WO2017101872A1 - 一种用于预防和治疗宫颈糜烂的方法 - Google Patents

一种用于预防和治疗宫颈糜烂的方法 Download PDF

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WO2017101872A1
WO2017101872A1 PCT/CN2016/110454 CN2016110454W WO2017101872A1 WO 2017101872 A1 WO2017101872 A1 WO 2017101872A1 CN 2016110454 W CN2016110454 W CN 2016110454W WO 2017101872 A1 WO2017101872 A1 WO 2017101872A1
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plasminogen
drug
erosion
cervical
cervical erosion
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PCT/CN2016/110454
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English (en)
French (fr)
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李季男
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深圳瑞健生命科学研究院有限公司
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Priority to CN201680073634.7A priority Critical patent/CN108463235A/zh
Priority to CA3008495A priority patent/CA3008495A1/en
Priority to US16/062,410 priority patent/US10874721B2/en
Priority to EP16874929.9A priority patent/EP3395355A4/en
Priority to JP2018550639A priority patent/JP6876066B2/ja
Publication of WO2017101872A1 publication Critical patent/WO2017101872A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a novel method of preventing and/or treating cervical erosion using plasminogen or plasmin. Compared with the existing drugs for treating cervical erosion, the method can effectively promote the repair of damaged mucosa.
  • Chronic cervicitis is a common and frequently-occurring disease in married women, accounting for the first place in the incidence of women in China, accounting for about 50% of gynecological diseases. Cervical erosion is the most common pathological change in chronic cervicitis. The incidence of cervical cancer is 7.3 times higher than that of women without cervical erosion. It has been reported that about 80% of simple squamous cell carcinomas occur in the cervical canal or erosion area, ie, the columnar epithelial area, and most of them occur in the "smashed" area [1] . The main cause of cervical erosion is usually the injury of the cervix caused by postpartum or postoperative women, and is subsequently accompanied by the invasion of pathogens.
  • CT Chlamydia trachomatis
  • NG Neisseria gonorrhoeae
  • HSV Herpes simplex virus
  • Ureaplasma urealyticum Uu
  • Trichomonas vaginalis TV
  • CA Candida
  • cervical erosion The main symptoms of cervical erosion are increased vaginal discharge and often purulent, contact bleeding, lumbosacral pain, infertility and so on.
  • oral medicine vaginal medicine
  • vaginal medicine local physical therapy of the cervix
  • surgical treatment for patients with cervical erosion, no matter which treatment method, although the treatment time is different, they can receive better results.
  • patients with moderate or severe cervical erosion, oral medication, slow onset, low local blood drug concentration it is difficult to achieve the desired results; simple vaginal medication treatment long, poor efficacy, and the effect is unstable, the probability of recurrence is large.
  • Surgery treatment costs are high, trauma, and healing time is long, and it is difficult for patients to accept.
  • Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease that hydrolyzes several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans [6] .
  • ECM extracellular matrix
  • plasmin can Some metalloproteinase precursors (pro-MMP) are activated to form active metalloproteinases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis [7,8] .
  • Plasmin is formed by proteolytic plasminogen by two physiological PA: tissue 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 been traditionally believed that the regulation of the PA system is primarily achieved by the synthesis and activity levels of PA. The synthesis of components of the PA system is tightly regulated by various factors such as hormones, growth factors and cytokines. In addition, specific physiological inhibitors of plasmin and PA are also present. The main inhibitor of plasmin is ⁇ 2-antiplasmin. Some cell surface has direct hydrolysis activity of uPA-specific cell surface receptors (uPAR) [9,10] .
  • uPAR uPA-specific cell surface receptors
  • Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa [11,12] . Plasminogen is mainly synthesized in the liver and is abundantly present in the extracellular fluid. The plasma plasminogen content is approximately 2 ⁇ M. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids [13,14] . Plasminogen exists in two molecular forms: glutamate-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved forms of plasminogen have an amino terminal (N-terminal) glutamate and are therefore referred to as glutamate-plasminogen.
  • plasminogen in the presence of plasmin, glutamate-plasminogen is hydrolyzed to Lys-Lysinogen at Lys76-Lys77. Compared to glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PA at a higher rate.
  • the Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide-linked double-chain protease plasmin [15] .
  • the amino terminal portion of plasminogen contains five homologous tricycles, the so-called kringle, which contains a protease domain.
  • Some kringles contain a lysine binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor alpha2-AP.
  • the main substrate for plasmin is fibrin, which is the key to preventing pathological thrombosis [16] .
  • Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycans and gelatin, suggesting that plasmin also plays an important role in ECM reconstruction [12,17, 18] .
  • plasmin can also degrade other components of 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 [19] .
  • plasmin has the ability to activate certain potential forms of growth factors [20-22] .
  • plasminogen has an unexpected effect in preventing and/or treating cervical erosion, which is specifically manifested in promoting repair of damage and inflammation.
  • the use of plasminogen for the prevention and/or treatment of cervical erosion has an incomparable advantage in terms of efficacy, patient tolerance, and ease of treatment. Therefore, fibrinogen may be a novel strategy for the prevention and/or treatment of cervical erosion.
  • the present invention relates to the prevention and/or treatment of cervical erosion by plasminogen.
  • the present inventors have surprisingly found that plasminogen exhibits prophylactic and/or therapeutic effects in the prevention and/or treatment of cervical erosion and is effective in promoting the repair of damaged tissues.
  • the invention relates to a novel method of preventing and/or treating cervical erosion and related disorders, and the use of plasminogen or plasmin for preventing and/or treating cervical erosion and related disorders, Methods or uses include administering plasminogen or plasmin to a subject.
  • the cervical erosion described above includes true erosion and false erosion.
  • the subject is a mammal, preferably a human.
  • the cervical erosion may be cervical erosion caused by any cause, in particular, cervical erosion caused by damage such as inflammation.
  • the subject is plasminogen or plasmin is low.
  • the low is congenital, secondary and/or local.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 sexual, and still have plasminogen activity.
  • plasminogen is added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, 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.
  • the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, plasminogen, microplasminogen, delta-plasminogen or random combination.
  • plasminogen Is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, microplasminogen, delta-plasminogen or microplasminogen.
  • the plasminogen is a human native plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, fibrinolysis from a primate or rodent
  • the zymogen is a straight homologue, such as a troponogen-directed homologue from gorillas, rhesus monkeys, rats, cows, horses, and dogs.
  • the amino acid sequence of the plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • the plasminogen or plasmin is administered systemically or locally, preferably by the following routes: intravenous, intramuscular, subcutaneous, topical, by rectal, vaginal administration.
  • the topical administration is by applying a dressing containing plasminogen in the area of cervical erosion.
  • the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer.
  • the plasminogen is 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg per day, 10-100mg / kg (calculated per kg body weight) or 0.0001-2000mg / cm 2, 0.001-800mg / cm 2, 0.01-600mg / cm 2, 0.1-400mg / cm 2, 1-200mg / cm 2, 1- 100mg / cm 2, 10-100mg / cm 2 ( calculated per square centimeter of body surface area) of the dose administered, preferably repeated at least once, preferably at least daily administration.
  • the above dosages may be further adjusted as appropriate.
  • the above plasminogen or plasmin may be administered alone or in combination with other drugs or therapies including antibacterial agents, antiviral drugs, antifungals, and trichomoniasis drugs. , antithrombotic drugs, antidiabetic drugs, physical therapy, laser therapy, local surgery and so on.
  • the invention relates to the use of plasminogen or plasmin in the manufacture of a medicament for the prevention and/or treatment of cervical erosion in a subject.
  • the invention further relates to a method of preparing a medicament comprising preparing a plasminogen or plasmin and a pharmaceutically acceptable carrier into a medicament for treating cervical erosion in a subject.
  • the cervical erosion includes true erosion and false erosion.
  • the subject is a mammal, preferably a human.
  • the cervical erosion may be cervical erosion caused by any cause, in particular, cervical erosion caused by damage such as inflammation.
  • the subject is plasminogen or plasmin is low.
  • the low is congenital, secondary and/or local.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 sexual, and still have plasminogen activity.
  • plasminogen is added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, 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.
  • the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, plasminogen, microplasminogen, delta-plasminogen or random combination. In one embodiment, the plasminogen is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, plasminogen, delta-plasminogen or Microplasminogen.
  • the plasminogen is a human native plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, fibrinolysis from a primate or rodent
  • the zymogen is a straight homologue, such as a troponogen-directed homologue from gorillas, rhesus monkeys, rats, cows, horses, and dogs.
  • the amino acid sequence of the plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • the plasminogen or plasmin is administered systemically or locally, preferably by the following routes: intravenous, intramuscular, subcutaneous, topical, by rectal, vaginal administration.
  • the topical administration is by applying a dressing containing plasminogen in the area of cervical erosion.
  • the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer.
  • the plasminogen is 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg per day, 10-100mg / kg (calculated per kg body weight) or 0.0001-2000mg / cm 2, 0.001-800mg / cm 2, 0.01-600mg / cm 2, 0.1-400mg / cm 2, 1-200mg / cm 2, 1- 100mg / cm 2, 10-100mg / cm 2 ( calculated per square centimeter of body surface area) of the dose administered, preferably repeated at least once, preferably at least daily administration.
  • the above dosages may be further adjusted as appropriate.
  • the above plasminogen or plasmin may be administered alone or in combination with other drugs or therapies including antibacterial agents, antiviral drugs, antifungals, and trichomoniasis drugs. , antithrombotic drugs, antidiabetic drugs, physical therapy, laser therapy, local surgery and so on.
  • the present invention relates to plasminogen or plasmin for preventing and/or treating cervical erosion, and plasminogen or plasmin for preventing and/or treating cervical erosion Pharmaceutical composition.
  • the cervical erosion includes true erosion and false erosion.
  • the subject is a mammal, preferably a human.
  • the cervical erosion may be cervical erosion caused by any cause, in particular, cervical erosion caused by damage such as inflammation.
