶酶原组，每组各12只，并开始给药，记为第1天，给纤溶酶原组小鼠尾静脉注射人纤溶酶原1mg/0.1mL/只/天，给溶媒对照组尾静脉注射同体积的溶媒PBS，连续给药14天。于第1、4、8、11、15天测量肿瘤的体积并称量体重。第15天处死小鼠取材肿瘤组织称重。肿瘤系数＝肿瘤组织重量/体重*100。
肿瘤系数
结果显示，给纤溶酶原组肿瘤系数明显小于溶媒组，且统计差异接近显著(P＝0.09)(图10)。
肿瘤体积
结果显示，给纤溶酶原组小鼠肿瘤体积明显小于溶媒组，且在第4天统计差异极为显著(**表示P<0.01)(图11)。
以上结果表明纤溶酶原能够抑制肺癌细胞生长。
实施例10纤溶酶原促进肺癌模型小鼠活动能力恢复
13-14周龄的雄性C57小鼠30只，根据体重随机分为两组，空白对照组6只，模型组24只。两组小鼠按照使用2％异氟烷麻醉，小鼠麻醉后消毒腰椎4～6处背部皮肤并皮下接种1 x 10 6个LLC重悬液 [46,47]。细胞注射完成后注意观察小鼠肿瘤生长状态，肿瘤细胞接种七天后所有小鼠进行体重、肿瘤体积及旷场检测，模型组小鼠根据检测结果随机分为两组，溶媒对照组和给纤溶酶原组，每组各12只，并开始给药，记为第1天，给纤溶酶原组小鼠尾静脉注射人纤溶酶原1mg/0.1mL/只/天，给溶媒对照组尾静脉注射同体积的溶媒PBS，连续给药7天。于给药第8天进行旷场实验。
旷场实验
实验时，小鼠放入旷场(40x40x40cm)底面中心，同时进行摄像和计时，观察持续5分钟，每只小鼠进行3次实验。Smart系统是一个完整且方便使用的视频跟踪系统，用于评估实验动物的行为。它允许记录轨迹，活动，特定行为(如旋转，拉伸和饲养)和事件，并执行各种分析参数的计算。本实验使用Smart3.0系统记录分析小鼠的运动情况，记录小鼠的边界静息时间率。每次实验中采用70％酒精擦拭箱体防止嗅觉产生的偏好。
结果显示，空白对照小鼠有一定的边界静息时间率；溶媒组小鼠边界静息时间率明显多于空白对照组小鼠；给纤溶酶原组小鼠边界静息时间率明显少于溶媒组，且统计差异显著(*表示P<0.05)(图12)。该结果表明纤溶酶原能促进肺癌模型小鼠活动行为恢复。
实施例11纤溶酶原抑制肺癌模型小鼠肿瘤生长
取7～10周龄的SCID(重症联合免疫缺陷)小鼠(NOD.CB17-Prkdcscid/NcrCrl，简称NOD SCID，购自维通利华实验动物技术有限公司，品系代码为40624)24只，称重后随机分为2组，空白对照组小鼠8只，模型组小鼠16只。分组完成后，模型组所有小鼠使用2％异氟烷麻醉，消毒背部并皮下接种1×10 6个Lewis lung carcinoma cells-GFP细胞(稳转绿色荧光蛋白GFP的小鼠肺癌细胞，简称LLC-GFP细胞)(购自上海美轩生物科技有限公司)悬液，观察7天。肿瘤细胞接种第8天所有小鼠进行体重检测，模型组小鼠进行肿瘤体积检测，根据检测结果模型组随机分为2组，溶媒组8只小鼠、给药组8只小鼠，空白对照组小鼠不做处理。小鼠分组完成后，给药组小鼠按照1mg/0.1ml/只/天尾静脉注射人纤溶酶原，溶媒组小 鼠尾静脉注射相同体积溶媒PBS，空白对照组小鼠不做给药处理，连续给药10天。于给药第0天(给药前)、第4天、第7天和第10天用游标卡尺测量小鼠皮下肿瘤的体积(长x宽 2x 0.52) [40-41]。
结果显示，每个测量时间点给纤溶酶原组小鼠肿瘤体积明显小于溶媒组小鼠肿瘤体积，给药第4天统计P值为0.22，给药第7天统计P值为0.003，给药第10天P值为0.07(图13)。说明纤溶酶原能够明显抑制肺癌模型小鼠肿瘤生长。
实施例12纤溶酶原改善癌性疼痛模型小鼠痛觉
取8-9周龄Balb/c小鼠雌性和雄性小鼠各18只，称重后随机分为2组，空白对照组小鼠12只和模型组小鼠24只。分组完成后，小鼠使用2％异氟烷用呼吸麻醉机进行麻醉；左腿脱毛，在股骨远端髁突皮肤上加碘溶液消毒后，作0.5cm浅切口；然后在髌骨韧带上进行钝性解剖，暴露股骨远端髁部，损伤最小；模型组小鼠将5μl CT-26细胞悬液(2×10 4细胞/μl)用50μl无菌显微注射器缓慢注射至左股骨远端髓腔。对照组小鼠骨腔注射5μl无菌生理盐水；注射完闭后，注射器放置90s以允许细胞填充骨腔，注射孔用无菌骨蜡密封，以防止移除注射器后细胞渗漏，然后用5.0丝线缝合伤口；手术完成的小鼠放置与加热垫上保温，待小鼠苏醒后放回笼盒。手术造模第8天所有小鼠称量体重，模型组小鼠根据体重结果进行随机分组，分为溶媒组和给药组，每组12只。分组完成后，给药组小鼠按照50mg/kg/只/天尾静脉注射人纤溶酶原，溶媒组小鼠及空白对照组小鼠按照5ml/kg/只/天尾静脉注射溶媒PBS，给药第一天定义为第1天，连续给药7天。第8天进行冷热盘测痛检测，记录小鼠开始出现痛觉反应。检测前一天将设备温度设置20℃恒温，将小鼠逐个放入适应5min。检测当天选择冷热盘RAMP模式，设置初始温度20℃和最终温度4℃，设置初始温度到最终温度所用时间5min，待设备温度达到初始温度后，将待测小鼠置于冷板上面点击Start开始计时和降温，观察小鼠后脚收缩抬起时停止，记录小鼠后脚收缩抬起时的时间及显示温度，最长检测时间6min，连续测量3次。注意每只小鼠实验后需清洁冷热板并用消毒水擦拭后再开始下一只小鼠，避免相互干扰。
小鼠后脚收缩抬起时温度
结果显示，溶媒组小鼠后脚收缩抬起时温度明显高于空白对照组小鼠，给药组小鼠后脚收缩抬起时温度明显低于溶媒组小鼠且接近于空白对照组小鼠。统计学分析结果显示，雌性给药组和溶媒组小鼠之间比较P＝0.007，雄性给药组和溶媒组之间P＝0.043(图14A)。
小鼠后脚收缩抬起时时间
结果显示，溶媒组小鼠后脚收缩抬起时间明显早于空白对照组小鼠，给药组小鼠后脚收缩抬起时时间明显晚于溶媒组小鼠且接近于空白对照组小鼠。统计学分析结果显示，雌性给药组和溶媒组小鼠之间比较P＝0.004，雄性给药组和溶媒组之间P＝0.023(图14B)。
