WO2021227417A1 - 一种治疗脊髓性肌萎缩症的方法和药物 - Google Patents

一种治疗脊髓性肌萎缩症的方法和药物 Download PDF

Info

Publication number
WO2021227417A1
WO2021227417A1 PCT/CN2020/129461 CN2020129461W WO2021227417A1 WO 2021227417 A1 WO2021227417 A1 WO 2021227417A1 CN 2020129461 W CN2020129461 W CN 2020129461W WO 2021227417 A1 WO2021227417 A1 WO 2021227417A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasminogen
sma
mice
plasmin
pathway activator
Prior art date
Application number
PCT/CN2020/129461
Other languages
English (en)
French (fr)
Inventor
李季男
Original Assignee
泰伦基国际有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 泰伦基国际有限公司 filed Critical 泰伦基国际有限公司
Priority to CA3182911A priority Critical patent/CA3182911A1/en
Priority to KR1020227041422A priority patent/KR20230005298A/ko
Priority to CN202080099318.3A priority patent/CN115697385A/zh
Priority to US17/924,617 priority patent/US20230181699A1/en
Priority to JP2022567202A priority patent/JP2023525257A/ja
Priority to EP20935975.1A priority patent/EP4140498A4/en
Publication of WO2021227417A1 publication Critical patent/WO2021227417A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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 a method for treating spinal muscular atrophy (SMA) and related disorders, comprising administering an effective amount of plasminogen to a subject suffering from spinal muscular atrophy (SMA) and related disorders Components of the activation pathway or related compounds, such as plasminogen, to repair damaged nerves and improve clinical symptoms and signs.
  • SMA spinal muscular atrophy
  • Components of the activation pathway or related compounds, such as plasminogen to repair damaged nerves and improve clinical symptoms and signs.
  • SMA Spinal Muscular Atrophy
  • SMA spinal muscular atrophy
  • spinal muscular atrophy also known as spinal muscular atrophy or spinal muscular atrophy. It is a type of disease in which muscle weakness and muscle atrophy are caused by the degeneration of the motor neurons in the anterior horn of the spinal cord. It is an autosomal recessive genetic disease.
  • SMA survival motor neuron
  • Symptoms include muscle weakness, hypotonia, weakness in crying, limp or falling tendency, difficulty sucking or swallowing, accumulation of secretions from the lungs or throat, difficulty eating, and susceptibility to respiratory infections.
  • the legs are often weaker than the arms and fail to reach developmental signs, such as raising the head or sitting up. Generally, the earlier the symptoms appear, the shorter the life span.
  • SMA Stret al. The process of SMA is directly related to the deterioration speed of motor neuron cells and the degree of weakness caused by it. Babies with severe forms of SMA often die from respiratory diseases due to weakness of the muscles that support breathing. Children with milder forms of SMA survive longer, but they may require extensive medical support.
  • SMA is an autosomal recessive genetic disease. About 95% of SMA is caused by the mutation of SMN1 (Survival Motor Neuron 1) gene on chromosome 5, so it is also called 5q type SMA.
  • Type 5q SMA is divided into 5 subtypes according to the patient's age of onset and the severity of the disease: Type 0 patients: generally more common in fetuses or newborns, the onset of fetal period is manifested as decreased fetal movement, and neonatal manifestations are Loss of muscle reflexes, facial paralysis, atrial septal defect and joint contractures. The most serious manifestations are respiratory failure. The life expectancy of sick children is greatly shortened, and most of them survive within 6 months.
  • Type I patients infantile, also known as Werdnig-Hoffman disease accounts for 50% of SMA patients.
  • the patient developed hypotonia, poor head control, and weakened or disappeared tendon reflexes within 6 months after birth.
  • Severe hypotonia manifested as "frog leg" posture when lying down, lack of head control, unable to sit upright, weak intercostal muscles, relatively small diaphragm, patients often suffer from weakened swallowing function, respiratory muscle weakness and respiratory failure .
  • 92% of children with type I SMA usually die of respiratory failure 20 months ago; type II patients: intermediate type, accounting for about 20% of SMA patients, usually 6-18 after birth
  • the patient can sit alone at a certain stage in the development process, but cannot walk independently.
  • Type III patients Juvenile type, also known as Kugelberg-Welander disease, accounts for about 30% of SMA patients. Patients usually develop onset within 18 months to 5 years after birth. It can walk with the help of material support. Unlike type II SMA, most of these people do not have complications such as scoliosis and respiratory muscle weakness. The cognition and life expectancy of this group are generally not affected by the disease; type IV patients: onset after adolescence, exercise ability gradually Decrease, accounting for about 5% of the total number of SMA patients.
  • SMAs are not caused by mutations in the SMN1 gene. They are called non-5q SMAs, which means that their pathogenic genes are not located in the SMN region of chromosome 5. Similar to 5q type SMA, children with non-5q type SMA will also have symptoms of muscle weakness very early, but there will be some differences, including the distal muscle weakness rather than the proximal muscle weakness, and the earlier occurrence of distant muscle weakness.
  • SMA is caused by inactivating mutations or deletions of telomere copies of genes (SMN1) on two chromosomes, resulting in loss of function of the SMN1 gene.
  • SMN1 protein functions as a cofactor in RNA maturation and is required for the viability of all eukaryotic cells (Talbot and Tizzano (2017) Gene Ther 24(9):529-533).
  • the SMN2 protein is almost the same as SMN1 except for a single mutation that plays a role in the splicing of RNA messages.
  • SMA is usually diagnosed by a test that combines clinical symptoms with at least one copy of SMN1 gene.
  • other tests such as electromyography (EMG) or muscle biopsy can also assist in the diagnosis.
  • EMG electromyography
  • the treatment of SMA is limited to supportive therapy, including breathing, nutrition and rehabilitation treatment and care. There is no medicine that can effectively treat this disease.
  • plasminogen pathway activators such as plasminogen can significantly improve the symptoms of nerve injury in SMA subjects, improve lung function, prolong survival, promote the transcription and expression of SMN genes, and increase brain and muscle tissue.
  • the level of SMN protein can promote the expression of NF- ⁇ B protein in brain tissue and muscle tissue, promote the formation of mature NGF in brain tissue, and improve lung tissue damage, thereby effectively preventing and treating SMA.
  • the present invention relates to a method for treating spinal muscular atrophy (SMA) (including type 0, type I, type II, type III, type IV, and non-5q SMA), comprising administering a motor neuron disease
  • spinal muscular atrophy SMA
  • subjects with spinal muscular atrophy (SMA) have a therapeutically effective amount of one or more plasminogen pathway activators selected from the following: components of the plasminogen activation pathway, capable of directly activating fibrin Lysinogen or compounds that indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, and can up-regulate plasminogen Or plasminogen activator-expressed compounds, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.
  • plasminogen pathway activators selected from the following: components of the plasminogen activation pathway, capable of directly activating fibrin Lysinogen or compounds that
  • the plasminogen pathway activator is effective against spinal muscular atrophy (SMA) (including type 0, type I, type II, type III, type IV, and non-5q SMA).
  • SMA spinal muscular atrophy
  • the subject has one or more activities selected from: 1. Reduce or improve the severity of SMA; 2. Delay the onset of SMA; 3. Inhibit the progression of SMA; 4. Prolong the survival time of the subject; 5. Improve the quality of life of the subject and/or improve the mental state of the subject; 6. Reduce the number of SMA-related symptoms; 7. Reduce or improve the severity of one or more symptoms related to SMA; 8. Shorten the duration of SMA-related symptoms; 9. Prevent the recurrence of SMA-related symptoms; 10. Inhibit the development or onset of SMA symptoms; 11.
  • the plasminogen pathway activator improves muscle atrophy, increases muscle strength, and/or improves muscle tone in the subject. In some specific embodiments, the plasminogen pathway activator prolongs the survival of the subject.
  • the plasminogen pathway activator promotes the transcription and/or expression of the SMN gene. In some specific embodiments, the plasminogen pathway activator promotes the recovery of muscle function in the subject. In some specific embodiments, the plasminogen pathway activator promotes the repair of spinal cord anterior horn neurons in the subject. In some specific embodiments, the plasminogen pathway activator promotes the expression of NF- ⁇ B protein in the subject. In some specific embodiments, the plasminogen pathway activator promotes the formation of mature NGF in the subject. The plasminogen pathway activator promotes the formation of mature NGF in the subject.
  • the plasminogen pathway activator is administered in combination with one or more other drugs and/or treatment methods.
  • the treatment methods include cell therapy (eg stem cell therapy) and gene therapy , Such as antisense RNA, small molecule splicing modifiers.
  • the plasminogen pathway activator is a component of the plasminogen activation pathway.
  • the components of the plasminogen activation pathway are selected from plasminogen (abbreviation: plasminogen), recombinant human plasmin, Lys-plasminogen, Glu -Plasminogen, plasmin, plasminogen and plasmin variants and the like containing one or more kringle domains and protease domains of plasminogen and plasmin Substances, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasmin Enzyme, delta-plasmin, plasminogen activator, tPA and uPA.
  • plasminogen abbreviation: plasminogen
  • the antagonist of the fibrinolysis inhibitor is PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin antagonist, such as PAI-1, complement C1 inhibitor, ⁇ 2 anti-plasmin or ⁇ 2 macroglobulin antibody.
  • the component of the plasminogen activation pathway is plasminogen.
  • the plasminogen comprises or has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96% identical to the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12. , 97%, 98% or 99% sequence identity of the amino acid sequence, and has plasminogen activity.
  • the plasminogen activity is the proteolytic activity of plasminogen.
  • the plasminogen activity is the lysine binding activity of plasminogen to the substrate molecule.
  • the plasminogen activity is the proteolytic activity of plasminogen and the lysine binding activity of plasminogen to the substrate molecule.
  • the plasminogen is added, deleted and/or substituted 1-100, 1-90, 1-80, 1-70 on the basis of sequence 2, 6, 8, 10 or 12. , 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1 -3, 1-2, 1 amino acid, and has plasminogen proteolytic activity and/or lysine binding activity protein.
  • the plasminogen activity is the proteolytic activity of plasminogen.
  • the plasminogen is a protein containing plasminogen active fragments and having the proteolytic activity and/or lysine binding activity of plasminogen.
  • the active fragment of plasminogen comprises or has a serine protease domain of plasminogen or is called a plasminogen protease domain.
  • the amino acid sequence of the active fragment of plasminogen is shown in SEQ ID NO: 14.