  • the subject is plasminogen or plasmin is low.
  • the low is congenital, secondary and/or local.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 sexual, and still have plasminogen activity.
  • plasminogen is added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, 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.
  • the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, plasminogen, microplasminogen, delta-plasminogen or random combination. In one embodiment, the plasminogen is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, plasminogen, delta-plasminogen or Microplasminogen.
  • the plasminogen is a human native plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, fibrinolysis from a primate or rodent Direct zymogen homologues, such as plasmin from gorillas, rhesus monkeys, rats, cattle, horses, and dogs Original straight to homologues.
  • a primate or rodent Direct zymogen homologues such as plasmin from gorillas, rhesus monkeys, rats, cattle, horses, and dogs Original straight to homologues.
  • the amino acid sequence of the plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • the plasminogen or plasmin is administered systemically or locally, preferably by the following routes: intravenous, intramuscular, subcutaneous, topical, by rectal, vaginal administration.
  • the topical administration is by applying a dressing containing plasminogen in the area of cervical erosion.
  • the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In one embodiment, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In one embodiment, the plasminogen is 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg per day, 10-100mg / kg (calculated per kg body weight) or 0.0001-2000mg / cm 2, 0.001-800mg / cm 2, 0.01-600mg / cm 2, 0.1-400mg / cm 2, 1-200mg / cm 2, 1- 100mg / cm 2, 10-100mg / cm 2 ( calculated per square centimeter of body surface area) of the dose administered, preferably repeated at least once, preferably at least daily administration. In the case of topical application, the above dosages may be further adjusted as appropriate.
  • the above plasminogen or plasmin may be administered alone or in combination with other drugs or therapies including antibacterial agents, antiviral drugs, antifungals, and trichomoniasis drugs. , antithrombotic drugs, antidiabetic drugs, physical therapy, laser therapy, local surgery and so on.
  • the invention in another aspect, relates to an article or kit comprising plasminogen or plasmin for use in preventing and/or treating cervical erosion in a subject.
  • the article or kit further comprises a container containing one or more other drugs.
  • the article or kit may further comprise instructions for use, wherein the plasminogen or plasmin may be used to prevent and/or treat the cervical erosion, and may further illustrate the plasminogen or The plasmin may be administered prior to, concurrently with, and/or after administration of the other drug or therapy.
  • the other drug or therapy comprises an antibacterial infection drug, an antiviral drug, an antifungal drug, an anti-triceps drug, an anti-thrombotic drug, an anti-diabetic drug, a physical therapy, a laser therapy, a local surgery therapy, etc. .
  • the instructions further clarify that the plasminogen or plasmin may be administered systemically or locally, preferably by the following routes: intravenous, muscular Internal, subcutaneous, local injection, administration through the rectum, vagina.
  • the topical administration is by applying a dressing containing plasminogen in the area of cervical erosion.
  • the cervical erosion includes true erosion and false erosion.
  • the subject is a mammal, preferably a human.
  • the cervical erosion may be cervical erosion caused by any cause, in particular, cervical erosion caused by damage such as inflammation.
  • the subject is plasminogen or plasmin is low.
  • the low is congenital, secondary and/or local.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 sexual, and still have plasminogen activity.
  • plasminogen is added, deleted and/or substituted on the basis of sequence 2, 6, 8, 10 or 12, 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.
  • the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, plasminogen, microplasminogen, or any combination thereof. In one embodiment, the plasminogen is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, plasminogen, delta-plasminogen or Microplasminogen.
  • the plasminogen is a human native plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, fibrinolysis from a primate or rodent
  • the zymogen is a straight homologue, such as a troponin-directed homologue from gorillas, rhesus monkeys, rats, cows, horses, and dogs.
  • the amino acid sequence of the plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • the present invention expressly covers all combinations of the technical features between the embodiments of the present invention, and these combined technical solutions are explicitly disclosed in the present application, just as the above technical solutions have been separately and explicitly disclosed.
  • the present invention also expressly encompasses all subcombinations of the various embodiments and elements thereof, and is disclosed herein as if each such subcombination is separately and explicitly disclosed herein.
  • Cervical erosion is the most common form of chronic cervical inflammation.
  • the common surface of the cervix is detached or replaced by another tissue of the cervix. You can even see the blood vessels underneath and the red tissue, forming a true erosion or False erosion.
  • True erosion is caused by long-term stimulation of the secretion of the cervical surface, impregnation of the squamous epithelium around the external cervix, accompanied by inflammatory infiltration, so that the squamous epithelium covering the surface of the cervix falls off and forms an ulcer.
  • “Pseudo-eating” refers to the replacement of columnar epithelial hyperplasia and extravasation of the cervical mucosa after squamous epithelial damage of the cervix. Because the covered single-layer columnar epithelium is thin, the lower blood vessels are clearly visible and the naked eye looks like It seems to be gorgeous, actually it is fake. False erosion is the most common cervical erosion in the clinic.
  • Cervical erosion can be divided into 3 types according to the surface condition of erosion:
  • the erosion surface is covered by a single layer of columnar epithelium, and the surface is flat, which is called simple erosion;
  • Columal epithelial cells are cervical columnar epithelial cells.
  • the single-layer columnar epithelium consists of a layer of prismatic cells. The nucleus is elliptical and is located at the base of the cell.
  • the single-layer columnar epithelium is distributed in the luminal surface of the stomach, intestine, uterus and fallopian tubes, and its function is mainly absorption and secretion.
  • Epithelial cells are a type of epithelial cell tissue.
  • Epithelial tissue also called epithelium, is an important structure that lining or covering other tissues. It consists of dense epithelial cells and a small amount of intercellular substance. The structural feature is that the cells are tightly bound and the intercellular substance is small. It usually has the functions of protection, absorption, secretion and excretion.
  • Epithelial tissue can be divided into three types: covered epithelium, glandular epithelium and sensory epithelium.
  • the coated epithelium is divided into a squamous epithelium, a columnar epithelium, a cubic epithelium, and a transitional epithelium according to the shape exhibited by the cells in a section perpendicular to the epithelial surface.
  • cervical erosion due to the low columnar epithelial resistance of the cervical canal, the pathogen is easily invaded and inflammation occurs.
  • the columnar epithelium When the columnar epithelium is damaged, the columnar epithelium of the cervical mucosa proliferates and the defect of the squamous epithelium of the uterus is vaginal. Extend, cover the wound, replacing the original phosphorus In the area of skin defect, because the columnar epithelium is thin, the capillaries under the mucosa are obviously visible. Therefore, the mucosa of the external cervix is marked with a bright red erosion-like area. Therefore, internationally, cervical erosion is also called "cervical epithelial differentiation.” Bit".
  • Plasmid is a very important enzyme found in the blood that hydrolyzes fibrin clots into fibrin degradation products and D-dimers.
  • Plasinogen is a zymogen form of plasmin, which is composed of 810 amino acids, based on the sequence in swiss prot, based on the native human plasminogen amino acid sequence (sequence 4) containing the signal peptide. 90 kD, a glycoprotein synthesized mainly in the liver and capable of circulating in the blood, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO:3.
  • Full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle 1-5).
  • the signal peptide includes the residue Met1-Gly19
  • PAp includes the residue Glu20-Val98
  • Kringle1 includes the residue Cys103-Cys181
  • Kringle2 includes the residue Glu184-Cys262
  • Kringle3 includes the residue Cys275-Cys352
  • Kringle4 Including the residue Cys377-Cys454
  • Kringle5 includes the residue Cys481-Cys560.
  • the serine protease domain includes the residues Val581-Arg804.
  • Glu-plasminogen is a natural full-length plasminogen consisting of 791 amino acids (not containing a 19 amino acid signal peptide), and the cDNA sequence encoding the sequence is shown in SEQ ID NO: 1, and its amino acid sequence is sequence 2. Shown. In vivo, there is also a Lys-plasminogen which is hydrolyzed from amino acids 76-77 of Glu-plasminogen, and as shown in SEQ ID NO: 6, the cDNA sequence encoding the amino acid sequence is as shown in SEQ ID NO: 5 Shown.
  • ⁇ -plasminogen is a fragment of full-length plasminogen lacking the structure of Kringle2-Kringle5, containing only Kringle1 and serine protease domains [23,24] .
  • ⁇ -plasminogen has been reported in the literature.
  • the amino acid sequence (SEQ ID NO: 8) [24] the cDNA sequence encoding the amino acid sequence is shown in Sequence 7.
  • Mini-plasminogen consists of Kringle5 and a serine protease domain, which has been reported in the literature to include the residue Val443-Asn791 (starting amino acid with a Glu residue of Glu-plasminogen sequence not containing a signal peptide) [25] , the amino acid sequence thereof is shown in SEQ ID NO: 10, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 9.
  • Micro-plasminogen contains only the serine protease domain, and its amino acid sequence has been reported to include the residue Ala543-Asn791 (from the Glu residue of the Glu-plasminogen sequence containing no signal peptide).
  • Plasin of the present invention is used interchangeably with “fibrinolytic enzyme” and “fibrinolytic enzyme”, and has the same meaning; “plasminogen” and “plasminogen”, “fibrinolytic enzyme” "Original” is used interchangeably and has the same meaning.
  • the present invention can prevent other diseases caused by cervical erosion by preventing and/or treating cervical erosion, such as cervical cancer, cervicitis, salpingitis, annexitis, pelvic inflammatory disease and the like. Therefore, prevention of these diseases is also encompassed within the scope of the present invention.
  • plasminogen adopts a closed inactive conformation, but when bound to the surface of a thrombus or cell, it is converted to openness mediated by plasminogen activator (PA).
  • PA plasminogen activator
  • Conformational active plasmin The active plasmin further hydrolyzes the fibrin clot into a fibrin degradation product and a D-dimer, thereby dissolving the thrombus.