以上结果说明，纤溶酶原能够缓解癌性疼痛模型小鼠痛觉。
实施例13纤溶酶原改善抑制癌性疼痛模型小鼠痛觉
取8-9周龄的C57小鼠30只，称重后随机分为2组，空白对照组小鼠6只，模型组小鼠24只。分组完成后，小鼠使用2％异氟烷用呼吸麻醉机进行麻醉；左腿脱毛，在股骨远端髁突皮肤上加碘溶液消毒后，作0.5cm浅切口；然后在髌骨韧带上进行钝性解剖，暴露股骨远端髁部，损伤最小；模型组小鼠将10ul LLC细胞悬液(2×10 5细胞/ul)用50ul无菌显微注射器缓慢注射至左股骨远端髓腔。对照组小鼠骨腔注射10ul无菌生理盐水；注射完闭后，注射器放置90s以允许细胞填充骨腔，注射孔用无菌骨蜡密封，以防止移除注射器后细胞渗漏，然后用5.0丝线缝合伤口；手术完成的小鼠放置与加热垫上保温，待小鼠苏醒后放回笼盒。手术造模第8天所有小鼠称量体重及电子疼痛检测，模型组小鼠根据疼痛及体重结果进行分组，溶媒组12只，给药组12只。分组完成后，给药组小鼠按照50mg/kg/只/天尾静脉注射人纤溶酶原，溶媒组小鼠及空白对照组小鼠按照5ml/kg/只/天尾静脉注射溶媒，给药第一天定义为给药第1天，连续给药20天。第20天行钳式压痛仪检测。具体步骤为：将待测动物置于固定架上，露出左、右爪子。用钳子夹子去夹老鼠的后爪，测量其所受压力的最大值。记录所需测量压力值。每只小鼠左、右后爪各重复测量4次。
结果显示，给药组小鼠疼痛阈值明显高于溶媒组，且统计分析P值为0.006(图15)。提示纤溶酶原能够缓解癌性疼痛模型小鼠痛觉。
实施例14纤溶酶原能够抑制肺癌模型小鼠肿瘤生长
取7-10周龄的SCID(重症联合免疫缺陷)小鼠(NOD.CB17-Prkdcscid/NcrCrl，简称NOD SCID，购自维通利华实验动物技术有限公司，品系代码为40624)21只，称重后随机分为两组，空白对照组小鼠7只，模型组小鼠14只。分组完成后，模型组所有小鼠使用2％异氟烷(购自鲁南贝特制药有限公司)麻醉，消毒背部并皮下注射1×10 6个Lewis lung carcinoma cells-GFP细胞(稳转绿色荧光蛋白GFP的小鼠肺癌细胞，简称LLC-GFP细胞)(购自上海美轩生物科技有限公司)悬液，观察7天。肿瘤细胞接种第8天所有小鼠进行体重检测，模型组小鼠进行肿瘤体积检测，根据检测结果模型组随机分为2组，溶媒组7只小鼠、给药组7只小鼠，空白对照组小鼠不做处理。分组完成后，给药组小鼠按照1mg/0.1ml/只/天尾静脉注射人纤溶酶原，溶媒组小鼠尾静脉注射相同体积溶媒PBS，空白对照组小鼠不做给药处理，连续给药10天。开始给药第1天定为第0天，于第0天、第4天、第7天和第10天测量肿瘤体积。
结果显示，给药组小鼠肿瘤体积在第0天时与溶媒组无差别，在第4天、第7天和第10天时给药组肿瘤体积明显小于溶媒组，且统计分析P值分别为0.22、0.003和0.07(图16)。说明纤溶酶原能够明显抑制肺癌细胞生长。
实施例15纤溶酶原对食道癌患者的治疗作用
患者男性，72岁，意识清楚，无糖尿病和心脏病史，有高血压病史。半年前诊断为食道癌并开始化疗。该患者签署知情同意书自愿接受人纤溶酶原治疗，并获得医院伦理委员会批准。第四次化疗结束后开始使用人纤溶酶原，并在第五次、第六次和第七次化疗后也使用了人纤溶酶原治疗。
第1疗程
患者化疗后，有身体乏力和恶心症状。下唇两处破溃，双手掌颜色发红。每天服用的其它药品：血速升颗粒，升血调元颗粒，盐酸昂丹司琼片。
人纤溶酶原给药方案：给药方式为静脉推注。给药21天为一个疗程，给药频率为前5天每天两次，以后为每天一次。每天给药剂量：第1天为40mg，之后逐渐增加，第21天剂量为190mg。
给药后对患者的疗效以1-10评分的方式记录疗效，第一天没有给药时的状况记录为10，若症状加重评分增加，症状减轻评分降低，1为最轻。
给药第3天患者情况好转，总体印象评分为7分，精神状态评分为8分，食欲评分为7分，恶心状况消退评分为0分，下唇破溃处已结痂，头发基本无脱落。给药第6天患者情况进一步好转，总体印象评分为4分，精神状态评分为5分，食欲评分为6分，左右手掌颜色评分均为8分，下唇破溃基本痊愈，头发基本无脱落。第21天总体印象评分为1分，精神状态评分为0分，食欲评分为0分，左右手掌颜色评分分别为3和4分。
第2疗程
人纤溶酶原第1疗程治疗结束10天后进行了第五次化疗，化疗1天结束，感觉身体发软。同时服用的其它药品为：兰索拉唑肠溶片。
人纤溶酶原第2疗程的给药方案：给药方式为静脉推注。给药为期21天，给药频率为每天一次，剂量为100-120mg。
疗效：患者精神状态好，食欲好，睡眠好，身体发软好转，无其他不适。
第3疗程
第2疗程给药结束5天后进行了第六次化疗，化疗结束后，患者自诉没有不适。同时服用的其它药品为：胸腺肽肠溶片。
人纤溶酶原第3疗程的给药方案：给药方式为静脉推注。给药为期7天，给药频率为每天一次，剂量为100-115mg。
疗效：患者情况有所好转，相对于此次给药前总体印象评分为6分。
第1疗程用药前和第3疗程结束4周后，患者进行电子胃镜检测。结果显示，给药前患者自距门齿20cm-30cm可见食道肿物(箭头标识)，累及食道3/4周，肿物脆易出血，食道狭窄，胃镜镜身勉强通过。第3个疗程结束4周后，胃镜检测结果显示，距离门齿21cm见一0.8cm大小的隆起型病变(箭头标识)，黏膜较光滑，22-24cm处可见一2cm大小的溃疡隆起型病变，占2/5周；27cm处可见一约0.3cm息肉，粘膜光滑，三个病灶互不相连(图17)。患者经人纤溶酶原和化疗治疗后食道癌肿物体积明显缩小。
参考文献
[1]Alexander CM and Werb,Z.(1991).Extracellular matrix degradation.In Cell Biology of Extracellular Matrix,Hay ED,ed.(New York:Plenum Press),pp.255-302.