  • the plasminogen is selected from Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (after cleavage between amino acids 76-77) Human full-length plasminogen), microplasminogen (including Kringle 5 (K5) and serine protease domain), microplasminogen (including serine protease domain), delta-plasminogen (including Kringle 1 and serine protease domain) or their variants that retain plasminogen activity.
  • the plasminogen is human full-length plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • the plasminogen activity is the proteolytic activity of plasminogen.
  • the plasminogen activity is the lysine binding activity of plasminogen to the substrate molecule. In some embodiments, the plasminogen activity is the proteolytic activity of plasminogen and the lysine binding activity of plasminogen to the substrate molecule. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or rodent or it still retains the proteolytic activity of plasminogen and/or lysine Variants or fragments of binding activity. In some embodiments, the plasminogen comprises an amino acid sequence as shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is natural human plasminogen.
  • the plasminogen pathway activator is administered systemically or locally, for example in the form of intravenous, intramuscular, intrathecal, nasal inhalation, nebulization, nasal drops, or eye drops Administration.
  • the subject is a human.
  • the subject lacks or lacks plasminogen.
  • the deficiency or deletion is congenital, secondary, and/or local.
  • the plasminogen is 0.0001-2000mg/kg, 0.001-800mg/kg, 0.01-600mg/kg, 0.1-400mg/kg, 1-200mg/kg, 1-100mg/kg, daily 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 per day, administration every two days or every three days continuously.
  • the aforementioned SMA is type 0, type I, type II, type III, type IV, or non-5q type SMA.
  • this application also relates to pharmaceutical compositions, drugs, preparations, kits, and products for the treatment of spinal muscular atrophy (SMA), including the above-mentioned plasminogen pathway activator, such as the above-mentioned Components of the plasminogen activation pathway, such as the plasminogen described above.
  • SMA spinal muscular atrophy
  • the pharmaceutical composition, medicament, or formulation comprises a pharmaceutically acceptable carrier and a plasminogen pathway activator, such as the components of the plasminogen activation pathway described above, such as those described above Plasminogen.
  • the kits and articles of manufacture include one or more containers that contain the pharmaceutical composition, drug, or formulation.
  • the kit or product further comprises a label or instructions for use, the label or instructions for use indicate the use of a plasminogen pathway activator, such as the components of the plasminogen activation pathway described above, for example The above-mentioned method for treating spinal muscular atrophy with plasminogen.
  • the kit or article of manufacture further comprises one or more additional containers containing one or more other drugs.
  • the aforementioned SMA is type 0, type I, type II, type III, type IV or non-5q type SMA.
  • the present application also relates to the above-mentioned plasminogen pathway activator for the treatment of spinal muscular atrophy (SMA), such as the above-mentioned plasminogen.
  • SMA spinal muscular atrophy
  • the aforementioned SMA is type 0, type I, type II, type III, type IV, or non-5q type SMA.
  • the present application also relates to the above-mentioned plasminogen pathway activator, such as the use of the above-mentioned plasminogen for the treatment of spinal muscular atrophy (SMA).
  • SMA spinal muscular atrophy
  • the aforementioned SMA is type 0, type I, type II, type III, type IV, or non-5q type SMA.
  • the present application also relates to a therapeutically effective amount of the above-mentioned plasminogen pathway activator (for example, the components of the above-mentioned plasminogen activation pathway, such as the above-mentioned plasminogen) for preparing the treatment of spinal cord.
  • plasminogen pathway activator for example, the components of the above-mentioned plasminogen activation pathway, such as the above-mentioned plasminogen
  • SMA muscular dystrophy
  • the plasminogen pathway activator is selected from one or more of the following plasminogen pathway activators: components of the plasminogen activation pathway, capable of directly activating plasmin Pro or compounds that indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, and can up-regulate plasminogen or fiber Compounds expressed by plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.
  • the components of the plasminogen activation pathway are selected from plasminogen, recombinant human plasmin, Lys-plasminogen, Glu-plasminogen, Plasmin, plasminogen and plasmin variants and analogs, microplasmin containing one or more kringle domains and protease domains of plasminogen and plasmin Mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin Enzyme (delta-plasmin), plasminogen activator, tPA and uPA.
  • the antagonist of the fibrinolysis inhibitor is PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin antagonist, such as PAI-1, complement C1 inhibitor, ⁇ 2 anti-plasmin or ⁇ 2 macroglobulin antibody.
  • the plasminogen pathway activator is a component of the plasminogen activation pathway.
  • the components of the plasminogen activation pathway are selected from plasminogen (abbreviation: plasminogen), recombinant human plasmin, Lys-plasminogen, Glu -Plasminogen, plasmin, plasminogen and plasmin variants and the like containing one or more kringle domains and protease domains of plasminogen and plasmin Substances, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasmin Enzyme, delta-plasmin, plasminogen activator, tPA and uPA.
  • plasminogen abbreviation: plasminogen
  • the antagonist of the fibrinolysis inhibitor is PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin antagonist, such as PAI-1, complement C1 inhibitor, ⁇ 2 anti-plasmin or ⁇ 2 macroglobulin antibody.
  • the component of the plasminogen activation pathway is plasminogen.
  • the plasminogen comprises or has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96% identical to the amino acid sequence shown in SEQ ID NO: 2, 6, 8, 10 or 12. , 97%, 98% or 99% sequence identity of the amino acid sequence, and has plasminogen activity.
  • the plasminogen activity is the proteolytic activity of plasminogen.
  • the plasminogen activity is the lysine binding activity of plasminogen to the substrate molecule.
  • the plasminogen activity is the proteolytic activity of plasminogen and the lysine binding activity of plasminogen to the substrate molecule.
  • the plasminogen is added, deleted and/or substituted 1-100, 1-90, 1-80, 1-70 on the basis of sequence 2, 6, 8, 10 or 12. , 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1 -3, 1-2, 1 amino acid, and has plasminogen proteolytic activity and/or lysine binding activity protein.
  • the plasminogen activity is the proteolytic activity of plasminogen.
  • the plasminogen is a protein containing plasminogen active fragments and having the proteolytic activity and/or lysine binding activity of plasminogen.
  • the active fragment of plasminogen comprises or has a serine protease domain of plasminogen or is called a plasminogen protease domain.
  • the amino acid sequence of the active fragment of plasminogen is shown in SEQ ID NO: 14.
  • the plasminogen is selected from Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (after cleavage between amino acids 76-77) Human full-length plasminogen), microplasminogen (including Kringle 5 (K5) and serine protease domain), microplasminogen (including serine protease domain), delta-plasminogen (including Kringle 1 and serine protease domain) or their variants that retain plasminogen activity.
  • the plasminogen is human full-length plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • the plasminogen activity is the proteolytic activity of plasminogen.
  • the plasminogen activity is the lysine binding activity of plasminogen to the substrate molecule. In some embodiments, the plasminogen activity is the proteolytic activity of plasminogen and the lysine binding activity of plasminogen to the substrate molecule. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or rodent or it still retains the proteolytic activity of plasminogen and/or lysine Variants or fragments of binding activity. In some embodiments, the plasminogen comprises an amino acid sequence as shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is natural human plasminogen.
  • the plasminogen pathway activator such as a component of the above-mentioned plasminogen activation pathway, for example, the above-mentioned plasminogen is administered in combination with one or more other drugs and/or treatment methods.
  • the plasminogen pathway activator for example, a component of the plasminogen activation pathway, such as plasminogen through intravenous, intramuscular, intrathecal, nasal inhalation, aerosol inhalation, drip It is administered in the form of nasal fluid or eye drops.
  • the pharmaceutical composition, medicament, or formulation includes a pharmaceutically acceptable carrier and a plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as plasminogen.
  • the kits and articles of manufacture include one or more containers that contain the pharmaceutical composition, drug, or formulation.
  • the kit or product further comprises a label or instructions for use, the label or instructions for use indicate the use of a plasminogen pathway activator, such as a component of the plasminogen activation pathway, such as plasminogen Methods of treating spinal muscular atrophy.
  • the kit or article of manufacture further comprises one or more additional containers containing one or more other drugs.
  • the aforementioned SMA is type 0, type I, type II, type III, type IV, or non-5q type SMA.
  • the present invention clearly covers all combinations of technical features belonging to the embodiments of the present invention, and the technical solutions after these combinations have been clearly disclosed in this application, just as the above-mentioned technical solutions have been separately and clearly disclosed.
  • the present invention also clearly covers the combinations between the various embodiments and their elements, and the technical solutions after the combination are clearly disclosed herein.
  • Figure 1 shows the amplitude results of the motor EMG before and after treatment in the type II SMA patient described in Example 1.
  • the results showed that the action potential amplitudes of the tibial nerve and common peroneal nerve of the patients were improved to varying degrees compared with before the medication.
  • the results show that plasminogen can improve the conduction function of peripheral neurons in patients with type II SMA and improve neuromuscular damage.
  • Figure 2 shows the results of the electromyogram amplitudes of the upper and lower limbs of the patient described in Example 2 before and after treatment. Compared with before medication, the action potential amplitudes of the patients' left femoral nerve, right ulnar nerve, bilateral common peroneal nerve and tibial nerve all improved to varying degrees. The results show that plasminogen can improve the conduction function of peripheral neurons in patients with type II SMA and improve neuromuscular damage.
  • Figure 3 shows the results of the electromyogram amplitudes of the upper and lower limbs of the patient described in Example 3 before and after treatment. Compared with before medication, the patient's bilateral median nerve, tibial nerve, common peroneal nerve, and ulnar nerve action potential increased to varying degrees. The results show that plasminogen can improve the conduction function of peripheral neurons in patients with type II SMA and improve neuromuscular damage.
  • FIG. 4A-4B Survival curve and survival time statistics of SMN ⁇ 7 SMA mice after plasminogen administration.
  • A is the statistical result of survival curve
  • B is the statistical result of survival time.
  • FIG. 5 qPCR results of SMN gene in the spinal cord of SMN ⁇ 7 SMA mice after administration of plasminogen.
  • the results showed that the spinal cord of the blank control group mice had a certain level of SMN gene transcription, the SMN gene transcription level of the mice in the solvent group was lower than that of the blank control mice, and the SMN gene transcription level of the mice in the administration group was significantly higher than that of the mice in the vehicle group. Blank control mice. This result suggests that plasminogen can promote SMN gene transcription.