  • the PAp domain of plasminogen contains an important determinant that maintains plasminogen in an inactive blocking conformation, while the KR domain is capable of binding to lysine residues present on the receptor and substrate.
  • plasminogen activators include tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hag Mann factor) and so on.
  • a "plasminogen active fragment” refers to an active fragment that binds to a target sequence in a substrate and exerts a proteolytic function in a plasminogen protein.
  • the technical solution of the present invention relating to plasminogen covers the technical solution of replacing plasminogen with a plasminogen active fragment.
  • the plasminogen active fragment of the present invention is a protein comprising a serine protease domain of plasminogen.
  • the plasminogen active fragment of the present invention comprises the sequence 14, and the sequence 14 has at least 80%, 90.
  • the plasminogen of the present invention comprises a protein comprising the plasminogen active fragment and still retaining the plasminogen activity.
  • methods for measuring plasminogen activity and activity in blood include: detection of tissue plasminogen activator activity (t-PAA), plasma tissue plasminogen activator Detection of antigen (t-PAAg), detection of plasma plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity , detection of plasma tissue plasminogen activator inhibitor antigen, plasma plasmin-anti-plasmin complex assay (PAP).
  • t-PAA tissue plasminogen activator activity
  • plgA plasma tissue plasminogen activator Detection of antigen
  • plgA plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma plasmin-anti-plasmin complex assay
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the plasma to be tested, and the PLG in the tested plasma is converted into PLM under the action of SK, and the latter acts on The chromogenic substrate is then measured spectrophotometrically and the increase in absorbance is directly proportional to the plasminogen activity.
  • plasminogen activity in blood can also be measured by immunochemical methods, gel electrophoresis, immunoturbidimetry, and radioimmunoassay.
  • ortholog or ortholog refers to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs, orthologs. It specifically refers to a protein or gene that has 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 of plasminogen activity derived from different species.
  • Constant substitution variant refers to a change in one of the given amino acid residues without altering the overall conformation and function of the protein or enzyme, including but not limited to similar properties (eg, acidic, basic, hydrophobic, etc.)
  • the amino acid replaces the amino acid in the amino acid sequence of the parent protein.
  • Amino acids having 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 that can be replaced by leucine, methionine or valine. Therefore, the similarity of two protein or amino acid sequences of similar function may be different.
  • Constant substitution variants also includes determining polypeptides or enzymes having more than 60% amino acid identity by BLAST or FASTA algorithm. If it is more than 75%, preferably more than 85%, or even more than 90%. Optimal and have the same or substantially similar properties or functions as the native or parent protein or enzyme.
  • Isolated plasminogen refers to a plasminogen protein that is isolated and/or recovered from its natural environment.
  • the plasminogen will purify (1) to a purity (by weight) greater than 90%, greater than 95%, or greater than 98%, as determined by the Lowry method, eg, over 99 % (by weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, the homogeneity It was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced or non-reducing conditions using Coomassie blue or silver staining.
  • Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.
  • polypeptide peptide
  • protein protein
  • fusion proteins including, but not limited to, fusion proteins having a heterologous amino acid sequence, fusions having heterologous and homologous leader sequences (with or without an N-terminal methionine residue);
  • percent amino acid sequence identity with respect to a reference polypeptide sequence is defined as the introduction of a gap as necessary to achieve maximum percent sequence identity, and without any conservative substitution being considered as part of sequence identity, in the candidate sequence The percentage of amino acid residues that are identical in amino acid residues in the reference polypeptide sequence. Comparisons for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum contrast over the full length of the sequences being compared. However, for the purposes of the present invention, amino acid sequence identity percent values are 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 may be expressed as having or comprising relative to, and, or for a given amino acid sequence)
  • a given amino acid sequence A of a certain % amino acid sequence identity of B is calculated as follows:
  • X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 in the A and B alignments of the program, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. All % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless explicitly stated otherwise.
  • the terms “treating,” “treating,” and “eliminating” refer to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be to completely or partially prevent the disease or its symptoms, and/or to partially or completely cure the disease and/or its symptoms, and includes: (a) preventing the disease from occurring in the subject, the subject may have The cause of the disease, but not yet diagnosed as having a disease; (b) inhibiting the disease, ie, retarding its formation; and (c) reducing the disease and/or its symptoms, ie causing the disease and/or its symptoms to subside.
  • the terms "individual”, “subject” and “patient” are used interchangeably herein to refer to a mammal, including but not limited to a mouse (rat, mouse), a non-human primate, a human, a dog, a cat. Hoofed animals (such as horses, cattle, sheep, pigs, goats).
  • “Therapeutically effective amount” or “effective amount” refers to an amount of plasminogen sufficient to effect such prevention and/or treatment of a disease when administered to a mammal or other subject to treat the disease.
  • the “therapeutically effective amount” will vary depending on the plasminogen used, the severity of the disease and/or its symptoms of the subject to be treated, and the age, weight, and the like.
  • Plasminogen can be isolated and purified from nature for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is chemically synthesized, it can be synthesized in a liquid phase or a solid phase.
  • Solid phase polypeptide synthesis SPPS
  • Fmoc and Boc Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen.
  • the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected to reveal a new N-terminal amine that can be attached to other amino acids.
  • the peptide remains immobilized on the solid phase and then cut off.
  • the plasminogen of the invention can be produced using standard recombinant methods. For example, inserting a nucleic acid encoding plasminogen into an expression vector to modulate it with a regulatory sequence in an expression vector Operable connection.
  • 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 regulation can be a eukaryotic promoter system in a vector that is 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 are typically replicated as an episome in the host organism or as an integral part of the host chromosomal DNA.
  • expression vectors typically contain a selection marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate transformation of the desired DNA sequence with foreign sources. Those cells are tested.
  • a selection 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 antibody-encoding polynucleotide.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Genus (Pseudomonas) species.
  • expression vectors can also be generated which will typically contain expression control sequences (e.g., 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 phage lambda. Promoters typically control expression, optionally in the context of manipulating a gene sequence, and have a ribosome binding site sequence, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast e.g., S. cerevisiae
  • Pichia are examples of suitable yeast host cells in which a suitable vector has expression control sequences (e.g., a promoter), an origin of replication, a termination sequence, and the like, as desired.
  • a typical promoter comprises 3-phosphoglycerate kinase and other saccharolytic enzymes.
  • Inducible yeast is initiated by a promoter specifically comprising an alcohol dehydrogenase, an isocytochrome C, and an enzyme responsible for the utilization of maltose and galactose.
  • mammalian cells e.g., mammalian cells cultured in in vitro cell culture
  • an anti-Tau antibody of the invention e.g., a polynucleotide encoding a subject anti-Tau antibody.
  • 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 these cells may contain expression control sequences such as origins of replication, promoters and enhancers (Queen et al, Immunol. Rev.
  • RNA splice sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences.
  • suitable expression control sequences are promoters derived from the white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol. 148: 1149 (1992).
  • the invention may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like.
  • Plasminogen is substantially pure, such as 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 more pure, for example, free of contaminants, such as cellular debris, macromolecules other than the subject antibody, and the like.
  • Therapeutic formulations are prepared as a lyophilized formulation or as an aqueous solution.
  • Acceptable carriers, excipients, and stabilizers are non-toxic to the recipient 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; chlorinated hexane diamine; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoic acid Esters such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptide (less than about 10 residues) Protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • the formulations of the invention may also contain more than one active compound as required for the particular condition being treated, preferably those having complementary activities and no side effects to each other.
  • active compound for example, anti-infective drugs and the like.
  • the plasminogen of the present invention may be encapsulated in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, for example, may be placed in a glial drug delivery system (eg, liposome, albumin microspheres, microemulsion) , nanoparticles and nanocapsules) or in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • glial drug delivery system eg, liposome, albumin microspheres, microemulsion
  • nanoparticles and nanocapsules nanoparticles and nanocapsules
  • hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • the plasminogen of the invention for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after lyophilization and reconstitution.
  • the plasminogen of the present invention can prepare a sustained release preparation.
  • sustained release formulations include solid hydrophobic polymeric semi-permeable matrices having a shape and containing glycoproteins, such as films or microcapsules.
  • sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al, J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem .Tech., 12: 98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S.
  • Patent 3,739,919, EP 58,481 copolymer of L-glutamic acid and ethyl-L-glutamic acid ( Sidman, et al, Biopolymers 22: 547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al, supra), or degradable lactic acid-glycolic acid copolymer such as Lupron DepotTM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly D-(-)-3-hydroxybutyric acid.
  • Lupron DepotTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • Polymers such as ethylene-acetic acid Vinyl ester and lactic acid-glycolic acid can release molecules for more than 100 days, while some hydrogels release proteins for a short time.
  • a reasonable strategy for protein stabilization can be designed according to the relevant mechanism. For example, if the mechanism of aggregation is found When thiodisulfide bonds are exchanged to form intermolecular SS bonds, the sulfhydryl residues can be modified, lyophilized from acidic solutions, and humidity controlled. Stabilization is achieved with suitable additives and the development of specific polymer matrix compositions.
  • the invention may be practiced in various ways, for example by intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (for example via carotid), intramuscular, intranasal, topical or intradermal administration or spinal or brain delivery.
  • Administration of the pharmaceutical composition
  • Aerosol formulations such as nasal spray formulations comprise purified aqueous or other solutions of the active agents and preservatives and isotonic agents. Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.
  • the plasminogen pharmaceutical compositions of the invention may be modified or formulated in such a manner as to provide their ability to cross the blood brain barrier.
  • Such plasminogen compositions can be administered to individuals suffering from thrombotic and/or thrombotic related diseases by a variety of enteral and parenteral routes of administration, including oral, intravenous, and the like.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous 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 buffering media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils.
  • Intravenous vehicles contain liquid and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.
  • the medical staff will determine the dosage regimen based on various clinical factors. As is well known in the medical arts, the dosage of any patient depends on a variety of factors, including the patient's size, body surface area, age, specific compound to be administered, sex, number and route of administration, overall health, and other medications administered simultaneously. .