[2]Werb,Z.,Mainardi,C.L.,Vater,C.A.,and Harris,E.D.,Jr.(1977).Endogenous activiation of latent collagenase by rheumatoid synovial cells.Evidence for a role of plasminogen activator.N.Engl.J.Med.296,1017-1023.
[3]He,C.S.,Wilhelm,S.M.,Pentland,A.P.,Marmer,B.L.,Grant,G.A.,Eisen,A.Z.,and Goldberg,G.I.(1989).Tissue cooperation in a proteolytic cascade activating human interstitial collagenase.Proc.Natl.Acad.Sci.U.S.A 86,2632-2636.
[4]Stoppelli,M.P.,Corti,A.,Soffientini,A.,Cassani,G.,Blasi,F.,and Assoian,R.K.(1985).Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes.Proc.Natl.Acad.Sci.U.S.A 82,4939-4943.
[5]Vassalli,J.D.,Baccino,D.,and Belin,D.(1985).A cellular binding site for the Mr55,000form of the human plasminogen activator,urokinase.J.Cell Biol.100,86-92.
[6]Wiman,B.and Wallen,P.(1975).Structural relationship between"glutamic acid"and"lysine"forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography.Eur.J.Biochem.50,489-494.
[7]Saksela,O.and Rifkin,D.B.(1988).Cell-associated plasminogen activation:regulation and physiological functions.Annu.Rev.Cell Biol.4,93-126.
[8]Raum,D.,Marcus,D.,Alper,C.A.,Levey,R.,Taylor,P.D.,and Starzl,T.E.(1980).Synthesis of human plasminogen by the liver.Science 208,1036-1037.
[9]Castellino FJ.Biochemistry of human plasminogen.Semin Thromb Hemost.1984 Jan；10(1):18-23.
[10]Sottrup-Jensen,L.,Zajdel,M.,Claeys,H.,Petersen,T.E.,and Magnusson,S.(1975).Amino-acid sequence of activation cleavage site in plasminogen:homology with"pro"part of prothrombin.Proc.Natl.Acad.Sci.U.S.A 72,2577-2581.
[11]Collen,D.and Lijnen,H.R.(1991).Basic and clinical aspects of fibrinolysis and thrombolysis.Blood 78,3114-3124.
[12]Alexander,C.M.and Werb,Z.(1989).Proteinases and extracellular matrix remodeling.Curr.Opin.Cell Biol.1,974-982.
[13]Mignatti,P.and Rifkin,D.B.(1993).Biology and biochemistry of proteinases in tumor invasion.Physiol Rev.73,161-195.
[14]Collen,D.(2001).Ham-Wasserman lecture:role of the plasminogen system in fibrin-homeostasis and tissue remodeling.Hematology.(Am.Soc.Hematol.Educ.Program.)1-9.
[15]Rifkin,D.B.,Moscatelli,D.,Bizik,J.,Quarto,N.,Blei,F.,Dennis,P.,Flaumenhaft,R.,and Mignatti,P.(1990).Growth factor control of extracellular proteolysis.Cell Differ.Dev.32,313-318.
[16]Andreasen,P.A.,Kjoller,L.,Christensen,L.,and Duffy,M.J.(1997).The urokinase-type plasminogen activator system in cancer metastasis:a review.Int.J.Cancer 72,1-22.
[17]Rifkin,D.B.,Mazzieri,R.,Munger,J.S.,Noguera,I.,and Sung,J.(1999).Proteolytic control of growth factor availability.APMIS 107,80-85.
[18]Marder V J,Novokhatny V.Direct fibrinolytic agents:biochemical attributes,preclinical foundation and clinical potential[J].Journal of Thrombosis and Haemostasis,2010,8(3):433-444.
[19]Hunt J A,Petteway Jr S R,Scuderi P,et al.Simplified recombinant plasmin:production and fu-nctional comparison of a novel thrombolytic molecule with plasma-derived plasmin[J].Thromb Haemost,2008,100(3):413-419.
[20]Duboscq C1,Genoud V,Parborell MF et al.Impaired clot lysis by rt-PA catalyzed mini-plasminogen activation.Thromb Res.1997Jun 15；86(6):505-13.
[21]Nagai N,Demarsin E,Van Hoef B,et al.Recombinant human microplasmin:production and potential therapeutic properties[J].Journal of Thrombosis and Haemostasis,2003,1(2):307-313.
[22]Cao Y,Ji RW,Davidson D et al.Kringle domains of human angiostatin.Characterization of the anti-proliferative activity on endothelial cells.J Biol Chem.1996 Nov 15；271(46):29461-7.
[23]O'Reilly MS,Holmgren L,Shing Y et al.Angiostatin:a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma.Cell.1994 Oct 21；79(2):315-28.
[24]O'Reilly MS,Wiederschain D,Stetler SW,Folkman J,Moses MA.Regulation of angiostatin production by matrix metalloproteinase-2in a model of concomitant resistance.J Biol Chem.1999；274:29568–29571.
[25]Paleari L,Brigati C,Anfosso L,Del'Eva R,Albini A,Noonan DM.Anti-angiogenesis in Search of Mechanisms:Angiostatin as a Prototype.In:Weber GF,editor.Cancer Therapy:Molecular Targets in Tumor-Host Interactions.Norfolk:Horizon Scientific Press；2005.pp.143–168.