  • FIG. 6 Western blot detection results and optical density quantitative analysis results of SMN ⁇ 7 SMA mice brain NF- ⁇ B protein after plasminogen administration.
  • FIG. 7 Western blot detection results and optical density quantitative analysis results of representative hindlimb muscle NF- ⁇ B protein of SMN ⁇ 7 SMA mice after administration of plasminogen.
  • the results showed that the muscles of the blank control group mice had a certain amount of NF- ⁇ B protein, the muscle NF- ⁇ B protein levels of the mice in the vehicle group were lower than those of the blank control group, and the muscle NF- ⁇ B protein levels of the mice in the administration group were significantly higher than those of the control group. Mice in the vehicle group, and statistically significant differences (* means P ⁇ 0.05). This result suggests that plasminogen can promote the increase of muscle NF- ⁇ B protein level in SMN ⁇ 7 SMA mice.
  • Figure 8 Western blot detection results and optical density (OD) value quantitative analysis results of representative brain SMN proteins in SMN ⁇ 7 SMA mice after plasminogen administration.
  • the results showed that the brain of the blank control group mice expressed a certain amount of SMN protein, the SMN protein expression level of the mice in the vehicle group was lower than that of the blank control mice, and the SMN protein expression level of the mice in the administration group was significantly higher than that of the mice in the vehicle group. This result suggests that plasminogen can promote SMN protein expression in the brain of SMN ⁇ 7 SMA mice.
  • FIG. 9 Western blot detection results and optical density (OD) quantitative analysis results of representative hindlimb muscle SMN protein of SMN ⁇ 7 SMA mice after plasminogen administration.
  • the results showed that the muscles of the blank control group mice expressed a certain amount of SMN protein, the muscle SMN protein expression level of mice in the vehicle group was lower than that of the blank control group mice, and the muscle SMN protein expression level of the mice in the administration group was significantly higher than that of the vehicle group mice .
  • This result suggests that plasminogen can promote the expression of SMN protein in the muscle of SMN ⁇ 7 SMA mice.
  • FIG 10 Western blot detection results and the quantitative analysis results of the NGF/Pro-NGF optical density (OD) ratio of the brain tissue of SMA mice after administration of plasminogen.
  • the results showed that the brain tissue of the blank control group had a certain ratio of NGF/ProNGF, the ratio of NGF/ProNGF in the brain tissue of the mice in the administration group was significantly higher than that of the mice in the vehicle group, and the statistical difference was extremely significant (*** means P ⁇ 0.001) . It is suggested that plasminogen can promote the transformation of ProNGF into NGF in the brain tissue of SMA model mice, and promote the formation of mature NGF.
  • FIG. 11 H&E staining of representative lung tissue of SMA mice after administration of plasminogen.
  • the results show that the terminal bronchiolar epithelial cells of the lung tissue of the blank control group are arranged neatly and clearly; the alveolar cavity is uniform in size and the alveolar space is not There is thickening, no inflammatory cell infiltration around the blood vessels; respiratory bronchiole epithelium in lung tissue of mice in the vehicle group falls off, alveolar ducts and alveolar sacs are enlarged, alveolar septums are widened, alveoli collapse to structural disorders, and eosinophils around the pulmonary vessels Cells, foam cells, lymphocytes; respiratory bronchiole epithelium in the lung tissue of mice in the administration group is arranged in an orderly manner, alveolar ducts and alveolar sacs are enlarged, and alveolar cavity is evenly enlarged, but alveolar walls composed of a single layer of alveolar epithelium can be seen. It is suggested
  • spinal muscular atrophy refers to a disease caused by inactivating mutations or deletions of the SMN1 gene on two chromosomes, resulting in the loss of SMN1 gene function.
  • Symptoms of SMA include muscle weakness, hypotonia, weak crying, weak coughing, limp or falling tendency, difficulty sucking or swallowing, difficulty breathing, accumulation of secretions in the lungs or throat, clenched fists and sweaty hands, and tongue shaking/ Vibration, head that tends to one side (even when lying down), legs that tend to be weaker than arms, legs that are often "frog legs", difficulty eating, increased sensitivity to respiratory infections, bowel/ Bladder weakness, lower than normal weight, inability to sit without support, inability to walk, inability to crawl, and hypotonia, loss of reflexes, and multiple congenital contractures (joint contractures) associated with loss of pre-hom cells.
  • treatment of spinal muscular atrophy (SMA) or “treatment of spinal muscular atrophy (SMA)” in this application includes obtaining one or more of the following effects: 1. Reduce or improve the severity of SMA; 2. Delay The onset of SMA; 3. Inhibit the progression of SMA; 4. Prolong the survival time of the subject; 5. Improve the quality of life of the subject and/or improve the mental state of the subject; 6. Reduce the number of SMA-related symptoms; 7 Reduce or improve the severity of one or more symptoms associated with SMA; 8. Shorten the duration of symptoms associated with SMA; 9. Prevent the recurrence of symptoms associated with SMA; 10. Inhibit the development of SMA symptoms or Onset; 11. Inhibit the progression of symptoms related to SMA; 12. Improve lung function; 13.
  • SMN gene Increase blood oxygen saturation; 14. Promote the transcription and expression of SMN gene; 15. Increase the level of SMN protein in brain tissue and muscle tissue; 16. Promote the expression of NF- ⁇ B protein in brain tissue and muscle tissue; 17. Promote the formation of mature NGF in brain tissue; 18. Reduce lung tissue damage; 19. Increase muscle strength; 20. Reduce muscle atrophy; 21. Reduce motor nerves Meta loss; 22. Promote growth and development; and/or 23. Improve motor function.
  • components of the plasminogen activation pathway of the present application or related compounds thereof, such as the plasminogen described above, enhance the transcription and/or expression of SMN genes.
  • the components of the plasminogen activation pathway of the present application or related compounds, such as the plasminogen described above, increase the expression of SMN protein in human subjects in need thereof.
  • the components of the plasminogen activation pathway of the present application or related compounds, such as plasminogen can be used alone or in combination with other drugs to treat or prevent inactivating mutations in the SMN gene Or diseases caused by deletion and/or related to loss or defect of SMN gene function. These diseases include but are not limited to spinal muscular atrophy (SMA).
  • SMA spinal muscular atrophy
  • the application relates to a method for treating diseases caused by inactivating mutations or deletions of SMN gene and/or related to the loss or defect of SMN gene function, such as SMA, comprising administering to a subject a therapeutically effective amount A component of the plasminogen activation pathway or its related compounds, such as plasminogen.
  • the application relates to a method of treating SMA, comprising administering to a subject a therapeutically effective amount of plasminogen.
  • the present application relates to a method of treating SMA, comprising administering to a subject a therapeutically effective amount of plasminogen, the plasminogen having one or more activities selected from the following: 1. Less Reduce or improve the severity of SMA; 2. Delay the onset of SMA; 3. Inhibit the progression of SMA; 4. Prolong the survival time of the subject; 5. Improve the quality of life of the subject and/or improve the mental state of the subject 6. Reduce the number of SMA-related symptoms; 7. Reduce or improve the severity of one or more symptoms related to SMA; 8. Shorten the duration of symptoms related to SMA; 9. Prevent symptoms related to SMA Recurrence; 10. Inhibit the development or onset of SMA symptoms; 11.
  • Fibrinolytic system also known as fibrinolytic system, is a system composed of a series of chemical substances involved in the process of fibrinolysis (fibrinolysis), mainly including fibrinolytic enzyme (plasminogen) and plasmin , Plasminogen activator, fibrinolysis inhibitor.
  • Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA).
  • t-PA tissue-type plasminogen activator
  • u-PA urokinase-type plasminogen activator
  • t-PA activates plasminogen
  • fibrin urokinase-type plasminogen activator
  • u-PA urokinase-type plasminogen activator
  • PLG Plasminogen
  • Plasminase is a serine protease, which has the following functions: degrades fibrin and fibrinogen; hydrolyzes a variety of coagulation factors V, VIII, X, VII, XI, II, etc.; turns plasminogen into fibrinolysis Enzymes; hydrolysis of complement, etc.
  • Fibrinolytic inhibitors including plasminogen activator inhibitor (PAI) and ⁇ 2 antiplasmin ( ⁇ 2-AP).
  • PAI mainly has two forms, PAI-1 and PAI-2, which can specifically bind to t-PA in a ratio of 1:1 to inactivate it and activate PLG at the same time.
  • ⁇ 2-AP is synthesized by the liver and combined with PL in a ratio of 1:1 to form a complex, inhibiting PL activity; FXIII makes ⁇ 2-AP covalently bond with fibrin, reducing the sensitivity of fibrin to PL.
  • Substances that inhibit the activity of the fibrinolytic system in the body PAI-1, complement C1 inhibitor; ⁇ 2 anti-plasmin; ⁇ 2 macroglobulin.
  • plasminogen pathway activator or "plasminogen pathway activator” of the present invention encompasses components of the plasminogen activation pathway, capable of directly activating plasminogen or by activating plasmin Compounds that indirectly activate plasminogen by activating upstream components of the pathway, compounds that mimic the activity of plasminogen or plasmin, and those that can up-regulate the expression of plasminogen or plasminogen activator Compounds, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolytic inhibitors.
  • component of the plasminogen activation pathway or “component of the plasminogen activation pathway” of the present invention encompasses:
  • Plasminogen activators such as tPA and uPA, and tPA or uPA variants and analogs containing one or more domains of tPA or uPA (such as one or more kringle domains and proteolytic domains) .
  • antagonist of fibrinolysis inhibitor covers PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin antagonist, such as PAI-1, complement C1 inhibitor, ⁇ 2 antifibrosis Antibodies to lysozyme or ⁇ 2 macroglobulin.
  • variants of plasminogen, plasmin, tPA and uPA include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as by addition, deletion and/or substitution such as 1- 100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, Proteins of 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid that still have plasminogen, plasmin, tPA or uPA activity.
  • variants of plasminogen, plasmin, tPA, and uPA include those by, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1- 45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative Mutant variants of these proteins obtained by amino acid substitutions.
  • the "plasminogen variants" of the present invention encompasses those containing or having at least 75%, 80%, 85%, 90%, 95%, 96%, and the amino acid sequence shown in sequence 2, 6, 8, 10 or 12. A protein with 97%, 98%, or 99% sequence identity, and has plasminogen proteolytic activity and/or lysine binding activity.
  • the "plasminogen variant” of the present invention can be added, deleted and/or substituted 1-100, 1-90, 1-80, 1- on the basis of sequence 2, 6, 8, 10 or 12.