  • the pharmaceutical composition of the present invention comprising plasminogen may have a dose ranging, for example, from about 0.0001 to 2000 mg/kg per day, or from about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75). Mg/kg, 10 mg/kg, 50 mg/kg, etc.) Subject weight.
  • the dose can 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, particularly in view of the above factors. Intermediate doses in the above ranges are also included in the scope of the present invention.
  • the subject can administer such doses daily, every other day, every week, or according to any other schedule determined by empirical analysis.
  • An exemplary dosage schedule includes 1-10 mg/kg for several days. The therapeutic effect and safety of thrombus and thrombosis-related diseases need to be evaluated and periodically evaluated in the drug administration process of the present invention.
  • Gynecological examination Focus on the size, shape, texture, thickness of the cervical canal, and whether there is contact bleeding.
  • Cytological examination is a routine examination for gynecology. It is simple, economical and effective. It is the most important primary screening method for auxiliary examination and anti-cancer screening.
  • Cervical scraping refers to taking a small amount of cell sample from the cervix of the uterus, placing it on a glass slide, and then studying whether it is abnormal under a microscope.
  • Colposcopy Can quickly find invisible lesions, take a suspicious site biopsy in colposcopy, can significantly improve the accuracy of biopsy.
  • TCT is the abbreviation of liquid-based thin layer cell detection
  • TCT examination uses a liquid-based thin-layer cell detection system to detect cervical cells and perform cytological classification diagnosis. It is the most advanced cervical cancer cell examination technique in the world, and it is compared with the traditional cervical smear Pap smear test. The ratio significantly increased the satisfaction of the specimen and the abnormal cell detection rate of the cervix.
  • Cervical biopsy Pathological examination of cervical living tissue is the basis for the diagnosis of cervical cancer. Cervical biopsy is a biopsy of the cervix, that is, taking a small piece or pieces of tissue from the cervix for pathological examination to determine the diagnosis.
  • the article preferably includes a container, label or package insert.
  • Suitable containers are bottles, vials, syringes, and the like.
  • the container can be made of various materials such as glass or plastic.
  • the container contains a composition that is effective to treat a disease or condition of the invention and has a sterile access port (eg, the container can be an intravenous solution or vial containing a stopper that can be penetrated by a hypodermic needle) of).
  • At least one active agent in the composition is plasminogen or plasmin.
  • the label on or attached to the container indicates that the composition is used to treat cervical erosion as described herein.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • the article comprises a package insert with instructions for use, including, for example, a user instructing the composition to administer the plasminogen composition and other drugs to treat the accompanying disease.
  • Figure 1 shows the results of cervical HE staining on days 5 and 9 of plg +/+ cervical erosion model mice given plasminogen or PBS.
  • Figure 2 shows the results of cervical HE staining on days 5, 9 and 13 of plg -/- cervical erosion model mice given plasminogen or PBS.
  • Figure 3 shows the results of cervical fibrin immunostaining on days 5 and 9 of plg +/+ cervical erosion model mice given plasminogen or PBS.
  • Figure 4 shows the results of immunostaining of cervical fibrin on days 5, 9 and 13 of plg -/- cervical erosion model mice given plasminogen or PBS.
  • Figure 5 shows the results of cervical F4/80 immunostaining on days 5 and 9 of plg +/+ cervical erosion model mice given plasminogen or PBS.
  • Figure 6 shows the results of cervical F4/80 immunostaining on days 5, 9 and 13 of plg -/- cervical erosion model mice given plasminogen or PBS.
  • plasminogen was administered to the plasminogen group at a dose of 1 mg/0.1 mL/day/day via the tail vein, and the same volume of PBS was administered to the vehicle PBS control group.
  • day 0 plasminogen or vehicle PBS was administered on the first day, and the administration period was 8 days.
  • 3 mice were randomly selected, and the mice were sacrificed by eyeball removal.
  • the cervical tissue was fixed in 4% paraformaldehyde fixative for 24-48 hours.
  • the fixed cervical tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the tissue section was 5 ⁇ m, the sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), 1% hydrochloric acid alcohol was differentiated, ammonia water was returned to the blue, and the mixture was dehydrated with an alcohol gradient, and the sections were observed under a microscope at 200 times.
  • HE staining hematoxylin and eosin
  • the results of HE staining showed that the keratinized layer of the mucosa in the PBS control group was over-exposed, shedding ( ⁇ ), squamous epithelial mild hyperplasia ( ⁇ ), and the keratinized stratum corneum was almost detached on the 9th day. Unevenness ( ⁇ ), no epithelial repair, severe squamous epithelial hyperplasia (Fig. 1A, B); partial detachment of the stratum corneum disappeared ( ⁇ ) on the 5th day of the plasminogen group, and the damaged epithelial surface was regenerated by the epithelial surface.
  • plasminogen was administered to the plasminogen group at a dose of 1 mg/0.1 mL/day/day via the tail vein, and the same volume of PBS was administered to the vehicle PBS control group.
  • day 1 plasminogen or vehicle PBS was administered on the first day, and the administration period was 12 days.
  • day 9th, and 13th day 3 mice were randomly selected, and the mice were sacrificed by eyeball removal.
  • the cervical tissue was fixed in 4% paraformaldehyde fixative for 24-48 hours.
  • the fixed kidney was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, the sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), 1% hydrochloric acid alcohol was differentiated, ammonia water was returned to the blue, and the mixture was dehydrated with an alcohol gradient, and the sections were observed under a microscope at 200 times.
  • HE staining hematoxylin and eosin
  • plasminogen was administered to the plasminogen group at a dose of 1 mg/0.1 mL/day/day via the tail vein, and the same volume of PBS was administered to the vehicle PBS control group.
  • day 0 plasminogen or vehicle PBS was administered on the first day, and the administration period was 8 days.
  • 3 mice were randomly selected, and the mice were sacrificed by eyeball removal.
  • the cervical tissue was fixed in 4% paraformaldehyde fixative for 24-48 hours.
  • the fixed cervical tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the citric acid was repaired for 30 minutes, and after cooling at room temperature for 10 minutes, the water was gently rinsed. Incubate for 15 minutes in 3% hydrogen peroxide and circle the tissue with a PAP pen. 10% of normal goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after the time was over, the sheep serum was discarded.
  • Rabbit anti-mouse fibrin (pro) antibody (Abeam) was incubated overnight at 4 ° C and washed twice with TBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and twice with TBS for 5 minutes each time.
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • Fibrinogen is a precursor of fibrin. In the case of tissue damage, as a stress response to the body, fibrinogen is hydrolyzed to fibrin [28-30] . Fibrin levels can therefore be used as a marker of the extent of damage.
  • plasminogen was administered to the plasminogen group at a dose of 1 mg/0.1 mL/day/day via the tail vein, and the same volume of PBS was administered to the vehicle PBS control group.
  • day 0 plasminogen or vehicle PBS was administered on the first day, and the administration period was 12 days.
  • 3 mice were randomly selected, and the mice were sacrificed by eyeball removal.
  • the cervical tissue was fixed in 4% paraformaldehyde fixative for 24-48 hours.
  • the fixed cervical tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the citric acid was repaired for 30 minutes, and after cooling at room temperature for 10 minutes, the water was gently rinsed. Incubate for 15 minutes in 3% hydrogen peroxide and circle the tissue with a PAP pen. 10% of normal goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after the time was over, the sheep serum was discarded.
  • Rabbit anti-mouse fibrin antibody (Abeam) was incubated overnight at 4 °C and washed twice with TBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and twice with TBS for 5 minutes each time.
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • Fibrinogen is a precursor of fibrin. In the case of tissue damage, as a stress response to the body, fibrinogen is hydrolyzed to fibrin [28-30] . Fibrin levels can therefore be used as a marker of the extent of damage.
  • plasminogen was administered to the plasminogen group at a dose of 1 mg/0.1 mL/day/day via the tail vein, and the same volume of PBS was administered to the vehicle PBS control group.
  • the body weight of the mice was weighed and grouped one day before the model establishment, and the day of the modeling was day 0, and plasminogen or vehicle PBS was administered on the first day, and the administration period was 8 days.
  • the cervical tissue was fixed in 4% paraformaldehyde fixative for 24-48 hours.
  • the fixed cervical tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 10% normal goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour, after which time the serum was removed and the tissue was circled with a PAP pen. Rabbit polyclonal antibody (Abeam) against F4/80 was incubated overnight at 4 °C, and TBS was washed twice for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and twice with TBS.
  • HRP Goat anti-rabbit IgG
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 400 times.
  • the F4/80 macrophage marker can indicate the extent and stage of the inflammatory response.
  • the results showed that the positive expression of cervical F4/80 in the vehicle PBS control group (Fig. 5A, B) and the plasminogen group (Fig. 5C, D) was higher than the fifth day on day 9, but plasmin was given.
  • the original group should be significantly less than the vehicle PBS control group. It is indicated that plasminogen can reduce the inflammation of injured tissues, indicating that plasminogen can promote the repair of cervical inflammation in mice with plg+/+ cervical erosion.
  • plasminogen was administered to the plasminogen group at a dose of 1 mg/0.1 mL/day/day via the tail vein, and the same volume of PBS was administered to the vehicle PBS control group.
  • the mice were weighed and grouped one day before the model establishment, and the day of modeling was day 0, and plasminogen or vehicle PBS was administered on the first day, and the administration period was 12 days. On the 5th, 9th, and 13th day, 3 mice were randomly selected, and the mice were sacrificed by eyeball removal.
  • the cervical tissue was fixed in 4% paraformaldehyde fixative for 24-48 hours. The fixed cervical tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 10% normal goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour, after which time the serum was removed and the tissue was circled with a PAP pen. Rabbit polyclonal antibody (Abeam) against F4/80 was incubated overnight at 4 °C, and TBS was washed twice for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and twice with TBS.
  • HRP Goat anti-rabbit IgG
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 400 times.