[26]Gurpreet K,Aulakh,Yadu Balachandran et al.Angiostatin inhibits activation and migration of neutrophils.Cell Tissue Res.2014 Feb；355(2):375-96.
[27]Cao Y,Chen A,An SS et al.Kringle 5of plasminogen is a novel inhibitor of endothelial cell growth.J Biol Chem.1997 Sep 5；272(36):22924-8.
[28]Cao Y,Xue L.Angiostatin.Semin Thromb Hemost.2004Feb；30(1):83-93.Review.
[29]Soff GA.Angiostatin and angiostatin-related proteins.Cancer Metastasis Rev.2000；19(1-2):97-107.Review.
[30]Judah Folkman,Ezio Merler,Charles Abernathy et al.ISOLATION OF A TUMOR FACTOR RESPONSIBLE FOR ANGIOGENESIS.J Exp Med.1971 Jan 31；133(2):275–288.
[31]O'Reilly MS,Holmgren L,Shing Y et al.Angiostatin:a circulating endothelial cell inhibitor that suppresses angiogenesis and tumor growth.Cold Spring Harb Symp Quant Biol.1994；59:471-82.
[32]US5639725A.
[33]Kurup A,Lin C-,Murry DJ et al.Recombinant human angiostatin(rhAngiostatin)in combination with paclitaxel and carboplatin in patients with advanced non-small-cell lung cancer:a phase II study from Indiana University.Ann Oncol.2006 Jan；17(1):97-103.Epub 2005 Nov 9.
[34]Nussenbaum F,Herman IM.Tumor angiogenesis:insights and innovations.J Oncol.2010；2010:132641.
[35]Lisanne C,Hamming,Ben J et al.The clinical application of angiostatic therapy in combination with radiotherapy:past,present,future.Angiogenesis(2017)20:217–232.
[36]O'Reilly MS,Holmgren L,Chen C et al.Angiostatin induces and sustains dormancy of human primary tumors in mice.Nat Med.1996 Jun；2(6):689-92.
[37]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.
[38]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.
[39]HAGAN JJ,ABLONDI FB,DE RENZO EC.Purification and biochemical properties of human plasminogen.J Biol Chem.1960 Apr；235:1005-10.
[40]Tom A.Drixler,Inne H.M.Borel Rinkes,Ewan D.Ritchie et al.Continuous Administration of Angiostatin Inhibits Accelerated Growth of Colorectal Liver Metastases after Partial Hepatectomy1.CANCER RESEARCH 60,1761–1765,March 15,2000.
[41]ZHANGWEI XU,HAI SHI,QI LI et al.Mouse macrophage metalloelastase generates angiostatin from plasminogen and suppresses tumor angiogenesis in murine colon cancer.ONCOLOGY REPORTS 20:81-88,2008.[42]Majchrzak K,Kaspera W,
Figure PCTCN2021113850-appb-000001
J et al.Markers of angiogenesis(CD31,CD34,rCBV)and their prognostic value in low-grade gliomas.Neurol Neurochir Pol.2013 Jul-Aug；47(4):325-31.
[43]Lee C,Liu A,Miranda-Ribera A,Hyun SW et al.NEU1 sialidase regulates the sialylation state of CD31 and disrupts CD31-driven capillary-like tube formation in human lung microvascular endothelia.J Biol Chem.2014 Mar 28；289(13):9121-35.
[44]Schlereth SL,Neuser B,Caramoy A et al.Enrichment of lymphatic vessel endothelial hyaluronan receptor 1(LYVE1)-positive macrophages around blood vessels in the normal human sclera.Invest Ophthalmol Vis Sci.2014 Feb 10；55(2):865-72.
[45]Davis JM,Hyjek E,Husain AN et al.Lymphatic endothelial differentiation in pulmonary lymphangioleiomyomatosis cells.J Histochem Cytochem.2013 Aug；61(8):580-90.
[46]Poston GJ,Adam R,Alberts S,et al.OncoSurge:a strategy for improving resectability with curative intent in metastatic colorectal cancer.JClin Oncol 2005；23:7125–34.
[47]Karmen A,Wroblewski F,Ladue JS(Jan 1955).Transaminase activity in human blood.The Journal of Clinical Investigation.34(1):126–31.
[48]Wang CS,Chang TT,Yao WJ,Wang ST,Chou P(Apr 2012).Impact of increasing alanine aminotransferase levels within normal range on incident diabetes.Journal of the Formosan Medical Association＝Taiwan Yi Zhi.111(4):201-8.
[49]Moungjaroen J,Nimmannit U,Callery PS,Wang L,Azad N,Lipipun V,Chanvorachote P,Rojanasakul Y(2006).Reactive oxygen species mediate caspase activation and apoptosis induced by lipoic acid in human lung epithelial cancer cells through Bcl-2downregulation.J Pharmacol Exp Ther 319,1062–1069.
[50]Wang L,Chanvorachote P,Toledo D,Stehlik C,Mercer RR,Castranova V,Rojanasakul Y(2008).Peroxide is a key mediator of Bcl-2 down-regulation and  apoptosis induction by cisplatinin human lung cancer cells.Mol Pharmacol 73,119–127.
[51]Jae Kyu Ryu,Mark A.Petersen,Sara G.Murray et al.Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation.NATURE COMMUNICATIONS,2015,6:8164.
[52]Dimitrios Davalos,Katerina Akassoglou.Fibrinogen as a key regulator of inflammation in disease.Seminars in Immunopathology,2012.34(1):43-62.
[53]Michael S.C’Reilly,Lars Holmgren,’Yuen Shing et al.Angiostatin:A Novel Angiogenesis Inhibitor That Mediates the Suppression of Metastases by a Lewis Lung Carcinoma.Cell,Vol.79,315-328,October 21,1994.
[54]Zuo J,Wen M et al.PLGA-Der p1 Vaccine Inhibited Tumor Growth in a Murine Model of Lung Cancer,Arch Med Res.2015 Dec 16.
[55]Yoon Choi a,Young Wook Yoon a,Heung Sik Na,Sun Ho Kim,Jin MO Chung.Behavioral signs of ongoing g pain and cold allodynia in a rat model of neuropathic pain，Pain,59(1994)369-376.
[56]Takehiko Maeda,Daisuke Yamada,Kohichi Kawahara，Cancer pain relief achieved by disrupting tumor-driven semaphorin 3A signaling in mice，Neuroscience Letters 632(2016)147–151.
[57]Li B,Vanroey M,Wang C,et al.Anti-programmed death-1synergizes with granulocyte macrophage colony-stimulating factor--secreting tumor cell immunotherapy providing therapeutic benefit to mice with established tumors[J].Clinical Cancer Research An Official Journal of the American Association for Cancer Research,2009,15(5):1623.