  • the plasminogen variants of the present invention include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as through conservative amino acid substitutions such as 1-100, 1-90, 1-80, 1- 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, Mutant variants of these proteins obtained from 1-3, 1-2, 1 amino acids.
  • the plasminogen of the present invention may be a human plasminogen ortholog from a primate or rodent or a variant that still retains the proteolytic activity and/or lysine binding activity of plasminogen.
  • plasminogen, plasmin, tPA, and uPA include compounds that provide substantially similar effects to plasminogen, plasmin, tPA, or uPA, respectively.
  • variants and analogs of plasminogen, plasmin, tPA and uPA encompass fibers comprising one or more domains (for example, one or more kringle domains and proteolytic domains) "Variants” and “analogs" of plasminogen, plasmin, tPA and uPA.
  • the "variants” and “analogs” of plasminogen encompass the inclusion of one or more plasminogen domains (e.g., one or more kringle (k) domains and proteolytic domains (or serine)).
  • Protease domain, or plasminogen protease domain plasminogen variants and analogs, such as mini-plasminogen.
  • plasminogens encompasses "variants” and “analogs” of plasmin comprising one or more plasmin domains (for example, one or more kringle domains and proteolytic domains), such as fibrinolytic enzymes. Enzyme (mini-plasmin) and delta-plasmin (delta-plasmin).
  • plasminogen, plasmin, tPA or uPA have the activity of plasminogen, plasmin, tPA or uPA respectively, or whether they provide the same
  • the substantially similar effects of plasminogen, plasmin, tPA or uPA can be detected by methods known in the art, for example, by methods based on enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS ( Fluorescence-activated cell sorting method) is measured by the level of activated plasmin activity, for example, it can be measured with reference to a method selected from the following documents: Ny, A., Leonardsson, G., Hagglund, AC, Hagglof, P.
  • the "component of plasminogen activation pathway" of the present invention is plasminogen, selected from Glu-plasminogen, Lys-plasminogen, and microplasmin Pro, microplasminogen, delta-plasminogen or their variants that retain plasminogen activity.
  • the plasminogen is natural or synthetic human plasminogen, or a conservative mutant variant or fragment thereof that still retains plasminogen activity and/or lysine binding activity.
  • the plasminogen is a human plasminogen ortholog from a primate or rodent or one that still retains plasminogen activity and/or lysine binding activity Conservative mutant variants or fragments thereof.
  • the amino acid sequence of the plasminogen comprises or has an amino acid sequence as shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is human full-length plasminogen.
  • the plasminogen is human full-length plasminogen as shown in sequence 2.
  • a compound capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway refers to a compound capable of directly activating plasminogen or by activating plasminogen Any compound that activates upstream components of the pathway and indirectly activates plasminogen, such as tPA, uPA, streptokinase, saruplase,reteplase, reteplase, tenecteplase, aniplase, Monteplase, Lanoteplase, Pamideplase, Staphylokinase.
  • the "antagonist of the fibrinolysis inhibitor" of the present invention is a compound that antagonizes, weakens, blocks, and prevents the action of the fibrinolysis inhibitor.
  • the fibrinolysis inhibitors are, for example, PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, and ⁇ 2 macroglobulin.
  • the antagonist such as PAI-1, complement C1 inhibitor, ⁇ 2 anti-plasmin or ⁇ 2 macroglobulin antibody, or blocking or down-regulating such as PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin Antisense RNA or small RNA expressed by globulin, or occupy the binding site of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin but without PAI-1, complement C1 inhibitor, ⁇ 2 anti-fibrosis Compounds that function as lysozyme or ⁇ 2 macroglobulin", or compounds that block the binding domain and/or active domain of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin.
  • Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease that can hydrolyze several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycan. In addition, plasmin can activate some metalloprotease precursors (pro-MMPs) to form active metalloproteases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis. Plasmin is formed by proteolysis of plasminogen through two physiological PAs: tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA).
  • tPA tissue-type plasminogen activator
  • uPA urokinase-type plasminogen activator
  • PAs Due to the relatively high levels of plasminogen in plasma and other body fluids, it is traditionally believed that the regulation of the PA system is mainly achieved through the synthesis and activity levels of PAs.
  • the synthesis of PA system components is strictly regulated by different factors, such as hormones, growth factors and cytokines.
  • the main inhibitor of plasmin is ⁇ 2-antiplasmin ( ⁇ 2-antiplasmin).
  • the activity of PAs was inhibited by both uPA and tPA plasminogen activator inhibitor-1 (PAI-1) and mainly inhibited uPA lysinogen activator inhibitor-2 (PAI-2).
  • PAI-1 uPA and tPA plasminogen activator inhibitor-1
  • PAI-2 mainly inhibited uPA lysinogen activator inhibitor-2
  • Certain cell surfaces have uPA-specific cell surface receptors (uPAR) with direct hydrolytic activity.
  • Plasminogen is a single-chain glycoprotein consisting of 791 amino acids and a molecular weight of approximately 92kDa. Plasminogen is mainly synthesized in the liver and exists in large amounts in the extracellular fluid. Plasminogen content in plasma is about 2 ⁇ M. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids. Plasminogen exists in two molecular forms: Glu-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved form of plasminogen has an amino terminal (N-terminal) glutamate and is therefore called glutamate-plasminogen.
  • glutamate-plasminogen is hydrolyzed at Lys76-Lys77 to lysine-plasminogen.
  • lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate.
  • the Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide bond-linked double-chain protease plasmin.
  • the amino terminal part of plasminogen contains five homologous tricyclic rings, so-called kringles, and the carboxy terminal part contains the protease domain.
  • Some kringles contain lysine binding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor ⁇ 2-AP.
  • Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycan and gelatin, indicating that plasmin also plays an important role in ECM reconstruction.
  • plasmin can also degrade other components of ECM by converting certain protease precursors into active proteases, including MMP-1, MMP-2, MMP-3 and MMP-9. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis.
  • plasmin has the ability to activate certain latent forms of growth factors. In vitro, plasmin can also hydrolyze components of the complement system and release chemotactic complement fragments.
  • Pulminin is a very important enzyme present in the blood, which can hydrolyze fibrin clots into fibrin degradation products and D-dimers.
  • “Plasminogen” is the zymogen form of plasmin. According to the sequence in swiss prot, it is composed of 810 amino acids and the molecular weight is about 90kD, a glycoprotein mainly synthesized in the liver and able to circulate in the blood. The cDNA sequence encoding this amino acid sequence is shown in sequence 3. The full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle1-5).
  • PAp Pan Apple
  • Kringle1 includes residues Cys103-Cys181
  • Kringle2 includes residues Glu184-Cys262
  • Kringle3 includes residues Cys275-Cys352
  • Kringle4 Including residues Cys377-Cys454
  • Kringle5 includes residues Cys481-Cys560.
  • the serine protease domain includes residues Val581-Arg804.
  • Glu-plasminogen is a natural full-length plasminogen consisting of 791 amino acids (without the signal peptide of 19 amino acids).
  • the cDNA sequence encoding this sequence is shown in sequence 1, and its amino acid sequence is shown in sequence 2. Shown. In the body, there is also a Lys-plasminogen formed by hydrolysis from amino acids 76-77 of Glu-plasminogen. As shown in sequence 6, the cDNA sequence encoding this amino acid sequence is as shown in sequence 5. Shown.
  • Delta-plasminogen is a fragment of the full-length plasminogen without the Kringle2-Kringle5 structure, and only contains Kringle1 and serine protease (structure) domain (also called proteolytic domain, or fiber The lysinogen protease domain), the amino acid sequence of delta-plasminogen (sequence 8) has been reported in the literature, and the cDNA sequence encoding this amino acid sequence is as sequence 7.
  • Mini-plasminogen Mini-plasminogen is composed of Kringle5 and serine protease domain.
  • Micro-plasminogen (Micro-plasminogen) only contains the serine protease domain, and it has been reported in the literature that its amino acid sequence includes residues Ala543-Asn791 (starting with the Glu residue of the Glu-plasminogen sequence without the signal peptide).
  • CN102154253A also reported that its sequence includes residues Lys531-Asn791 (the Glu residue of the Glu-plasminogen sequence without the signal peptide is used as the starting amino acid).
  • residues Lys531-Asn791 the Glu residue of the Glu-plasminogen sequence without the signal peptide is used as the starting amino acid.
  • the amino acid sequence is shown in Sequence 12
  • the cDNA sequence encoding the amino acid sequence is shown in Sequence 11.
  • the meaning or activity of the "deficiency" of plasminogen means that the content of plasminogen in the subject is lower than that of a normal person, and is low enough to affect the normal physiological function of the subject;
  • the meaning or activity of "deletion" of plasminogen is that the content of plasminogen in the subject is significantly lower than that of normal people, even the activity or expression is minimal, and normal physiological functions can only be maintained through external sources.
  • plasminogen of the present invention covers both plasminogen and plasmin.
  • plasminogen activator PA
  • PA plasminogen activator
  • the active plasmin can further hydrolyze the fibrin clot into fibrin degradation products and D-dimers, and then dissolve the thrombus.
  • the PAp domain of plasminogen contains important determinants that maintain plasminogen in an inactive closed conformation, while the KR domain can bind to lysine residues present on the receptor and substrate.
  • a variety of enzymes that can act as plasminogen activators are known, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hager Man factor) and so on.
  • Plasminogen active fragments are included in this application: 1) In the plasminogen protein, active fragments capable of binding to the target sequence in the substrate, also known as lysine-binding fragments, such as Kringle 1, Fragments of Kringle 2, Kringle 3, Kringle 4, and/or Kringle 5 (for the plasminogen structure, see Aisina R B, Mukhametova L I. Structure and function of plasminogen/plasmin system [J].
  • the plasminogen is a protein comprising the active fragment of plasminogen shown in SEQ ID NO: 14.
  • the plasminogen is a protein comprising lysine binding fragments of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and/or Kringle 5.
  • the plasminogen active fragment of the present application comprises sequence 14, an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% homology with sequence 14 Protein. Therefore, the plasminogen of the present invention includes a protein that contains the active fragment of the plasminogen and still maintains the activity of the plasminogen.
  • the plasminogen of the present application includes Kringle 1, Kringle 2, Kringle 3, Kringle 4, and/or Kringle 5, or has at least 80 percent with Kringle 1, Kringle 2, Kringle 3, Kringle 4, or Kringle 5. Proteins with %, 90%, 95%, 96%, 97%, 98%, 99% homology and still have lysine binding activity.