  • the F4/80 macrophage marker can indicate the extent and stage of the inflammatory response.
  • the results showed that there was no significant change in the F4/80 positive expression on days 5, 9, and 13 in the vehicle PBS control group (Fig. 6A-C) and the plasminogen group (Fig. 6D-F), but The positive expression level in the plasminogen group was lower than that in the vehicle PBS control group.
  • the results showed that plasminogen can reduce the inflammation level of injured tissues, indicating that plasminogen can promote the inflammatory repair of injured cervical prg -/- cervical erosion model mice.

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Abstract

纤维蛋白溶酶原在治疗宫颈糜烂中的用途，与现有其它治疗宫颈糜烂的药物相比，该纤维蛋白溶酶原或纤维蛋白溶酶能促进损伤粘膜的炎症修复。

Description

一种用于预防和治疗宫颈糜烂的方法 技术领域
本发明涉及一种新的使用纤维蛋白溶酶原或纤溶酶预防和/或治疗宫颈糜烂的方法。与现有治疗宫颈糜烂的药物相比，该方法能有效促进损伤粘膜的修复。
背景技术
慢性宫颈炎是已婚妇女的常见病、多发病，在我国妇女发病中占首位，约占妇科疾患的50％。宫颈糜烂又是慢性宫颈炎中最常见的一种病理改变，其发生宫颈癌者要高于无宫颈糜烂妇女的7.3倍。有报道，单纯鳞状细胞癌中，约有80％是在颈管或糜烂区即柱状上皮区发生，而其中绝大多数是在“糜烂”区发生[1]。造成子宫颈糜烂的主因，通常是妇女在产后或者术后造成子宫颈的损伤，并随后伴随着病原体的侵入而形成的。20世纪80年代以前，导致慢性宫颈炎的病原体主要为葡萄球菌、链球菌、大肠杆菌及厌氧菌[2]。近年来，随着性病的发病率逐年上升和性传播疾病的增加，宫颈糜烂亦呈日益增多之趋势，严重影响妇女的生殖健康和生活质量，同时宫颈糜烂的病原体也发生了变化，越来越多的资料表明，沙眼衣原体(CT)、奈瑟淋病双球菌(NG)、单纯疱疹病毒(HSV)、解脲支原体(Uu)、阴道毛滴虫(TV)和念珠菌(CA)等感染均与宫颈炎有关[3-5]。
宫颈糜烂主要症状为白带增多且常为脓性、接触性出血、腰骶部疼痛、不孕等。目前，治疗宫颈糜烂的方法很多，包括口服药、阴道上药、宫颈局部物理疗法、手术治疗等。对于宫颈糜烂病情轻者，不论哪种治疗方法，虽治疗时间长短不同，都可收到较好的疗效。但宫颈糜烂中、重度患者，口服药物治疗，起效慢、局部血药浓度低，难以达到预期效果；单纯阴道上药治疗疗程长、疗效欠佳，且疗效不稳定，复发的几率大。手术治疗费用高、创伤大、愈合时间长，一般患者很难接受。
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[6]，此外，纤溶酶能将一些金属蛋白酶前体 (pro-MMP)激活形成具有活性的金属蛋白酶(MMP)。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[7，8]。纤溶酶是由纤溶酶原通过两种生理性的PA：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PA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PA的特定生理抑制剂。纤溶酶的主要抑制剂是α2-抗纤溶酶(α2-antiplasmin)。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)[9，10]。
纤溶酶原(plasminogen，plg)是一个单链糖蛋白，由791个氨基酸组成，分子量约为92kDa[11，12]。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[13，14]。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PA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[15]。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringle，羧基末端部分包含蛋白酶结构域。一些kringle含有介导纤溶酶原与纤维蛋白及其抑制剂α2-AP特异性相互作用的赖氨酸结合位点。最新发现一个纤维蛋白溶酶原为38kDa的片段，其中包括kringle1-4，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[16]。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[12，17，18]。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[19]。此外，纤溶酶具有激活某些潜在形式的生长因子的能力[20-22]。
本发明经过研究发现，纤维蛋白溶酶原在预防和/或治疗宫颈糜烂时具有意想不到的效果，其具体表现在能促进损伤和炎症的修复。使用纤维蛋白溶酶原预防和/或治疗宫颈糜烂，无论是从疗效上、患者承受能力上、治疗的简便性上，都具有不可比拟的优势。因此，纤维酶原可能成为一种新颖的预防和/或治疗宫颈糜烂的策略。
发明简述
本发明涉及纤溶酶原对宫颈糜烂的预防和/或治疗。本发明惊奇地发现，纤维蛋白溶酶原在预防和/或治疗宫颈糜烂时，表现出突出的预防和/或治疗效果，能有效促进损伤组织的修复。
一方面，本发明涉及一种新的预防和/或治疗宫颈糜烂及其相关病症的方法，以及纤溶酶原或纤溶酶用于预防和/或治疗宫颈糜烂及其相关病症的用途，该方法或用途包括向受试者体内施用纤溶酶原或纤溶酶。以上所述宫颈糜烂包括真性糜烂和假性糜烂。所述受试者为哺乳动物，优选为人。在一个实施方案中，所述宫颈糜烂可以是任何原因导致的宫颈糜烂，具体地，为炎症等损伤导致的宫颈糜烂。
在一个实施方案中，所述受试者纤溶酶原或纤溶酶低下。具体地，所述低下是先天的、继发的和/或局部的。
在一个实施方案中，纤维蛋白溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤维蛋白溶酶原活性。在一个实施方案中，纤维蛋白溶酶原是在序列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，并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原选自Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、微纤维蛋白溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案中，纤溶酶原 是选自如下的保守取代变体：Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、δ-纤溶酶原或微纤维蛋白溶酶原。在一个实施方案中，纤维蛋白溶酶原为人天然纤维蛋白溶酶原，例如序列2所示的纤溶酶原的直向同系物，例如，来自灵长类动物或啮齿类动物的纤维蛋白溶酶原直向同系物，例如来自大猩猩，恒河猴、鼠、牛、马，狗的纤维蛋白溶酶原直向同系物。最优选，本发明的纤维蛋白溶酶原的氨基酸序列如序列2、6、8、10或12所示。
在一个实施方案中，所述纤维蛋白溶酶原或纤维蛋白溶酶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、局部注射、通过直肠、阴道施用。在一个实施方案中，所述局部给药通过在宫颈糜烂区域应用含有纤维蛋白溶酶原的敷料来进行。
在一个实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在一个实施方案中，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。在局部施用的情况下，上述剂量还可以根据情况进一步调整。
上述纤维蛋白溶酶原或纤维蛋白溶酶可以单独施用，也可以与其它药物或疗法联合使用，所述其它药物或疗法包括抗细菌感染药物，抗病毒感染药物，抗真菌药物、抗滴虫药物、抗血栓药物、抗糖尿病药物、物理疗法、激光疗法、局部手术疗法等。
另一方面，本发明涉及纤维蛋白溶酶原或纤维蛋白溶酶在制备预防和/或治疗受试者宫颈糜烂的药物中的用途。本发明还涉及一种药物的制备方法，包括将纤溶酶原或纤溶酶与药学可接受载体制备成治疗受试者宫颈糜烂的药物。在一个实施方案中，所述宫颈糜烂包括真性糜烂和假性糜烂。所述受试者为哺乳动物，优选为人。在一个实施方案中，所述宫颈糜烂可以是任何原因导致的宫颈糜烂，具体地，为炎症等损伤导致的宫颈糜烂。
在一个实施方案中，所述受试者纤溶酶原或纤溶酶低下。具体地，所述低下是先天的、继发的和/或局部的。
在一个实施方案中，纤维蛋白溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤维蛋白溶酶原活性。在一个实施方案中，纤维蛋白溶酶原是在序列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，并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原选自Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、微纤维蛋白溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、δ-纤溶酶原或微纤维蛋白溶酶原。在一个实施方案中，纤维蛋白溶酶原为人天然纤维蛋白溶酶原，例如序列2所示的纤溶酶原的直向同系物，例如，来自灵长类动物或啮齿类动物的纤维蛋白溶酶原直向同系物，例如来自大猩猩，恒河猴、鼠、牛、马，狗的纤维蛋白溶酶原直向同系物。最优选，本发明的纤维蛋白溶酶原的氨基酸序列如序列2、6、8、10或12所示。
在一个实施方案中，所述纤维蛋白溶酶原或纤维蛋白溶酶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、局部注射、通过直肠、阴道施用。在一个实施方案中，所述局部给药通过在宫颈糜烂区域应用含有纤维蛋白溶酶原的敷料来进行。
在一个实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在一个实施方案中，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。在局部施用的情况下，上述剂量还可以根据情况进一步调整。
上述纤维蛋白溶酶原或纤维蛋白溶酶可以单独施用，也可以与其它药物或疗法联合使用，所述其它药物或疗法包括抗细菌感染药物，抗病毒感染药物，抗真菌药物、抗滴虫药物、抗血栓药物、抗糖尿病药物、物理疗法、激光疗法、局部手术疗法等。
另一方面，本发明涉及用于预防和/或治疗宫颈糜烂的纤溶酶原或纤溶酶，以及用于预防和/或治疗宫颈糜烂的、包含纤维蛋白溶酶原或纤维蛋白溶酶的药物组合物。在一个实施方案中，所述宫颈糜烂包括真性糜烂和假性糜烂。所述受试者为哺乳动物，优选为人。在一个实施方案中，所述宫颈糜烂可以是任何原因导致的宫颈糜烂，具体地，为炎症等损伤导致的宫颈糜烂。
在一个实施方案中，所述受试者纤溶酶原或纤溶酶低下。具体地，所述低下是先天的、继发的和/或局部的。