[58]Sabino M A C,Luger N M,Mach D B,et al.Different tumors in bone each give rise to a distinct pattern of skeletal destruction,bone cancer-related pain behaviors and neurochemical changes in the central nervous system[J].International Journal of Cancer,2003,104(5):550-558.
[59]Rama Krishna Nimmakayala,Surinder K.Batra,Moorthy P.Ponnusamy.Unraveling the Journey of Cancer Stem Cells from Origin to Metastasis.Biochim Biophys Acta Rev Cancer.2018 Nov 9；1871(1):50-63.

Claims (14)

  1. 一种治疗肿瘤的方法，包括给药肿瘤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。
  2. 权利要求1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或2的方法，其中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，其中所述化合物为纤溶酶原。
  5. 权利要求1-4任一项的方法，其中其中所述肿瘤为癌症。
  6. 权利要求1-5任一项的方法，其中所述肿瘤选自如下的一种或多种：口腔癌、食管癌、胃癌、小肠癌、结肠癌、直肠癌、肺癌、肝癌、肝细胞癌、胰腺癌、胆囊癌、非小细胞肺(NSCL)癌、支气管肺泡细胞肺癌、乳腺癌、卵巢癌、宫颈癌，输卵管癌、子宫内膜癌、阴道癌、前列腺癌、尿道癌、阴茎癌、肾癌、输尿管癌、肾细胞癌、肾盂癌、膀胱癌、头颈部癌、皮肤癌、黑素瘤、间皮瘤、骨癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、胶质瘤、多形性成胶质细胞瘤、星形细胞瘤、施旺细胞瘤、室管膜瘤、髓母细胞瘤、脑脊膜瘤、鳞状细胞癌、垂体腺瘤。
  7. 权利要求1-6任一项的方法，其中所述纤溶酶原具有选自如下的一种或多种作用：减小肿瘤体积、改善肿瘤受试者的一般生存状况、延迟肿瘤的进展、抑制肿瘤细胞的生长、提高存活率、延长肿瘤受试者生存期、减轻癌性疼痛、抑制肿瘤血管形成、促进肿瘤细胞坏死或凋亡、促进抗肿瘤免疫应 答、调节肿瘤相关抗原或淋巴细胞表面分子的表达、减轻癌细胞对组织器官的损伤、促进肿瘤损伤组织结构或功能恢复。
  8. 权利要求1-7任一项的方法，其中所述受试者血纤溶酶原水平或肿瘤组织中或未患肿瘤的组织中纤溶酶原水平高于、等于或低于健康受试者血纤溶酶原水平或未患肿瘤的相应组织中的纤溶酶原水平。
  9. 权利要求1-8任一项的方法，其中所述受试者血纤维蛋白水平或肿瘤组织中或未患肿瘤的组织中纤维蛋白水平高于、等于或低于健康受试者血纤维蛋白水平或未患肿瘤的相应组织中的纤维蛋白水平。
  10. 权利要求1-9任一项的方法，本申请所述纤溶酶原与选自如下的一项或多项联合施用：化疗、放疗、手术疗法、细胞疗法和免疫疗法。
  11. 权利要求1-10任一项的方法，其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守取代变体。
  12. 权利要求1-10任一项的方法，其中所述纤溶酶原是包含丝氨酸蛋白酶结构域和/或赖氨酸结合结构域的纤溶酶原蛋白。
  13. 权利要求1-10任一项的方法，其中所述纤溶酶原是包含与序列14具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并具有蛋白水平活性的纤溶酶原蛋白。
  14. 权利要求1-10任一项的方法，其中所述纤溶酶原与序列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。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12576139B2 (en) 2020-05-11 2026-03-17 Talengen International Limited Method and drug for treating spinal muscular atrophy

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220127880A (ko) * 2020-01-17 2022-09-20 탈렌젠 인터내셔널 리미티드 신경 손상 및 이의 관련 병증 치료 방법

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
CN102154253A (zh) 2011-01-06 2011-08-17 郑州大学 具有抑制血小板凝集功能的微小纤溶酶原突变体及其制备方法和用途
CN104789544A (zh) * 2014-01-16 2015-07-22 中国科学院福建物质结构研究所 重组pai-1抑制剂、包含其的组合物及其用于治疗和检测用途
CN105008323A (zh) * 2012-10-31 2015-10-28 密执安大学评议会 纤溶酶原激活物抑制因子-1抑制剂和其使用方法
CN109925507A (zh) * 2017-12-15 2019-06-25 深圳瑞健生命科学研究院有限公司 一种预防或治疗骨关节炎的方法和药物
CN110066783A (zh) * 2019-05-16 2019-07-30 重庆派金生物科技有限公司 一种无自切形式的微纤溶酶制备方法
CN110869346A (zh) * 2017-07-27 2020-03-06 密歇根大学董事会 纤维蛋白溶酶原活化物抑制因子-1(pai-1)抑制剂和使用方法

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE355379T1 (de) * 1994-04-26 2006-03-15 Childrens Medical Center Angiostatin und verfahren zu seiner nutzung zur vermeidung der angiogenese
US5639725A (en) * 1994-04-26 1997-06-17 Children's Hospital Medical Center Corp. Angiostatin protein
US5801146A (en) * 1996-05-03 1998-09-01 Abbott Laboratories Compound and method for inhibiting angiogenesis
US20020031518A1 (en) * 1996-10-09 2002-03-14 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Plasminogen fragment having activity to inhibit tumor metastasis and growth and process for preparing same technical field
JP3806471B2 (ja) * 1996-10-09 2006-08-09 財団法人化学及血清療法研究所 腫瘍転移増殖抑制効果を有するプラスミノーゲン断片および該断片の調製方法
WO2000010506A2 (en) * 1998-08-20 2000-03-02 University Of Vermont And State Agriculture College Angiogenesis inhibitors and uses thereof
CN1110319C (zh) * 1999-12-22 2003-06-04 上海国源生物技术有限公司 含蚯蚓纤溶酶的抗肿瘤药剂和保健品
US7285277B2 (en) * 2002-03-15 2007-10-23 Mogam Biotechnology Research Institute Anticancer agent
US7919103B2 (en) * 2003-05-09 2011-04-05 Transfert Plus Compound and method for regulating plasminogen activation and cell migration
JP7213552B2 (ja) * 2016-12-15 2023-01-27 タレンゲン インターナショナル リミテッド 肝の繊維化を予防及び治療するための方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
CN102154253A (zh) 2011-01-06 2011-08-17 郑州大学 具有抑制血小板凝集功能的微小纤溶酶原突变体及其制备方法和用途
CN105008323A (zh) * 2012-10-31 2015-10-28 密执安大学评议会 纤溶酶原激活物抑制因子-1抑制剂和其使用方法
CN104789544A (zh) * 2014-01-16 2015-07-22 中国科学院福建物质结构研究所 重组pai-1抑制剂、包含其的组合物及其用于治疗和检测用途
CN110869346A (zh) * 2017-07-27 2020-03-06 密歇根大学董事会 纤维蛋白溶酶原活化物抑制因子-1(pai-1)抑制剂和使用方法
CN109925507A (zh) * 2017-12-15 2019-06-25 深圳瑞健生命科学研究院有限公司 一种预防或治疗骨关节炎的方法和药物
CN110066783A (zh) * 2019-05-16 2019-07-30 重庆派金生物科技有限公司 一种无自切形式的微纤溶酶制备方法

Non-Patent Citations (77)

* Cited by examiner, † Cited by third party
Title
ALEXANDER CMWERB, Z.: "Cell Biology of Extracellular Matrix", 1991, PLENUM PRESS, article "Extracellular matrix degradation", pages: 255 - 302