  • the methods for measuring plasminogen and its activity in blood include: the detection of tissue plasminogen activator activity (t-PAA), the detection of plasma tissue plasminogen activator antigen (t-PAAg), Detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator inhibition Detection of substance antigens, plasma plasmin-antiplasmin complex detection (PAP).
  • t-PAA tissue plasminogen activator activity
  • t-PAAg the detection of plasma tissue plasminogen activator antigen
  • plgA Detection of plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma tissue plasminogen activator inhibition
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the tested plasma, the PLG in the tested plasma is transformed into PLM under the action of SK, and the latter acts on The chromogenic substrate is subsequently measured with a spectrophotometer, and the increase in absorbance is proportional to the activity of plasminogen.
  • SK streptokinase
  • immunochemical methods, gel electrophoresis, immunoturbidimetry, radioimmuno-diffusion methods, etc. can also be used to determine the plasminogen activity in the blood.
  • orthologs or orthologs refer to homologs between different species, including both protein homologs and DNA homologs, and are also called orthologs and vertical homologs. It specifically refers to proteins or genes in different species that evolved from the same ancestor gene.
  • the plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs derived from different species that have plasminogen activity.
  • Constant substitution variant refers to a given amino acid residue that changes but does not change the overall conformation and function of the protein or enzyme. This includes, but is not limited to, those with similar characteristics (such as acidic, basic, hydrophobic, etc.) Amino acids replace amino acids in the amino acid sequence of the parent protein. Amino acids with similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and can be interchanged. Similarly, isoleucine is a hydrophobic amino acid and can be replaced by leucine, methionine or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different.
  • Constant substitution variants also include polypeptides or enzymes that are determined by BLAST or FASTA algorithms to have more than 60% amino acid identity. If it can reach more than 75%, it is better, preferably more than 85%, or even more than 90%. It is the best, and has the same or substantially similar properties or functions compared with the natural or parent protein or enzyme.
  • isolated plasminogen refers to plasminogen protein separated and/or recovered from its natural environment.
  • the plasminogen will be purified (1) to a purity (by weight) greater than 90%, greater than 95%, or greater than 98%, as determined by the Lowry method, for example, greater than 99% (By weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, which is achieved by using Coomassie blue or silver staining is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions.
  • the isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering technology and separated by at least one purification step.
  • polypeptide refers to polymerized forms of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived Modified amino acids, and polypeptides with modified peptide backbones.
  • the term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without an N-terminal methionine residue); and so on.
  • the “percent amino acid sequence identity (%)" with respect to the reference polypeptide sequence is defined as when gaps are introduced when necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity, the candidate sequence is Refers to the percentage of amino acid residues that are identical to amino acid residues in the polypeptide sequence.
  • the comparison for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment of the sequences, including any algorithm that achieves the maximum alignment requirements over the entire length of the sequence being compared. However, for the purposes of the present invention, the percent amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2.
  • the% amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or can be expressed as having or containing relative to, with, or against a given amino acid sequence)
  • a given amino acid sequence A) of a certain% amino acid sequence identity of B is calculated as follows:
  • X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's A and B alignment
  • Y is the total number of amino acid residues in B. It should be appreciated that in the case where the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% amino acid sequence identity of A relative to B may not be equal to the% amino acid sequence identity of B relative to A. Unless expressly stated otherwise, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.
  • mice rats, mice
  • non-human primates humans
  • dogs and cats
  • Hoofed animals such as horses, cows, sheep, pigs, goats
  • a “therapeutically effective amount” or “effective amount” refers to an amount of plasminogen sufficient to achieve the prevention and/or treatment of the 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 symptoms of the subject to be treated, age, weight, and the like.
  • treatment of a disease state includes inhibiting or preventing the development of the disease state or its clinical symptoms, or alleviating the disease state or symptoms, so that the disease state or its clinical symptoms are temporarily or permanently regressed.
  • muscle strength refers to the force of muscle contraction during voluntary movement of the limbs or the force of active muscle movement. According to the situation of muscle strength, muscle strength is usually divided into the following 0-5 grades: grade 0, completely paralyzed, muscle contraction cannot be measured; grade 1, only muscle contraction is measured, but no movement can be produced; grade 2, limb physical ability Move in parallel on the bed, but cannot resist its own gravity, that is, cannot lift off the bed; level 3, the limb can overcome gravity, can lift off the bed, but cannot resist resistance; level 4, the limb can do exercises against external resistance , But not complete; grade 5, normal muscle strength.
  • muscle tone refers to the degree of tension of a muscle in a resting and relaxed state. Muscle tone is the basis for maintaining various postures and normal movements of the body. Muscle tension manifests in many forms. For example, when a person is lying and resting, the tension of various muscles of the body is called resting muscle tension. When the body is standing, although no significant muscle contraction is seen, the muscles in the front and back of the body also maintain a certain tension to maintain the standing posture and body stability, which is called postural muscle tension. The tension of muscles during exercise, called motor muscle tension, is an important factor to ensure continuous and smooth muscle movement (without tremor, twitching, or spasm). Under pathological conditions, muscle tension increases or decreases, which affects the normal posture or movement of the human body.
  • Plasminogen can be isolated from nature and purified for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When a polypeptide is synthesized chemically, it can be synthesized via a liquid phase or a solid phase.
  • Solid phase peptide synthesis (SPPS) (where the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence) is a suitable method for the chemical synthesis of plasminogen.
  • SPPS Solid phase peptide synthesis
  • Various forms of SPPS, such as Fmoc and Boc can be used to synthesize plasminogen.
  • the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. Then, the unit is deprotected, exposing a new N-terminal amine that can be attached to other amino acids.
  • the peptide remains immobilized on the solid phase, after which it is cut off.
  • Standard recombinant methods can be used to produce the plasminogen of the present invention.
  • a nucleic acid encoding plasminogen is inserted into an expression vector so that it is operably linked to the regulatory sequence in the expression vector.
  • Expression control sequences include, but are not limited to, promoters (such as naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression control can be a eukaryotic promoter system in a vector that can transform or transfect eukaryotic host cells (such as COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of plasminogen.
  • Suitable expression vectors are usually replicated in the host organism as an episome or as an integrated part of the host chromosomal DNA.
  • the expression vector contains a selection marker (for example, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate the transformation of the desired DNA sequence for exogenous use Those cells are tested.
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone plasminogen-encoding polynucleotides.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various pseudomonas. Genus (Pseudomonas) species.
  • expression vectors can also be produced, which usually contain expression control sequences compatible with the host cell (for example, an origin of replication).
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from bacteriophage lambda. Promoters usually control expression, optionally in the case of manipulating gene sequences, and have ribosome binding site sequences, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast such as S. cerevisiae
  • Pichia Pichia
  • suitable yeast host cells in which suitable vectors have expression control sequences (such as promoters), origins of replication, termination sequences, etc., as required.
  • suitable promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeasts are initiated by specifically including promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose.
  • mammalian cells e.g., mammalian cells cultured in an in vitro cell culture
  • the plasminogen of the present invention e.g., a polynucleotide encoding plasminogen.
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas.
  • Expression vectors used in these cells may contain expression control sequences such as an origin of replication, promoters and enhancers (Queen et al., Immunol. Rev.
  • ribosome binding Site RNA splice site
  • polyadenylation site RNA splice site
  • transcription terminator sequence RNA splice site
  • suitable expression control sequences are promoters derived from white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149 (1992).
  • Plasminogen is substantially pure, for example at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure Or purer, for example free of contaminants such as cell debris, macromolecules other than plasminogen, etc.
  • a freeze-dried formulation can be formed by mixing plasminogen with the required purity with optional pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.ed. (1980)) Or aqueous solutions to prepare therapeutic formulations.
  • Acceptable carriers, excipients, and stabilizers are non-toxic to recipients at the dose and concentration used, and include buffers such as phosphate, citrate and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (such as Octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoic acid Esters such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues) ; Proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • the formulations of the present invention may also contain more than one active compound required for the specific condition to be treated, preferably those with complementary activities and no side effects between each other.
  • active compound required for the specific condition to be treated, preferably those with complementary activities and no side effects between each other.
  • the plasminogen of the present invention can be encapsulated in microcapsules prepared by techniques such as coacervation or interfacial polymerization, for example, can be placed in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules) or placed in hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a coarse emulsion.
  • colloidal drug delivery system e.g., liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules
  • hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a coarse emulsion.
  • the plasminogen of the present invention for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after freeze-drying and reformulation.
  • the plasminogen of the present invention can be used to prepare sustained-release preparations.
  • sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers having a certain shape and containing glycoproteins, such as films or microcapsules.
  • sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.
  • Polymers such as ethylene- Vinyl acetate and lactic-glycolic acid can continue to release molecules for more than 100 days, but some hydrogels release proteins for a short time.
  • a reasonable strategy for stabilizing the protein can be designed according to the relevant mechanism. For example, if the mechanism of aggregation is found to be The formation of intermolecular SS bonds through the exchange of thiodisulfide bonds can be stabilized by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions .
  • the administration of the pharmaceutical composition of the present invention can be achieved in different ways, such as intravenously, intraperitoneally, subcutaneously, intracranially, intrathecal, intraarterial (e.g. via carotid artery), intramuscular.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, and so on. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, among others.
  • the dosage range of the pharmaceutical composition containing plasminogen of the present invention can be about 0.0001 to 2000 mg/kg, or about 0.001 to 500 mg/kg (for example, 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg per day). /kg, 10mg/kg, 50mg/kg, etc.) the weight of the subject.
  • the dosage may be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Doses above or below this exemplified range are also covered, especially taking into account the factors mentioned above.
  • the intermediate dose in the above range is also included in the scope of the present invention.
  • the subject can administer such doses every day, every other day, every week, or according to any other schedule determined through empirical analysis.
  • An exemplary dosage schedule includes 1-10 mg/kg for consecutive days. During the administration of the drug of the present invention, it is necessary to evaluate the therapeutic effect and safety in real time.
  • One embodiment of the present invention relates to a product or a kit containing the plasminogen or plasmin of the present invention that can be used to treat cardiovascular diseases and related disorders caused by diabetes.
  • the article preferably includes a container, label or package insert. Suitable containers are bottles, vials, syringes, etc.