在一个实施方案中，纤维蛋白溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤维蛋白溶酶原活性。在一个实施方案中，纤维蛋白溶酶原是在序列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，并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原选自Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、微纤维蛋白溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、δ-纤溶酶原或微纤维蛋白溶酶原。在一个实施方案中，纤维蛋白溶酶原为人天然纤维蛋白溶酶原，例如序列2所示的纤溶酶原的直向同系物，例如，来自灵长类动物或啮齿类动物的纤维蛋白溶酶原直向同系物，例如来自大猩猩，恒河猴、鼠、牛、马，狗的纤维蛋白溶酶 原直向同系物。最优选，本发明的纤维蛋白溶酶原的氨基酸序列如序列2、6、8、10或12所示。
在一个实施方案中，所述纤维蛋白溶酶原或纤维蛋白溶酶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、局部注射、通过直肠、阴道施用。在一个实施方案中，所述局部给药通过在宫颈糜烂区域应用含有纤维蛋白溶酶原的敷料来进行。
在一个实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在一个实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在一个实施方案中，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。在局部施用的情况下，上述剂量还可以根据情况进一步调整。
上述纤维蛋白溶酶原或纤维蛋白溶酶可以单独施用，也可以与其它药物或疗法联合使用，所述其它药物或疗法包括抗细菌感染药物、抗病毒感染药物、抗真菌药物、抗滴虫药物、抗血栓药物、抗糖尿病药物、物理疗法、激光疗法、局部手术疗法等。
另一个方面，本发明涉及用于预防和/或治疗受试者宫颈糜烂的、包含纤维蛋白溶酶原或纤维蛋白溶酶的制品或药盒。优选，该制品或药盒进一步包含含有一种或多种其它的药物的容器。该制品或药盒还可包含使用说明书，说明所述纤维蛋白溶酶原或纤维蛋白溶酶可以用于预防和/或治疗所述宫颈糜烂，并且可以进一步说明，所述纤维蛋白溶酶原或纤维蛋白溶酶可以在其它药物或疗法施用之前，同时，和/或之后施用。在一个实施方案中，所述其它药物或疗法包括抗细菌感染药物，抗病毒感染药物，抗真菌药物、抗滴虫药物、抗血栓药物、抗糖尿病药物、物理疗法、激光疗法、局部手术疗法等。
在一个实施方案中，所述说明书进一步说明所述纤维蛋白溶酶原或纤维蛋白溶酶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、局部注射、通过直肠、阴道施用。在一个实施方案中，所述局部给药通过在宫颈糜烂区域应用含有纤维蛋白溶酶原的敷料来进行。
在一个实施方案中，所述宫颈糜烂包括真性糜烂和假性糜烂。所述受试者为哺乳动物，优选为人。在一个实施方案中，所述宫颈糜烂可以是任何原因导致的宫颈糜烂，具体地，为炎症等损伤导致的宫颈糜烂。
在一个实施方案中，所述受试者纤溶酶原或纤溶酶低下。具体地，所述低下是先天的、继发的和/或局部的。
在一个实施方案中，纤维蛋白溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤维蛋白溶酶原活性。在一个实施方案中，纤维蛋白溶酶原是在序列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，并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原选自Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、微纤维蛋白溶酶原、或其任意组合。在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、δ-纤溶酶原或微纤维蛋白溶酶原。在一个实施方案中，纤维蛋白溶酶原为人天然纤维蛋白溶酶原，例如序列2所示的纤溶酶原的直向同系物，例如，来自灵长类动物或啮齿类动物的纤维蛋白溶酶原直向同系物，例如来自大猩猩、恒河猴、鼠、牛、马，狗的纤维蛋白溶酶原直向同系物。最优选，本发明的纤维蛋白溶酶原的氨基酸序列如序列2、6、8、10或12所示。
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所有亚组合，并且在本文中公开，就像每一个此类亚组合单独且明确在本文中公开一样。
发明详述
1.定义
“宫颈糜烂”是慢性宫颈炎症最常见的一种表现形式，常见宫颈口表面上皮脱落或是被宫颈口另外一种组织所代替，甚至可以看到下方的血管和红色的组织，形成真性糜烂或假性糜烂。
“真性糜烂”是由于宫颈表面分泌物长期刺激、浸渍宫颈外口周围的鳞状上皮，伴随炎性浸润，使覆在宫颈表面的鳞状上皮脱落，形成溃疡。
“假性糜烂”是指宫颈鳞状上皮损伤脱落后，由子宫颈管粘膜的柱状上皮增生、外移所取代，由于所覆盖的单层柱状上皮很薄，其下血管清晰可见，肉眼看起来状似糜烂，实际上为假性糜烂。假性糜烂是临床上最常见的宫颈糜烂。
宫颈糜烂根据糜烂表面状况可以分为3型：
1).在炎症初期，糜烂面仅为单层柱状上皮所覆盖，表面平坦，称单纯型糜烂；
2).随后由于腺上皮过度增生并伴有间质增生，糜烂面凸凹不平呈颗粒状，称颗粒型糜烂；
3).当间质增生显著，表面不平现象更加明显呈乳突状，称乳突型糜烂。
“柱状上皮细胞”为宫颈柱状上皮细胞。其中单层柱状上皮由一层棱柱形细胞组成。细胞核呈椭圆形，位于细胞基底部。单层柱状上皮分布于胃、肠、子宫和输卵管的内腔面，其功能主要是吸收和分泌。
“鳞状上皮细胞”是上皮细胞组织的一种。上皮组织也叫做上皮，它是衬贴或覆盖在其它组织上的一种重要结构。由密集的上皮细胞和少量细胞间质构成。结构特点是细胞结合紧密，细胞间质少。通常具有保护、吸收、分泌、排泄的功能。上皮组织可分成被覆上皮、腺上皮和感觉上皮三类。被覆上皮按其细胞在与上皮表面垂直的切面中所呈现的形状分为鳞状上皮、柱状上皮、立方上皮、移行上皮。
就宫颈糜烂的病理表现而言，由于宫颈管柱状上皮抵抗力低，病原体容易侵入发生炎症，当柱状上皮损伤后，由子宫颈管粘膜的柱状上皮增生，并向子宫阴道部磷状上皮的缺损处延伸，覆盖创面，取代了原磷状上 皮缺损的区域，由于柱状上皮较薄，粘膜下方充血的毛细血管明显易见，所以肉眼见宫颈外口病变粘膜呈鲜红色糜烂样区，因此，国际上，宫颈糜烂也称为“宫颈上皮异位”。
“纤溶酶”是存在于血液中的一种非常重要的酶，能将纤维蛋白凝块水解为纤维蛋白降解产物和D-二-聚体。
“纤溶酶原”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源纤溶酶原氨基酸序列(序列4)计算由810个氨基酸组成，分子量约为90kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的纤溶酶原包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基Cys103-Cys181，Kringle2包括残基Glu184-Cys262，Kringle3包括残基Cys275-Cys352，Kringle4包括残基Cys377-Cys454，Kringle5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。Δ-纤溶酶原(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶域[23，24]，有文献报道了δ-纤溶酶原的氨基酸序列(序列8)[24]，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)[25]，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)[26]，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。
本领域技术人员可以理解，本发明通过预防和/或治疗宫颈糜烂可以预防由于宫颈糜烂引发的其它疾病，例如宫颈癌、宫颈炎、输卵管炎、附件炎、盆腔炎等疾病的发生。因此，对于这些疾病的预防也涵盖在本发明的范围内。
在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时，在纤溶酶原激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性纤溶酶。具有活性的纤溶酶可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中纤溶酶原的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为纤溶酶原激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”是指在纤溶酶原蛋白中，能够与底物中的靶序列结合并发挥蛋白水解功能的活性片段。本发明涉及纤溶酶原的技术方案涵盖了用纤溶酶原活性片段代替纤溶酶原的技术方案。本发明所述的纤溶酶原活性片段为包含纤溶酶原的丝氨酸蛋白酶域的蛋白质，优选，本发明所述的纤溶酶原活性片段包含序列14、与序列14具有至少80％、90％、95％、96％、97％、98％、99％同源性的氨基酸序列的蛋白质。因此，本发明所述的纤溶酶原包括含有该纤溶酶原活性片段、并且仍然保持该纤溶酶原活性的蛋白。
目前，对于血液中纤维蛋白溶酶原及其活性测定方法包括：对组织纤维蛋白溶酶原激活剂活性的检测(t-PAA)、血浆组织纤维蛋白溶酶原激活剂 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)、血浆组织纤维蛋白溶酶原激活剂抑制物活性的检测、血浆组织纤维蛋白溶酶原激活剂抑制物抗原的检测、血浆纤维蛋白溶酶-抗纤维蛋白溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤维蛋白溶酶原活性成正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤维蛋白溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤维蛋白溶酶原包括人的天然纤维蛋白溶酶原，还包括来源于不同物种的、具有纤维蛋白溶酶原活性的纤维蛋白溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性，碱性，疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸，蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤维蛋白溶酶原是指从其天然环境分离和/或回收的纤维蛋白溶酶原蛋白。在一些实施方案中，所述纤维蛋白溶酶原会纯化(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。分离的纤维蛋白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维蛋白溶酶原。
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”、“处理”和“消除”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。
2.本发明纤维蛋白溶酶原的制备
纤维蛋白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)是适合纤维蛋白溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤维蛋白溶酶原。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.2006Mini Rev.Med Chem.6：3-10和Camarero JA等2005Protein 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维蛋白溶酶原。例如，将编码纤维蛋白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维蛋白溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆主题抗体编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(Enterobacteriaceae)，诸如沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物种。在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子系统，色氨酸(trp)启动子系统，β-内酰胺酶启动子系统，或来自噬菌体λ的启动子系统。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