ALEXANDER, C.M.WERB, Z.: "Proteinases and extracellular matrix remodeling", CURR. OPIN. CELL BIOL., vol. 1, 1989, pages 974 - 982, XP000872201, DOI: 10.1016/0955-0674(89)90068-9
ANDREASEN, P.A.KJOLLER, L.CHRISTENSEN, L.DUFFY, M.J.: "The urokinase-type plasminogen activator system in cancer metastasis: a review.", INT. J. CANCER, vol. 72, 1997, pages 1 - 22, XP002210773, DOI: 10.1002/(SICI)1097-0215(19970703)72:1<1::AID-IJC1>3.0.CO;2-Z
BARANY, SOLID-PHASE PEPTIDE SYNTHESIS, pages 3 - 284
CAMARERO JA ET AL., PROTEIN PEPT LETT, vol. 12, 2005, pages 723 - 8
CAO YCHEN AAN SS ET AL.: "Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth", J BIOL CHEM., vol. 272, no. 36, 5 September 1997 (1997-09-05), pages 22924 - 8, XP002205624, DOI: 10.1074/jbc.272.36.22924
CAO YJI RWDAVIDSON D ET AL.: "Kringle domains of human angiostatin. Characterization of the anti-proliferative activity on endothelial cells", J BIOL CHEM., vol. 271, no. 46, 15 November 1996 (1996-11-15), pages 29461 - 7, XP002086445, DOI: 10.1074/jbc.271.46.29461
CAO YXUE L. ANGIOSTATIN, SEMIN THROMB HEMOST, vol. 30, no. 1, February 2004 (2004-02-01), pages 83 - 93
CASTELLINO FJ: "Biochemistry of human plasminogen", SEMIN THROMB HEMOST, vol. 10, no. 1, January 1984 (1984-01-01), pages 18 - 23
CO ET AL., J. IMMUNOL., vol. 148, 1992, pages 1149
COLLEN, D.: "Ham-Wasserman lecture: role of the plasminogen system in fibrin-homeostasis and tissue remodeling", HEMATOLOGY. (AM. SOC. HEMATOL. EDUC. PROGRAM., 2001, pages 1 - 9
COLLEN, D.LIJNEN, H.R.: "Basic and clinical aspects of fibrinolysis and thrombolysis", BLOOD, vol. 78, 1991, pages 3114 - 3124
DAVIS JMHYJEK EHUSAIN AN ET AL.: "Lymphatic endothelial differentiation in pulmonary lymphangioleiomyomatosis cells", J HISTOCHEM CYTOCHEM, vol. 61, no. 8, August 2013 (2013-08-01), pages 580 - 90
DIMITRIOS DAVALOSKATERINA AKASSOGLOU: "Fibrinogen as a key regulator of inflammation in disease", SEMINARS IN IMMUNOPATHOLOGY, vol. 34, no. 1, 2012, pages 43 - 62, XP019988748, DOI: 10.1007/s00281-011-0290-8
DUBOSCQ C1GENOUD VPARBORELL MF ET AL.: "Impaired clot lysis by rt-PA catalyzed mini-plasminogen activation", THROMB RES, vol. 86, no. 6, 15 June 1997 (1997-06-15), pages 505 - 13
FOLKMAN ET AL., CELL, 1994
GANESAN A, MINI REV. MED CHEM., vol. 6, 2006, pages 3 - 10
GEIGER MHUBER KWOJTA JSTINGL LESPANA FGRIFFIN JHBINDER BR: "Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo", BLOOD, vol. 74, no. 2, August 1989 (1989-08-01), pages 722 - 8
GRAVANIS ITSIRKA SE: "Tissue-type plasminogen activator as a therapeutic target in stroke", EXPERT OPINION ON THERAPEUTIC TARGETS, vol. 12, no. 2, February 2008 (2008-02-01), pages 159 - 70, XP093006015, DOI: 10.1517/14728222.12.2.159
GURPREET K, AULAKHYADU BALACHANDRAN ET AL.: "Angiostatin inhibits activation and migration of neutrophils", CELL TISSUE RES, vol. 355, no. 2, February 2014 (2014-02-01), pages 375 - 96, XP035331633, DOI: 10.1007/s00441-013-1753-0
HAGAN JJABLONDI FBDE RENZO EC: "Purification and biochemical properties of human plasminogen", J BIOL CHEM., vol. 235, April 1960 (1960-04-01), pages 1005 - 10
HE, C.S.WILHELM, S.M.PENTLAND, A.P.MARMER, B.L.GRANT, G.A.EISEN, A.Z.GOLDBERG, G.I.: "Tissue cooperation in a proteolytic cascade activating human interstitial collagenase", PROC. NATL. ACAD. SCI. U. S. A, vol. 86, 1989, pages 2632 - 2636
HUNT J APETTEWAY JR S RSCUDERI P ET AL.: "Simplified recombinant plasmin: production and fu-nctional comparison of a novel thrombolytic molecule with plasma-derived plasmin [J", THROMB HAEMOST, vol. 100, no. 3, 2008, pages 413 - 419, XP009113156
JAE KYU RYUMARK A. PETERSENSARA G. MURRAY ET AL.: "Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation", NATURE COMMUNICATIONS, vol. 6, 2015, pages 8164
JUDAH FOLKMANEZIO MERLERCHARLES ABERNATHY ET AL.: "ISOLATION OF A TUMOR FACTOR RESPONSIBLE FOR ANGIOGENESIS", J EXP MED., vol. 133, no. 2, 31 January 1971 (1971-01-31), pages 275 - 288
KARMEN AWROBLEWSKI FLADUE JS: "Transaminase activity in human blood", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 34, no. 1, January 1955 (1955-01-01), pages 126 - 31
KENNETH C. ROBBINSLOUIS SUMMARIADAVID ELWYN ET AL.: "Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 240, no. 1, 1965, pages 541 - 550
KURUP ALIN CMURRY DJ ET AL.: "Recombinant human angiostatin (rhAngiostatin) in combination with paclitaxel and carboplatin in patients with advanced non-small-cell lung cancer: a phase II study from Indiana University", ANN ONCOL, vol. 17, no. 1, 9 November 2005 (2005-11-09), pages 97 - 103