  • the container can be made of various materials such as glass or plastic.
  • the container contains a composition that can effectively treat the disease or condition of the present invention and has a sterile access (for example, the container may be an intravenous solution pack or a vial, which contains a stopper that can be penetrated by a hypodermic injection needle of). At least one active agent in the composition is plasminogen/plasmin.
  • the label on or attached to the container indicates that the composition is used for the treatment of cardiovascular disease and related disorders caused by diabetes according to the present invention.
  • the preparation may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further contain other substances required from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.
  • the article contains a package insert with instructions for use, including, for example, instructing the user of the composition to administer the plasminogen composition and other medications for the treatment of concomitant diseases to the patient.
  • the human plasminogen used in all the following examples is derived from donor plasma, based on the method described in the literature [1-3] and optimized by the process, purified from human donor plasma, of which human Lys-plasminogen (Lys-plasminogen) and Glu-plasminogen (Glu-plasminogen)>98%.
  • nebulized inhalation or intravenous injection All the patients in the following Examples 1-7 signed informed consent, voluntarily accepted the treatment of plasminogen purified from human plasma, and were approved by the hospital ethics committee. According to the severity and course of the disease, adjust the usage and dosage.
  • the mode of administration is nebulized inhalation or intravenous injection.
  • the drug concentration of nebulized inhalation and intravenous injection are both 5mg/ml, and physiological saline is used as the solvent.
  • Intravenous injection 100-200 mg/time; frequency: once every 1 day, every 2 days or every 3 days; 2 weeks is a course of treatment; each course is 2-3 weeks apart. A total of 6 courses of treatment.
  • the Hammersmith Functional Motor Scale (Expanded Hammersmith Functional Motor Scale, HFMSE) is specifically used to assess the motor function of patients with type II and type III SMA, reflecting the severity of the disease. It is defined compared to the change in the baseline, assessing the changes in the children's motor function, the higher the score, the better the motor function [4-6].
  • Neuro-EMG examination is the main diagnosis and identification method of motor neuron disease.
  • the amplitude of compound muscle action potential reflects neuronal axon damage.
  • SMA is a motor neuron degenerative disease, a large number of motor neurons die, muscle weakness and compound muscle action potential amplitude is reduced or even undetectable [7].
  • HFMSE score 20 points before medication, 21 points after the first course of treatment. For the second course of treatment, the score was 23 points before treatment and 24 points after treatment. After the 6th course of treatment, the score was 25 points.
  • the patient Before treatment, the patient cannot stand without assistance. After 2 courses of treatment, the patient can assist standing. After 3 courses of treatment, the patient achieves assisted walking, and the patient's head control ability is significantly improved. As the treatment progresses, the patient's motor function is further improved, including the extension of the auxiliary standing time and the increase of the auxiliary walking distance.
  • Electromyography The amplitude of the action potentials of the bilateral tibial nerve, common peroneal nerve and femoral nerve increased significantly after treatment compared with that before treatment ( Figure 1).
  • plasminogen can improve the HFMSE score of patients with type II SMA, improve the patient's motor function, and improve the patient's neuromuscular function and mental state.
  • the first time intravenous injection, 50mg
  • the second time intravenous injection, 50mg
  • the third and fourth time intravenous injection, 100mg.
  • the frequency of medication is once every 2 days or once every 3 days, sharing the medicine for 2 weeks.
  • the HFMSE score sheet before the medication is 2 points, and the HFMSE score is 8 points after the fourth medication.
  • the patient can sit alone and raise his hands.
  • the EMG results showed that the action potential amplitude of the left femoral nerve, right ulnar nerve, common peroneal nerve, and tibial nerve increased after treatment compared with before treatment ( Figure 2).
  • plasminogen can improve the HFMSE score of patients with type II SMA, improve the patient's motor function, and improve the neuromuscular function, and there are no drug-related side effects during the treatment.
  • Intravenous injection dose: 50mg-100mg each time; frequency: once every 1 day or once every 3 days; 2 weeks is a course of treatment, each course is 3-4 weeks apart, a total of 8 courses of treatment.
  • HFMSE score 23 points before treatment, 24 points after the first course of treatment. There was an interval of approximately two months between the patient's first course of treatment and the second course of treatment. The second course of treatment is 23 minutes before treatment and 24 minutes after treatment. After the 8th course of treatment, the score was 28 points.
  • Electromyography After treatment, the amplitude of the action potentials of the median nerve, tibial nerve, common peroneal nerve, and ulnar nerve increased to varying degrees (Figure 3).
  • plasminogen can increase the HFMSE score of patients with type II SMA, improve the patient's motor function, and improve neuromuscular function.
  • the first time intravenous injection, 50mg; the second time: intravenous injection, 50mg; the third time: intravenous injection, 100mg; the fourth time: intravenous injection, 150mg, each time interval of 3 days, medication for 2 weeks.
  • the leg strength increased significantly, the breathing improved, the voice became louder, the pectus excavatum improved significantly, the signs of rib valgus improved, the strength of the arms and hands increased, but he could not grasp the adult's hand and was pulled up.
  • the arm elevation has been degraded. Previously, the arm could be raised to the top of the head by itself, but it could only reach the face in the following treatments.
  • the patient male, 11 months old, was diagnosed as type I SMA by genetic testing at 6 months; when the doctor was diagnosed, he informed that the average life cycle of patients with this type of disease was 2 years old, and the family did not take any treatment measures.
  • Symptoms Weak head support; Weak upper limbs and arms, unable to lift, less swinging hands, weak grasping, weak middle finger; Weak lower limbs, less swinging, movable toes; unable to sit alone, involuntary turning over, low sucking power, difficulty swallowing , Perform blood oxygen monitoring 24 hours a day, the blood oxygen saturation is 92-97%, and the chest fluctuates weakly when breathing.
  • the dose of nebulized inhalation is 5-10mg, and the dose of intravenous injection is 50-200mg.
  • the CHOP INTEND scale (Children's Hospital of Philadelphia Infant Neuromuscular Disease Test Scale) was used to evaluate the improvement of motor function in patients with type I SMA. A higher score indicates better motor function [8].
  • CHOP INTEND score Before treatment, the CHOP INTEND score was 30 points, and the score increased to 50 points after 5 courses of treatment. For some reasons, the interval between the 5th and 6th courses of treatment was about 2 months, and the score dropped to 36 points. After the 6th course of treatment, the score was 44 points. The interval between the 6th course of treatment and the 7th course of treatment is approximately 2 months. The 7th course of treatment is 44 minutes before medication and 45 minutes after medication. After the 10th course of treatment, the score was 46 points.
  • Swallowing function After the medication, the patient's coughing frequency is reduced when eating and drinking, and the speech function is good.
  • plasminogen can improve the CHOP INTEND score of patients with type I SMA, improve the patient’s motor function, and achieve a milestone improvement of 30 seconds of sitting without assisted support and 30 seconds of head support; it can also improve the patient’s swallowing function and Speaking ability; improve the patient's chest collapse signs, improve lung function, increase blood oxygen saturation; and improve the patient's mental state and sleep.
  • the second course of treatment will be carried out 2 months after the end of the first course of treatment.
  • the first course of treatment the time of hanging and shaking both hands is increased, the amplitude is increased, and the strength is increased.
  • the left upper arm can move inward autonomously under the condition of supporting it.
  • the lower limbs assisted with bending the knees and standing up for 30 minutes, the facial expressions increase, the eyes can be blinked, and the mouth can twitch spontaneously.
  • Second course of treatment occasionally swallowing soup, sleep improvement
  • the third course of treatment normal bowel movements, the head can be swayed left and right, with auxiliary support, the head can be raised for a few seconds.
  • the wrist is slightly stronger, the fingertips can be rotated cyclically, and the left arm can be swayed autonomously for a greater range.
  • the blood oxygen was maintained at 97%, and no oxygen was given.
  • the fourth course of treatment the left arm has better coordination, the right arm has a small movement range, but the swing frequency is fast.
  • the muscles of the lower limbs are soft and not stiff, the facial expressions increase, and they can defecate spontaneously.
  • plasminogen can improve the motor function of patients with non-5q SMA, including increasing the strength, amplitude and range of limb movement, enriching the patient’s facial expression; improving the patient’s lung function and respiratory function, reducing oxygen transfusion, and increasing blood oxygen saturation To improve the patient’s swallowing function; to improve the patient’s sleep quality.
  • Examples 8-15 are drug studies on animal models, and the plasminogen is still the above-mentioned plasminogen protein purified from human donor plasma.
  • FVB.Cg-Grm7Tg(SMN2)89AhmbSmn1tm1MsdTg(SMN2*delta7)4299Ahmb/J gene mutant mice (hereinafter referred to as SMN ⁇ 7SMA mice) have homozygous mutations in the SMN1 gene and express the human SMN2 gene. The clinical and pathological manifestations of this mouse Similar to human SMA.
  • the breeding rat was purchased from the Jackson Laboratory in the United States (pedigree number: 005025).
  • mice SMN ⁇ 7 SMA mice were weighed at birth and randomly divided into vehicle group (6 mice) and drug group (5 mice) according to their body weight. The mice were administered 3 days after birth. The mice in the vehicle group were injected intraperitoneally with 6ml/kg of vehicle per day, and the mice in the administration group were injected with 60mg/kg intraperitoneally per day of plasminogen. Record the survival of the mice.
  • mice Take out 7 SMN ⁇ 7 SMA mice 3 days old, 4 mice in the vehicle group, and give 6 ⁇ l bovine serum albumin solution (5mg/ml) by intraperitoneal injection every morning and afternoon every day for the first 9 days, starting from day 10, every day 6 ⁇ l bovine serum albumin solution (10mg/ml) was given by intraperitoneal injection once; 3 mice in the administration group were given plasminogen by 30 ⁇ g/6 ⁇ l intraperitoneal injection once a day in the morning and afternoon for the first 9 days, starting on the 10th day , Plasminogen was injected intraperitoneally at 60 ⁇ g/6 ⁇ l once a day; 4 wild-type mice were taken as blank control group, and 6 ⁇ l bovine serum albumin solution (5mg/ml ), starting from day 10, 6 ⁇ l bovine serum albumin solution (10mg/ml) was given by intraperitoneal injection once a day.
  • 6 ⁇ l bovine serum albumin solution 5mg/ml
  • mice On the 12th day, the mice were sacrificed and brain tissues were collected, brain tissue homogenates were prepared, and Western blot detection of NF- ⁇ B protein was performed.