在微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达并生成本发明的抗-Tau抗体(例如编码主题抗-Tau抗体的多核苷酸)。参见Winnacker，From Genes to Clones，VCH Publishers，N.Y.，N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起点，启动子和增强子 (Queen等，Immunol.Rev.89：49(1986))，以及必需的加工信息位点，诸如核糖体结合位点、RNA剪接位点、多聚腺苷酸化位点，和转录终止子序列。合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛乳头瘤病毒、巨细胞病毒等衍生的启动子。参见Co等，J.Immunol.148：1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀、亲和柱、柱层析、高效液相层析(HPLC)、凝胶电泳等来纯化本发明所述的纤维蛋白溶酶原。该纤维蛋白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，除主题抗体以外的大分子，等等。
3.药物配制剂
可以通过将具有所需纯度的纤维蛋白溶酶原与可选的药用载体、赋形剂，或稳定剂(Remington′s Pharmaceutical Sciences，16版，Osol，A.ed.(1980))混合形成冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。优选冻干的抗-VEGF抗体配制剂在WO 97/04801中描述，其包含在本文中作为参考。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。例如，抗感染的药物等。
本发明的纤维蛋白溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送系统(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington′s Pharmaceutical Sciences 16th edition，Osol，A.Ed.(1980)。
用于体内给药的本发明的纤维蛋白溶酶原必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤维蛋白溶酶原可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J.Biomed.Mater.Res.，15：167-277(1981)；Langer，Chem.Tech.，12：98-105(1982))或聚(乙烯醇)、聚交酯(美国专利3773919，EP 58,481)、L-谷氨酸与乙基-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。
4.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、鼻内、表面或皮内施用或脊髓或脑投递来实现本发明药物组合物的施用。气溶胶制剂如鼻喷雾制剂包含活性剂的纯化的水性或其它溶液及防腐剂和等渗剂。将此类制剂调节至与鼻粘膜相容的pH和等渗状态。
在一些情况中，可以以下方式修饰或配制本发明的纤维蛋白溶酶原药物组合物，从而提供其穿过血脑屏障的能力。可以通过各种肠内和胃肠外施用路径，包括口服，静脉内等对患有血栓和/或血栓相关疾病的个体施用此类纤维蛋白溶酶原的组合物。
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，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、定期评估血栓和血栓相关疾病的治疗效果和安全性。
5.宫颈糜烂治疗效果的评价方法
1)妇科检查。重点检查宫颈的大小、外形、质地、宫颈管粗细，是否有接触性出血。
2)宫颈刮片。细胞学检查为妇科常规检查，简便易行，经济有效，是最重要的辅助检查及防癌普查首选的初筛方法。
宫颈刮片，是指从子宫颈部取少量的细胞样品，放在玻璃片上，然后在显微镜下研究是否异常。
3)阴道镜检查。能迅速发现肉眼看不见的病变，在阴道镜检查中取可疑部位活检，能显著提高活检的准确率。
4)TCT检查。TCT是液基薄层细胞检测的简称
TCT检查是采用液基薄层细胞检测系统检测宫颈细胞并进行细胞学分类诊断，它是目前国际上最先进的一种宫颈癌细胞学检查技术，与传统的宫颈刮片巴氏涂片检查相比明显提高了标本的满意度及宫颈异常细胞检出率。
5)宫颈活检。宫颈活体组织的病理检查是确诊宫颈癌的依据。宫颈活检就是子宫颈的活体组织检查，即从宫颈上取一小块或几块组织作病理检查，以确定诊断。
6.制品或药盒
本发明的一个实施方案涉及一种制品或药盒，其包含本发明纤维蛋白溶酶原或纤维蛋白溶酶。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维蛋白溶酶原或纤溶酶。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宫颈糜烂。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维蛋白溶酶原组合物以及治疗伴随的疾病的其它药物给药患者。
附图简述
图1显示plg+/+宫颈糜烂模型小鼠给予纤溶酶原或PBS的第5和第9天宫颈HE染色观察结果。
图2显示plg-/-宫颈糜烂模型小鼠给予纤溶酶原或PBS的第5、9和13天宫颈HE染色观察结果。
图3显示plg+/+宫颈糜烂模型小鼠给予纤溶酶原或PBS的第5和第9天宫颈纤维蛋白免疫染色观察结果。
图4显示plg-/-宫颈糜烂模型小鼠给予纤溶酶原或PBS的第5、9和13天宫颈纤维蛋白免疫染色观察结果。
图5显示plg+/+宫颈糜烂模型小鼠给予纤溶酶原或PBS的第5和第9天宫颈F4/80免疫染色观察结果。
图6显示plg-/-宫颈糜烂模型小鼠给予纤溶酶原或PBS的第5、9和13天宫颈F4/80免疫染色观察结果。
实施例
实施例1纤溶酶原对plg+/+宫颈糜烂模型小鼠的保护作用
本实验使用6-7周龄健康的雌性plg+/+小鼠12只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各6只。造模前一天称量小鼠体重并分组，分组完成后建立宫颈糜烂模型，小鼠宫颈口注入苯酚胶浆0.01mL/次/天，连续造模处理4次。酚胶的配制方案：苯酚于60℃熔融，取3mL，加入4g阿拉伯树胶粉及5mL蒸馏水，搅拌混匀得乳白色粘稠状的苯酚胶浆[27]。建立模型后，给纤溶酶原组按1mg/0.1mL/只/天经尾静脉注射给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。造模结束当天为第0天，第1天开始给予纤溶酶原或溶媒PBS，给药期为8天。两组分别于第5天、第9天，随机抓取3只小鼠，摘眼球取血处死小鼠，取宫颈组织在4％多聚甲醛固定液中固定24-48小时。固定后的宫颈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5μm，切片脱蜡复水并用苏木素和伊红染色(HE染色)，1％盐酸酒精分化，氨水返蓝，并酒精梯度脱水封片，切片在显微镜下200倍下观察。
HE染色结果显示，给溶媒PBS对照组小鼠第5天粘膜角质层角化过度，脱落(↓)，鳞状上皮轻度增生(Δ)，第9天时角化的角质层基本已脱落，表面不平整(↓)，未伴随上皮修复，鳞状上皮增生严重(图1A，B)；给纤溶酶原组第5天时部分角质层脱落消失(↓)，损伤的上皮表面被新生的上皮所 覆盖(◆)，而至第9天时新生的上皮进一步修复，发生鳞状上皮化生(↓)，覆盖至损伤的粘膜表面(图1C，D)。可见给溶媒PBS对照组小鼠宫颈损伤愈加严重，而给纤溶酶原组已经发生了修复，而且随着时间的推移，损伤的粘膜表面不断好转，说明纤溶酶原对宫颈糜烂组织有保护作用。
实施例2纤溶酶原促进plg-/-宫颈糜烂模型小鼠宫颈损伤的修复
本实验使用6-7周龄健康的雌性plg-/-小鼠18只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各9只。造模前一天称量小鼠体重并分组，分组完成后建立宫颈糜烂模型，小鼠宫颈口注入苯酚胶浆0.01mL/次/天，连续造模处理4次。酚胶的配制方案：苯酚于60℃熔融，取3mL，加入4g阿拉伯树胶粉及5mL蒸馏水，搅拌混匀得乳白色粘稠状的苯酚胶浆[27]。建立模型后，给纤溶酶原组按1mg/0.1mL/只/天经尾静脉注射给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。造模结束当天为第0天，第1天开始给予纤溶酶原或溶媒PBS，给药期为12天。两组分别于第5天、第9天、第13天，随机抓取3只小鼠，摘眼球取血处死小鼠，取宫颈组织在4％多聚甲醛固定液中固定24-48小时。固定后的肾脏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5μm，切片脱蜡复水并用苏木素和伊红染色(HE染色)，1％盐酸酒精分化，氨水返蓝，并酒精梯度脱水封片，切片在显微镜下200倍下观察。
HE染色结果显示，给溶媒PBS对照组在第5、9、13天鳞状上皮角质层均已脱落，表面形成糜烂，腔内可见有脱落的角质层(↓)，且见大量炎性细胞浸润(◆)，随着时间的推移，炎症不断加重，粘膜表面的溃疡也不断加重(图2A-C)；给纤溶酶原组(图2D-F)第5天虽然有粘膜变性坏死脱落形成(↓)，但表面已经发生了新生的上皮修复，第9天宫腔仅有少量的炎性细胞浸润，新生的上皮进一步增生，且在新生上皮下已经出现了鳞状上皮角质层(↓)，第13天宫腔内无异物，溃疡已经愈合，鳞状上皮表面已经被修复的角质层所覆盖。plg-/-小鼠体内缺乏纤溶酶原，所以给溶媒PBS对照组小鼠体内仍然缺乏纤溶酶原，而给纤溶酶原组小鼠纤溶酶原得到补充。给溶媒PBS对照组损伤严重，并且随着时间的延长损伤未见修复，给纤溶酶原组宫颈损伤较轻，并且随着给药时间的延长，损伤逐渐修复。意味着纤溶酶原能够显著的促进plg-/-宫颈糜烂模型小鼠宫颈损伤的修复。
实施例3纤溶酶原促进plg+/+宫颈糜烂模型小鼠宫颈纤维蛋白的降解
本实验使用6-7周龄健康的雌性plg+/+小鼠12只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各6只。造模前一天称量小鼠体重并分组，分组完成后建立宫颈糜烂模型，小鼠宫颈口注入苯酚胶浆0.01mL/次/天，连续造模处理4次。苯酚胶的配制方案：苯酚于60℃熔融，取3mL，加入4g阿拉伯树胶粉及5mL蒸馏水，搅拌混匀得乳白色粘稠状的苯酚胶浆[27]。建立模型后，给纤溶酶原组按1mg/0.1mL/只/天经尾静脉注射给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。造模结束当天为第0天，第1天开始给予纤溶酶原或溶媒PBS，给药期为8天。两组分别于第5天、第9天，随机抓取3只小鼠，摘眼球取血处死小鼠，取宫颈组织在4％多聚甲醛固定液中固定24-48小时。固定后的宫颈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5μm，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％的正常羊血清(Vector laboratories，Inc.，USA)封闭1小时；时间到后，弃除羊血清液。兔抗小鼠纤维蛋白(原)抗体(Abcam)4℃孵育过夜，TBS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，TBS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，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白[28-30]。因此可将纤维蛋白水平作为损伤程度的一个标志。