LANGER ET AL., J. BIOMED. MATER. RES., vol. 15, 1981, pages 167 - 277
LANGER, CHEM. TECH., vol. 12, 1982, pages 98 - 105
LEE CLIU AMIRANDA-RIBERA AHYUN SW ET AL.: "NEU1 sialidase regulates the sialylation state of CD31 and disrupts CD31-driven capillary-like tube formation in human lung microvascular endothelia", J BIOL CHEM., vol. 289, no. 13, 28 March 2014 (2014-03-28), pages 9121 - 35
LI BVANROEY MWANG C ET AL.: "Anti-programmed death-1 synergizes with granulocyte macrophage colony-stimulating factor--secreting tumor cell immunotherapy providing therapeutic benefit to mice with established tumors[J", CLINICAL CANCER RESEARCH AN OFFICIAL JOURNAL OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, vol. 15, no. 5, 2009, pages 1623, XP055166457, DOI: 10.1158/1078-0432.CCR-08-1825
LISANNE C, HAMMINGBEN J ET AL.: "The clinical application of angiostatic therapy in combination with radiotherapy: past, present, future", ANGIOGENESIS, vol. 20, 2017, pages 217 - 232, XP036238976, DOI: 10.1007/s10456-017-9546-9
MAJCHRZAK KKASPERA WSZYMAS J ET AL.: "Markers of angiogenesis (CD31, CD34, rCBV) and their prognostic value in low-grade gliomas", NEUROL NEUROCHIR POL, vol. 47, no. 4, pages 325 - 31
MARDER V JNOVOKHATNY V: "Direct fibrinolytic agents: biochemical attributes, preclinical foundation and clinical potential [J", JOURNAL OF THROMBOSIS AND HAEMOSTASIS, vol. 8, no. 3, 2010, pages 433 - 444
MERRIFIELD ET AL.: "Special Methods in Peptide Synthesis, Part A.", J. AM. CHEM. SOC., vol. 85, 1963, pages 2149 - 2156
MICHAEL S. C'REILLYLARS HOLMGRENYUEN SHING ET AL.: "Angiostatin: A Novel Angiogenesis Inhibitor That Mediates the Suppression of Metastases by a Lewis Lung Carcinoma", CELL, vol. 79, 21 October 1994 (1994-10-21), pages 315 - 328
MIGNATTI, P.RIFKIN, D.B.: "Biology and biochemistry of proteinases in tumor invasion", PHYSIOL REV, vol. 73, 1993, pages 161 - 195
MOUNGJAROEN JNIMMANNIT UCALLERY PSWANG LAZAD NLIPIPUN VCHANVORACHOTE PROJANASAKUL Y: "Reactive oxygen species mediate caspase activation and apoptosis induced by lipoic acid in human lung epithelial cancer cells through Bcl-2 downregulation", J PHARMACOL EXP THER, vol. 319, 2006, pages 1062 - 1069
NAGAI NDEMARSIN EVAN HOEF B ET AL.: "Recombinant human microplasmin: production and potential therapeutic properties [J", JOURNAL OF THROMBOSIS AND HAEMOSTASIS, vol. 1, no. 2, 2003, pages 307 - 313, XP055535850
NATURE MEDICINE, 1996
NUSSENBAUM FHERMAN IM: "Tumor angiogenesis: insights and innovations", J ONCOL, 2010
NY, A.LEONARDSSON, G.HAGGLUND, A.CHAGGLOF, P.PLOPLIS, V.A.CARMELIET, PNY, T.: "Ovulation inplasminogen-deficient mice", ENDOCRINOLOGY, vol. 140, 1999, pages 5030 - 5035
O'REILLY MSHOLMGREN LCHEN C ET AL.: "Angiostatin induces and sustains dormancy of human primary tumors in mice", NAT MED, vol. 2, no. 6, June 1996 (1996-06-01), pages 689 - 92, XP002925790, DOI: 10.1038/nm0696-689
O'REILLY MSHOLMGREN LSHING Y ET AL.: "Angiostatin: a circulating endothelial cell inhibitor that suppresses angiogenesis and tumor growth", COLD SPRING HARB SYMP QUANT BIOL., vol. 59, 1994, pages 471 - 82
O'REILLY MSHOLMGREN LSHING Y ET AL.: "Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma", CELL, vol. 79, no. 2, 21 October 1994 (1994-10-21), pages 315 - 28, XP024244979, DOI: 10.1016/0092-8674(94)90200-3
O'REILLY MSWIEDERSCHAIN DSTETLER SWFOLKMAN JMOSES MA: "Regulation of angiostatin production by matrix metalloproteinase-2 in a model of concomitant resistance", J BIOL CHEM., vol. 274, 1999, pages 29568 - 29571
PALEARI LBRIGATI CANFOSSO LDEL'EVA RALBINI ANOONAN DM: "Cancer Therapy: Molecular Targets in Tumor-Host Interactions", 2005, HORIZON SCIENTIFIC PRESS, article "Anti-angiogenesis in Search of Mechanisms: Angiostatin as a Prototype", pages: 143 - 168
POSTON GJADAM RALBERTS S ET AL.: "OncoSurge: a strategy for improving resectability with curative intent in metastatic colorectal cancer", JCLIN ONCOL, vol. 23, 2005, pages 7125 - 34
QUEEN ET AL., IMMUNOL. REV., vol. 89, 1986, pages 49
RAMA KRISHNA NIMMAKAYALASURINDER K. BATRAMOORTHY P. PONNUSAMY: "Unraveling the Journey of Cancer Stem Cells from Origin to Metastasis", BIOCHIM BIOPHYS ACTA REV CANCER, vol. 1871, no. 1, 9 November 2018 (2018-11-09), pages 50 - 63, XP085582029, DOI: 10.1016/j.bbcan.2018.10.006
RAUM, D.MARCUS, D.ALPER, C.A.LEVEY, R.TAYLOR, P.D.STARZL, T.E.: "Synthesis of human plasminogen by the liver", SCIENCE, vol. 208, 1980, pages 1036 - 1037
REMINGTON'S PHARMACEUTICAL SCIENCES, 1980