  • a 10% gel was prepared according to the gel preparation instructions of the SDS-PAGE gel preparation kit (Solarbio, P1320).
  • the samples of each group were mixed with 4 ⁇ loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 minutes, cooled and centrifuged for 2 minutes, and then 20 ⁇ L was loaded.
  • the electrophoresis conditions are 30V running gel for 30min, and then 100V electrophoresis to the bottom of the gel.
  • the gel was stripped and transferred to an activated PVDF membrane (GE, A29433753).
  • the electroporation conditions were 15V, 2.5h.
  • the transferred PVDF membrane was immersed in a blocking solution (5% degreasing emulsion) and sealed overnight in a refrigerator at 4°C.
  • TBST 0.01M Tris-NaCl, pH7.6 buffer
  • rabbit anti-mouse NF- ⁇ B antibody (Cell Signaling Technology, 8242) was incubated at room temperature for 3 hours, TBST was washed 4 times, and goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) was added to incubate at room temperature for 1 hour.
  • Nuclear factor kappa-B is a key nuclear transcription factor.
  • the members of the NF- ⁇ B family mainly include RelA (p65), c-Rel, RelB, NF- ⁇ B1 (p50 protein and its precursor p105) and NF- ⁇ B2 (p52 protein and its precursor p100), each member can form homology Or a heterodimer to perform its function.
  • the most common in mammalian cells is the combination of p65 and p50 to form a p65/p50 dimer.
  • the NF- ⁇ B transcription factor binds to the inhibitor of kappa B (Inhibitor of kappa B) protein and is thus trapped in the cytoplasm.
  • I ⁇ B protein The stimulation of the upstream signal causes I ⁇ B protein to be phosphorylated and modified under the action of IKK (I ⁇ B kinase), and then recognized by the ubiquitin ligase complex, which promotes the degradation of I ⁇ B protein in a proteasome-dependent manner, and NF- ⁇ B is released thereby , Enter the nucleus and initiate the expression of target genes [11].
  • IKK I ⁇ B kinase
  • NF- ⁇ B ubiquitin ligase complex
  • NF- ⁇ B can be found in almost all animal cells. They participate in the cell's response to external stimuli, and play a key role in the cell's inflammatory response, immune response and other processes. NF- ⁇ B is also related to synaptic plasticity and memory [12].
  • mice in the blank control group had a certain amount of NF- ⁇ B protein
  • the levels of NF- ⁇ B protein in the brains of mice in the vehicle group were lower than those in the control group
  • the levels of NF- ⁇ B protein in the brains of mice in the administration group were significantly higher than those in the control group.
  • the muscles were obtained from the sacrificed mice in the foregoing Example 10, and the Western blot detection of NF- ⁇ B protein was performed according to the method described in the foregoing Example 10.
  • Example 12 Plasminogen promotes the increase of SMN protein level in brain tissue of SMA model mice
  • mice Two SMN ⁇ 7 SMA mice aged 3 days were taken out, one was in the vehicle group, and 6 ⁇ l bovine serum albumin solution (5mg/ml) was given by intraperitoneal injection every morning and afternoon; one was in the administration group. According to 30 ⁇ g/6 ⁇ l intraperitoneal injection once a day in the morning and afternoon to give plasminogen; take 2 wild-type (FVB) mice as a blank control group, intraperitoneal injection once a day in the morning and afternoon to give 6 ⁇ l bovine serum albumin solution ( 5mg/ml). After 9 days of treatment, the mice were sacrificed and brain tissues were collected, and brain tissue homogenates were prepared for Western blot detection of SMN protein.
  • 6 ⁇ l bovine serum albumin solution 5mg/ml
  • mice The hind limb muscle tissues of the sacrificed mice were obtained as described in Example 12, and a tissue homogenate was prepared for Western blot detection of SMN protein. The detection method was as described in Example 12.
  • Brain tissues were taken from the sacrificed mice in Example 12, tissue homogenates were prepared, and Western blot detection of NGF protein was performed.
  • the samples of each group were mixed with 4 ⁇ loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 minutes, cooled and centrifuged for 2 minutes, and then 20 ⁇ L was loaded.
  • the electrophoresis conditions were 30V running for 30min, and then 100V electrophoresis to the bottom of the gel. After the electrophoresis, the gel was stripped and transferred to an activated PVDF membrane (GE, A29433753).
  • the electroporation conditions were 15V, 2.5h.
  • the transferred PVDF membrane is soaked in a blocking solution (5% degreasing emulsion) and sealed overnight in a refrigerator at 4°C. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), add rabbit anti-mouse NGF antibody Incubate at room temperature for 3 hours. After washing 4 times with TBST, add goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) and incubate at room temperature for 1 hour.
  • TBST 0.01M Tris-NaCl, pH7.6 buffer
  • Immobilon Western HRP Substrate develops color, takes pictures under a biomolecular imager and uses Image J software to obtain the optical density value of each band for quantitative analysis.
  • Lung tissues were taken from the sacrificed mice in Example 12 and fixed in 10% neutral formalin fixative for 24 hours.
  • the fixed lung tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the thickness of the tissue section was 5 ⁇ m.
  • the section was deparaffinized and rehydrated and stained with hematoxylin and eosin (H&E staining), differentiated with 1% hydrochloric acid and alcohol, turned blue with ammonia, and mounted with alcohol gradient dehydration. The section was observed under a microscope at 200 times.
  • the terminal bronchiole epithelial cells in the lung tissue of the blank control group were neatly arranged and clearly distinguishable; the alveolar cavity was uniform in size, the alveolar compartment was not thickened, and there was no inflammatory cell infiltration around the blood vessels; the lung tissue of the vehicle group was breathing Bronchiolar epithelium is shed, alveolar ducts and alveolar sacs are enlarged, alveolar septums are widened, alveoli collapse to structural disorders, and pulmonary blood vessels are surrounded by eosinophils, foam cells, and lymphocytes; respiratory bronchioles in lung tissue of mice in the administration group
  • the epithelium is arranged in an orderly manner, the alveolar ducts and alveolar sacs are enlarged, and the alveolar cavity is evenly enlarged, but the alveolar wall composed of a single layer of alveolar epithelium can be seen (Figure 11). It is suggested that plasminogen can improve lung tissue damage in SMA model mice.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Neurology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Neurosurgery (AREA)