结果显示，给溶媒PBS对照组(图3A、B)和给纤溶酶原组(图3C、D)小鼠宫颈纤维蛋白阳性着色均是第9天深于第5天，但是给溶媒PBS对照组的阳性着色均深于给纤溶酶原组，且均有统计学差异(图3E)。说明纤溶酶原能够使沉积的纤维蛋白减少，显示plg+/+宫颈糜烂模型小鼠宫颈的损伤减轻。
实施例4纤溶酶原促进plg-/-宫颈糜烂模型小鼠宫颈纤维蛋白的降解
本实验使用6-7周龄健康的雌性plg-/-小鼠18只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各9只。造模前一天称量小鼠体重并分组，分组完成后建立宫颈糜烂模型，小鼠宫颈口注入苯酚胶浆0.01mL/次/天，连续造模处理4次。苯酚胶的配制方案：苯酚于60℃熔融，取3mL，加入4g阿拉伯树胶粉及5mL蒸馏水，搅拌混匀得乳白色粘稠状的苯酚胶浆[27]。建立模型后，给纤溶酶原组按1mg/0.1mL/只/天经尾静脉注射给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。造模结束当天为第0天，第1天开始给予纤溶酶原或溶媒PBS，给药期为12天。两组分别于第5天、第9天第13天，随机抓取3只小鼠，摘眼球取血处死小鼠，取宫颈组织在4％多聚甲醛固定液中固定24-48小时。固定后的宫颈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5μm，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％的正常羊血清(Vector laboratories，Inc.，USA)封闭1小时；时间到后，弃除羊血清液。兔抗小鼠纤维蛋白抗体(Abcam)4℃孵育过夜，TBS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，TBS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，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白[28-30]。因此可将纤维蛋白水平作为损伤程度的一个标志。
结果显示，给溶媒PBS对照组纤维蛋白阳性着色逐渐加深(图4A-C)。给纤溶酶原组(图4D-F)阳性着色在逐渐变浅，且第13天与第5、9天有显著统计学差异；与给溶媒PBS对照组相比其着色较浅，并且在第13天二者有统计学差异(图4G)。plg-/-小鼠体内缺乏纤溶酶原，所以给溶媒PBS对照组小鼠体内仍然缺乏纤溶酶原，而给纤溶酶原组小鼠纤溶酶原得到补充。说明纤溶酶原显著减少沉积的纤维蛋白，显示纤溶酶原能促进plg-/-宫颈糜烂模型小鼠宫颈损伤的修复。
实施例5纤溶酶原促进plg+/+宫颈糜烂模型小鼠的炎症修复
本实验使用6-7周龄健康的雌性plg+/+小鼠12只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各6只。分组完成后，建立宫颈糜烂模型，小鼠宫颈口注入苯酚胶浆0.01mL/1次/天，连续造模给药4次。苯酚胶的配制方案：苯酚于60℃熔融，取3mL，加入4g阿拉伯树胶粉及5mL蒸馏水，搅拌混匀得乳白色粘稠状的苯酚胶浆[27]。建立模型后，给纤溶酶原组按1mg/0.1mL/只/天经尾静脉注射给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。造模前一天称量小鼠体重并分组，造模结束当天为第0天，第1天开始给予纤溶酶原或溶媒PBS，给药期为8天。两组分别于第5天、第9天，随机抓取3只小鼠，摘眼球取血处死小鼠，取宫颈组织在4％多聚甲醛固定液中固定24-48小时。固定后的宫颈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5μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟，水洗2次，每次5分钟。10％正常羊血清(Vector laboratories，Inc.，USA)封闭1小时，时间到后甩去血清，用PAP笔圈出组织。针对F4/80的兔多克隆抗体(Abcam)4℃孵育过夜，TBS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，TBS洗2次。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下400倍下观察。
F4/80巨噬细胞标志物，可以表示炎症反应的程度和阶段。结果显示，给溶媒PBS对照组(图5A、B)和给纤溶酶原组(图5C、D)宫颈F4/80阳性表达量均是第9天高于第5天，但是给纤溶酶原组要明显的少于给溶媒PBS对照组。说明纤溶酶原能够减轻损伤组织的炎症，显示纤溶酶原能促进plg+/+宫颈糜烂模型小鼠损伤宫颈炎症的修复。
实施例6纤溶酶原促进plg-/-宫颈糜烂模型小鼠的炎症修复
本实验使用6-7周龄健康的雌性plg-/-小鼠18只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各9只。分组完成后，建立宫颈糜烂模型，小鼠宫颈口注入苯酚胶浆0.01ml，1次/天，连续造模给药4次。苯酚胶的配制方案：苯酚于60℃熔融，取3mL，加入4g阿拉伯树胶粉及5mL蒸馏水，搅拌混匀得乳白色粘稠状的苯酚胶浆[27]。建立模型后，给纤溶酶原组按1mg/0.1mL/只/天经尾静脉注射给予纤溶酶原，给溶媒PBS对照 组给予相同体积的PBS。造模前一天称量小鼠体重并分组，造模结束当天为第0天，第1天开始给予纤溶酶原或溶媒PBS，给药期为12天。两组分别于第5天、第9天、第13天，随机抓取3只小鼠，摘眼球取血处死小鼠，取宫颈组织在4％多聚甲醛固定液中固定24-48小时。固定后的宫颈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5μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟，水洗2次，每次5分钟。10％正常羊血清(Vector laboratories，Inc.，USA)封闭1小时，时间到后甩去血清，用PAP笔圈出组织。针对F4/80的兔多克隆抗体(Abcam)4℃孵育过夜，TBS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，TBS洗2次。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下400倍下观察。
F4/80巨噬细胞标志物，可以表示炎症反应的程度和阶段。结果显示，给溶媒PBS对照组(图6A-C)和给纤溶酶原组(图6D-F)，在第5、9、13天F4/80阳性表达量均无没有明显的改变，但是给纤溶酶原组阳性表达量低于给溶媒PBS对照组。plg-/-小、鼠体内缺乏纤溶酶原，所以给溶媒PBS对照组小鼠体内仍然缺乏纤溶酶原，而给纤溶酶原组小鼠纤溶酶原得到补充。结果表明，纤溶酶原能够减轻损伤组织炎症水平，显示纤溶酶原能促进plg-/-宫颈糜烂模型小鼠损伤宫颈的炎症修复。
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Claims (14)

  1. 一种预防和/或治疗受试者宫颈糜烂及其相关病症的方法，包括给药受试者有效量的纤溶酶原。
  2. 根据权利要求1的方法，其中所述宫颈糜烂包括真性糜烂和假性糜烂。
  3. 根据权利要求1或2的方法，其中所述纤溶酶原与序列2、6、8、10或12具有至少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。
  4. 根据权利要求1-3任一项的方法，其中所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。
  5. 根据权利要求1-4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、δ-纤溶酶原或其任意组合。
  6. 根据权利要求1-5任一项的方法，其中所述在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、δ(delta)-纤溶酶原或微纤溶酶原。
  7. 根据权利要求1-6任一项的方法，其中所述纤溶酶原为人天然纤溶酶原，例如序列2所示的纤溶酶原的直向同系物。
  8. 权利要求1-7任一项的方法，其中所述纤溶酶原或纤维蛋白溶酶全身或局部施用，例如，通过静脉内、肌内、皮下、局部注射、直肠施用、阴道施用。
  9. 根据权利要求1-8任一项的方法，其中所述纤溶酶原或纤维蛋白溶酶可与其它药物或疗法组合施用。
  10. 根据权利要求9的方法，其中所述其它药物或疗法包括抗细菌感染药物、抗病毒感染药物、抗真菌药物、抗滴虫药物、抗血栓药物、抗糖尿病药物、物理疗法、激光疗法、局部手术疗法。
  11. 一种用于预防和/或治疗受试者宫颈糜烂及其相关病症的制品，其包含含有有效剂量的纤溶酶原的容器，和指导施用所述制品预防和/或治疗受试者宫颈糜烂及其相关病症的说明书。
  12. 权利要求11的制品，进一步包含含有一种或多种其它药物的容器。
  13. 权利要求12的制品，其中所述其它药物包括抗细菌感染药物、抗病毒感染药物、抗真菌药物、抗滴虫药物、抗血栓药物、抗糖尿病药物。
  14. 权利要求12或13的制品，其中所述说明书进一步说明所述纤溶酶原可以在所述其它药物施用之前，同时，和/或之后施用。
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LU XING-YAN; ZHANG TIAN-YUAN; LUO JIN-XIAN : "Antitumor Activity of Recombinant KI-3 Domain of Human Plasmihogen", AMINO ACIDS AND BIOTIC RESOURCES., vol. 27, no. 2, 31 December 2005 (2005-12-31), pages 55 - 57, XP009510893, ISSN: 1006-8376 *
See also references of EP3395355A4 *
XIE, XING ET AL.: "Obstetrics and Gynecology, edition 8", PEOPLE'S MEDICAL PUBLISHING HOUSE, 31 March 2013 (2013-03-31), pages 256, XP009511668, ISBN: 7117171804 *

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* Cited by examiner, † Cited by third party
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US10874721B2 (en) 2015-12-18 2020-12-29 Talengen International Limited Method for preventing and treating cervical erosion
US11007253B2 (en) 2015-12-18 2021-05-18 Talengen International Limited Method for preventing or treating radiation and chemical damage

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CA3008495A1 (en) 2017-06-22
US10874721B2 (en) 2020-12-29
US20190151422A1 (en) 2019-05-23
JP6876066B2 (ja) 2021-05-26
EP3395355A4 (en) 2019-06-12
JP2019500427A (ja) 2019-01-10
TWI623321B (zh) 2018-05-11
TW201722469A (zh) 2017-07-01
JP2021075573A (ja) 2021-05-20
CN108463235A (zh) 2018-08-28

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