RIFKIN, D.B.MAZZIERI, R.MUNGER, J.S.NOGUERA, I.SUNG, J: "Proteolytic control of growth factor availability", APMIS, vol. 107, 1999, pages 80 - 85
RIFKIN, D.B.MOSCATELLI, D.BIZIK, J.QUARTO, N.BLEI, F.DENNIS, P.FLAUMENHAFT, R.MIGNATTI, P.: "Growth factor control of extracellular proteolysis", CELL DIFFER. DEV., vol. 32, 1990, pages 313 - 318, XP024562018, DOI: 10.1016/0922-3371(90)90045-X
SABINO MACLUGER N MMACH D B ET AL.: "Different tumors in bone each give rise to a distinct pattern of skeletal destruction, bone cancer-related pain behaviors and neurochemical changes in the central nervous system[J", INTERNATIONAL JOURNAL OF CANCER, vol. 104, no. 5, 2003, pages 550 - 558
SAKSELA, O.RIFKIN, D.B.: "Cell-associated plasminogen activation: regulation and physiological functions", ANNU. REV. CELL BIOL., vol. 4, 1988, pages 93 - 126
SCHLERETH SLNEUSER BCARAMOY A ET AL.: "Enrichment of lymphatic vessel endothelial hyaluronan receptor 1 (LYVEl)-positive macrophages around blood vessels in the normal human sclera", INVEST OPHTHALMOL VIS SCI, vol. 55, no. 2, 10 February 2014 (2014-02-10), pages 865 - 72
SIDMAN ET AL., BIOPOLYMERS, vol. 22, 1983, pages 547
SILVERSTEIN RLLEUNG LLHARPEL PCNACHMAN RL: "Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator", J. CLIN. INVEST, vol. 74, no. 5, November 1984 (1984-11-01), pages 1625 - 33
SOFF GA: "Angiostatin and angiostatin-related proteins", CANCER METASTASIS REV, vol. 19, 2000, pages 97 - 107, XP001203669, DOI: 10.1023/A:1026525121027
SOTTRUP-JENSEN, L.ZAJDEL, M.CLAEYS, H.PETERSEN, T.E.MAGNUSSON, S.: "Amino-acid sequence of activation cleavage site in plasminogen: homology with ''pro'' part of prothrombin", PROC. NATL. ACAD. SCI. U. S. A, vol. 72, 1975, pages 2577 - 2581
STEWART ET AL.: "Solid Phase Peptide Synthesis", 1984, PIERCE CHEM. CO.
STOPPELLI, M.P.CORTI, A.SOFFIENTINI, A.CASSANI, G.BLASI, F.ASSOIAN, R.K.: "Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes", PROC. NATL. ACAD. SCI. U. S. A, vol. 82, 1985, pages 4939 - 4943
SUMMARIA LSPITZ FARZADON L ET AL.: "Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins", J BIOL CHEM., vol. 251, no. 12, 25 June 1976 (1976-06-25), pages 3693 - 9, XP001021272
TAKEHIKO MAEDADAISUKE YAMADAKOHICHI KAWAHARA: "Cancer pain relief achieved by disrupting tumor-driven semaphorin 3A signaling in mice", NEUROSCIENCE LETTERS, vol. 632, 2016, pages 147 - 151, XP029744353, DOI: 10.1016/j.neulet.2016.08.060
THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY., vol. 2
TOM A. DRIXLERINNE H. M. BOREL RINKESEWAN D. RITCHIE ET AL.: "Continuous Administration of Angiostatin Inhibits Accelerated Growth of Colorectal Liver Metastases after Partial Hepatectomyl", CANCER RESEARCH, vol. 60, 15 March 2000 (2000-03-15), pages 1761 - 1765
VASSALLI, J.D.BACCINO, D.BELIN, D.: "A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase", J. CELL BIOL., vol. 100, 1985, pages 86 - 92, XP001176730, DOI: 10.1083/jcb.100.1.86
WANG CSCHANG TTYAO WJWANG STCHOU P: "Impact of increasing alanine aminotransferase levels within normal range on incident diabetes", JOURNAL OF THE FORMOSAN MEDICAL ASSOCIATION = TAIWAN YI ZHI, vol. 111, no. 4, April 2012 (2012-04-01), pages 201 - 8, XP028416749, DOI: 10.1016/j.jfma.2011.04.004
WANG LCHANVORACHOTE PTOLEDO DSTEHLIK CMERCER RRCASTRANOVA VROJANASAKUL Y: "Peroxide is a key mediator of Bcl-2 down-regulation and apoptosis induction by cisplatinin human lung cancer cells", MOL PHARMACOL, vol. 73, 2008, pages 119 - 127
WERB, Z.MAINARDI, C.L.VATER, C.A.HARRIS, E.D., JR.: "Endogenous activiation of latent collagenase by rheumatoid synovial cells. Evidence for a role of plasminogen activator", N. ENGL. J. MED., vol. 296, 1977, pages 1017 - 1023
WIMAN, B.WALLEN, P.: "Structural relationship between ''glutamic acid'' and ''lysine'' forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography", EUR. J. BIOCHEM., vol. 50, 1975, pages 489 - 494
WINNACKER: "From Genes to Clones", 1987, VCH PUBLISHERS
YOON CHOI AYOUNG WOOK YOON AHEUNG SIK NASUN HO KIMJIN MO CHUNG: "Behavioral signs of ongoing g pain and cold allodynia in a rat model of neuropathic pain", PAIN, vol. 59, 1994, pages 369 - 376, XP024380595, DOI: 10.1016/0304-3959(94)90023-X
ZHANGWEI XUHAI SHIQI LI ET AL.: "Mouse macrophage metalloelastase generates angiostatin from plasminogen and suppresses tumor angiogenesis in murine colon cancer", ONCOLOGY REPORTS, vol. 20, 2008, pages 81 - 88
ZUO JWEN M ET AL.: "PLGA-Der pl Vaccine Inhibited Tumor Growth in a Murine Model of Lung Cancer", ARCH MED RES, 16 December 2015 (2015-12-16)

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