  • Pulmonology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Dermatology (AREA)
  • Otolaryngology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Steroid Compounds (AREA)

Abstract

一种治疗脊髓性肌萎缩症(SMA)的方法，包括给药受试者治疗有效量的纤溶酶原途径激活剂；以及用于治疗脊髓性肌萎缩症的包含纤溶酶原途径激活剂的药物组合物、制品和试剂盒。

Description

一种治疗脊髓性肌萎缩症的方法和药物 技术领域
本发明涉及一种治疗脊髓性肌萎缩症(SMA)及其相关病症的方法，包括给药患有脊髓性肌萎缩症(SMA)及其相关病症的受试者有效量的纤维蛋白溶酶原激活途径的组分或其相关化合物，例如纤溶酶原，以修复损伤神经，改善临床症状和体征。
背景技术
脊髓性肌萎缩症(Spinal Muscular Atrophy)简称SMA，又称脊髓性肌萎缩或脊肌萎缩症，是一类由脊髓前角运动神经元变性导致肌无力、肌萎缩的疾病。属常染色体隐性遗传病。SMA最常见的形式是由运动神经元生存(SMN)基因中的突变所引起的，婴儿SMA是这种神经退行性病症的最严重形式。症状包括肌无力、肌张力低下、哭泣无力、跛行或摔倒倾向、吮吸或吞咽困难、肺或咽喉分泌物积聚、摄食困难和易患呼吸道感染。腿部往往比手臂更无力，并且不能达到发育标志，如抬头或坐起。一般地，症状出现得越早，寿命就越短。
SMA的进程与运动神经元细胞恶化速度以及所造成的无力程度直接相关。患有严重形式的SMA的婴儿由于支持呼吸的肌肉无力而经常死于呼吸道疾病。患有较轻形式的SMA的儿童存活时间较长，但是他们可能需要广泛的医疗支撑。
SMA是一种常染色体隐性遗传疾病。约95％的SMA是由5号染色体上的SMN1(Survival Motor Neuron 1)基因突变所引起，因此也被称作5q型SMA。5q型SMA根据患者的发病年龄和疾病的严重程度，脊肌萎缩症分为5种亚型：0型患者：一般多见于胎儿或新生儿，胎儿期即发作表现为胎动减少，新生儿表现为肌肉反射消失、面部瘫痪、房间隔缺损和关节挛缩，最严重的表现为呼吸衰竭，病儿预期寿命大大缩短，大多数生存期在6个月以内；Ⅰ型患者：即婴儿型，也称为Werdnig-Hoffman病，占SMA患者的50％。患者出生后6个月内出现肌张力低下，头部控制不佳和肌腱反射减弱或消失。严重的肌张力低下，表现为躺倒时“蛙腿”姿势，缺乏头 部控制，不能端坐，肋间肌肉薄弱，膈肌相对较小，患者往往出现吞咽功能减弱，呼吸肌无力而产生呼吸衰竭。在无辅助通气情况下，92％的Ⅰ型SMA患儿通常在20个月前因呼吸衰竭而死亡；Ⅱ型患者：即中间型，约占SMA患者的20％，一般在出生后6-18个月内发病，患者可以在发育过程中的某一阶段独坐，但无法独立行走。该类患者多出现脊柱侧凸、关节挛缩和下颌关节强直等并发症，脊柱侧凸和肋间肌无力往往导致严重的肺部疾病。这些儿童的认知能力是正常的；Ⅲ型患者：即少年型，也称为Kugelberg-Welander病，约占SMA患者的30％，患者一般在出生后18个月-5岁内发病，在辅助物支撑的帮助下可行走。与Ⅱ型SMA不同的是，这些人大多没有脊柱侧弯和呼吸肌无力等并发症，这个群体的认知和预期寿命一般不受疾病的影响；Ⅳ型患者：在少年之后发病，运动能力逐渐降低，大约占SMA患者总数的5％。与Ⅲ型相似，但是发病在成年期，通常认为在30岁或更晚时候发病。4％的SMA不是由SMN1基因的突变所致，它们被称为非5q型SMA，意思就是说它们的致病基因并不位于5号染色体的SMN区域。和5q型SMA类似，非5q型SMA的患儿很早也就会出现肌肉无力的症状，但会有一些不同之处，其中包括是远端肌肉而非近端肌肉无力、较早出现的远端关节挛缩、隔肌麻痹伴随较早出现的呼吸衰竭及小脑变性等(Verhaart IEC,Robertson A,Wilson IJ,Aartsma-Rus A,Cameron S,Jones CC,Cook SF,Lochmüller H.Prevalence,incidence and carrier frequency of 5q-linked spinal muscular atrophy-a literature review.Orphanet J Rare Dis.2017Jul 4；12(1):124.；Sugarman EA,Nagan N,Zhu H,Akmaev VR,Zhou Z,Rohlfs EM,Flynn K,Hendrickson BC,Scholl T,Sirko-Osadsa DA,Allitto BA.Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy:clinical laboratory analysis of>72,400specimens.Eur J Hum Genet.2012Jan；20(1):27-32)。
SMA是由两个染色体上基因(SMN1)的端粒拷贝的失活突变或缺失，从而导致SMN1基因功能丧失所引起的。SMN1蛋白在RNA成熟中具有辅助因子的功能，是所有真核细胞存活力所需要的(Talbot and Tizzano(2017)Gene Ther 24(9):529-533)。SMN2蛋白除在RNA信息的剪接中起作用的单突变外与SMN1几乎相同。所有SMA患者保留了基因(SMN2) 的着丝粒拷贝，并且SMA患者中SMN2基因的拷贝数通常与疾病严重性负相关，即SMA不太严重的患者具有更多的SMN2拷贝。尽管如此，由于外显子7中翻译沉默的C向T的突变所引起的外显子7的选择性剪接，SMN2不能完全补偿SMN1功能的损失。因此，由SMN2产生的大部分转录物缺乏外显子7(Δ7 SMN2)并且编码具有受损的功能并且被快速降解为截短的SMN蛋白。
在临床上，通常通过临床症状结合至少一个SMN1基因拷贝的测试诊断SMA。在一些情况下，当SMN1基因测试未显示异常时，其他测试如肌电描记术(EMG)或肌肉活组织检查也可以辅助诊断。到目前为止，SMA的治疗仅限于支持疗法，包含呼吸、营养和康复的治疗和护理，尚无药物能够有效治疗这种疾病。
发明概述
本发明研究发现纤溶酶原途径激活剂例如纤溶酶原可以明显改善SMA受试者神经损伤症状、改善肺功能、延长生存期、促进SMN基因的转录和表达、提高脑组织和肌肉组织中SMN蛋白水平、促进脑组织和肌肉组织中NF-κB蛋白的表达、促进脑组织成熟NGF的形成、改善肺组织损伤，从而有效预防和治疗SMA。
一方面，本发明涉及一种治疗脊髓性肌萎缩症(SMA)(包括0型、I型、II型、III型、IV型和非5q型SMA)的方法，包括给药患运动神经元病例如脊髓性肌萎缩症(SMA)的受试者治疗有效量的选自如下的一种或多种纤溶酶原途径激活剂：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。
在一些具体实施方案中所述纤溶酶原途径激活剂对所述患脊髓性肌萎缩症(SMA)(包括0型、I型、II型、III型、IV型和非5q型SMA)的受试者具有选自如下的一种或多种活性：1.减小或改善SMA的严重程度； 2.延迟SMA的发作；3.抑制SMA的进展；4.延长受治者的存活时间；5.提高受治者的生活质量和/或改善受试者精神状态；6.减少SMA相关症状的数目；7.减小或改善与SMA相关的一种或多种症状的严重程度；8.缩短与SMA相关的症状的持续时间；9.防止与SMA相关的症状的复发；10.抑制SMA症状的发展或发作；11.抑制与SMA相关的症状的进展；12.改善肺功能；13.提高血氧饱和度；14.促进SMN基因的转录和表达；15.提高脑组织和肌肉组织中SMN蛋白水平；16.促进脑组织和肌肉组织中NF-κB蛋白的表达；17.促进脑组织成熟NGF的形成；18.减轻肺组织损伤；19.增加肌力；20.减少肌萎缩；21.减少运动神经元损失；22.促进生长、发育；和/或23.提高运动功能。在一些具体实施方案中，所述纤溶酶原途径激活剂改善受试者的肌肉萎缩、增强肌力、和/或改善肌张力。在一些具体实施方案中，所述纤溶酶原途径激活剂延长受试者生存期。在一些具体实施方案中，所述纤溶酶原途径激活剂促进SMN基因的转录和/或表达。在一些具体实施方案中，所述纤溶酶原途径激活剂促进受试者的肌肉功能恢复。在一些具体实施方案中，所述纤溶酶原途径激活剂促进受试者的脊髓前角神经元损伤修复。在一些具体实施方案中，所述纤溶酶原途径激活剂促进受试者NF-κB蛋白的表达。在一些具体实施方案中，所述纤溶酶原途径激活剂促进受试者成熟NGF的形成。所述纤溶酶原途径激活剂促进受试者成熟NGF的形成。
在一些具体实施方案中，所述纤溶酶原途径激活剂与一种或多种其它药物和/或治疗方法联合施用，优选地，所述治疗方法包括细胞疗法(例如干细胞疗法)和基因疗法，例如反义RNA、小分子剪接修饰剂。
在一些实施方案中，所述纤溶酶原途径激活剂为纤维蛋白溶酶原激活途径的组分。
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(简称：纤溶酶原)、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。在一些具体实施方案中，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。
在一些具体实施方案中，所述纤维蛋白溶酶原激活途径的组分为纤溶酶原。在一些具体实施方案中，所述纤溶酶原包含或具有与序列2、6、8、10或12所示氨基酸序列具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，并且具有纤溶酶原活性。在一些实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水解活性。在一些实施方案中，所述纤溶酶原活性为纤溶酶原与底物分子的赖氨酸结合活性。在一些实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水解活性和纤溶酶原与底物分子的赖氨酸结合活性。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且具有纤溶酶原的蛋白水解活性和/或赖氨酸结合活性的蛋白质。在一些具体实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水解活性。在一些具体实施方案中，所述纤溶酶原为包含纤溶酶原活性片段、并且具有纤溶酶原的蛋白水解活性和/或赖氨酸结合活性的蛋白质。在一些具体实施方案中，所述纤溶酶原活性片段包含或具有纤溶酶原的丝氨酸蛋白酶结构域或称纤溶酶原蛋白酶结构域。在一些具体实施方案中，所述纤溶酶原活性片段的氨基酸序列如序列14所示。在一些具体实施方案中，所述纤溶酶原选自Glu-纤溶酶原(人全长纤溶酶原)、Lys-纤溶酶原(在第76-77位氨基酸之间切割后的人全长纤溶酶原)、小纤溶酶原(包含Kringle 5(K5)和丝氨酸蛋白酶结构域)、微纤溶酶原(包含丝氨酸蛋白酶结构域)、delta-纤溶酶原(包含Kringle 1和丝氨酸蛋白酶结构域)或它们的保留纤溶酶原活性的变体。在一些具体实施方案中，所述纤溶酶原为人全长纤溶酶原、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，所述纤溶酶原活性为纤溶酶原的蛋白水解活性。在一些实施方案中，所述纤溶酶原活性为纤溶酶原与底物分子的赖氨酸结合活性。在一些实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水 解活性和纤溶酶原与底物分子的赖氨酸结合活性。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的蛋白水解活性和/或赖氨酸结合活性的变体或片段。在一些实施方案中，所述纤溶酶原包含如序列2、6、8、10或12所示的氨基酸序列。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。
在一些具体实施方案中，所述纤溶酶原途径激活剂以全身或局部方式给药，例如通过静脉内、肌肉内、鞘内、鼻腔吸入、雾化吸入、滴鼻液或滴眼液形式给药。在一些实施方案中，所述受试者是人。在一些实施方案中，所述受试者缺乏或缺失纤溶酶原。在一些实施方案中，所述缺乏或缺失是先天的、继发的和/或局部的。在一些实施方案中，所述纤溶酶原以每天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/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(以每平方厘米体表面积计算)的剂量，每天、每二天或每三天连续施用。
在一些实施方案中，上述SMA为0型、I型、II型、III型、IV型或非5q型SMA。
一方面，本申请还涉及用于治疗脊髓性肌萎缩症(SMA)的药物组合物、药物、制剂、试剂盒、制品，包含以上所述的纤溶酶原途径激活剂，例如以上所述的纤维蛋白溶酶原激活途径的组分，例如以上所述的纤溶酶原。
在一些实施方案中，所述药物组合物、药物、制剂包含药学上可接受的载体和纤溶酶原途径激活剂，例如以上所述的纤溶酶原激活途径的组分，例如以上所述的纤溶酶原。在一些实施方案中，所述试剂盒和制品包含一个或多个容器，所述容器中包含所述药物组合物、药物或制剂。在一些实施方案中，所述试剂盒或制品还包含标签或使用说明书，该标签或使用说明书指示使用纤溶酶原途径激活剂，例如以上所述的纤溶酶原激活途径的组分，例如以上所述的纤溶酶原治疗脊髓性肌萎缩症的方法。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容器，该容器中含有一种或多种其他药物。在一些实施方案中，上述SMA为0型、I型、II 型、III型、IV型或非5q型SMA。
一方面本申请还涉及用于治疗脊髓性肌萎缩症(SMA)的以上所述的纤溶酶原途径激活剂，例如以上所述的纤溶酶原。在一些实施方案中，上述SMA为0型、I型、II型、III型、IV型或非5q型SMA。
一方面，本申请还涉及以上所述的纤溶酶原途径激活剂，例如以上所述的纤溶酶原用于治疗脊髓性肌萎缩症(SMA)的用途。在一些实施方案中，上述SMA为0型、I型、II型、III型、IV型或非5q型SMA。
一方面，本申请还涉及治疗有效量的上述纤溶酶原途径激活剂(例如以上所述的纤溶酶原激活途径的组分，例如以上所述的纤溶酶原)在制备治疗脊髓性肌萎缩症(SMA)的药物组合物、药物、制剂、试剂盒、制品中的用途。
在一些实施方案中，所述纤溶酶原途径激活剂选自如下的一种或多种纤溶酶原途径激活剂：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。
在一些具体实施方案中，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。在一些具体实施方案中，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。
在一些实施方案中，所述纤溶酶原途径激活剂为纤维蛋白溶酶原激活途径的组分。
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(简称：纤溶酶原)、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。在一些具体实施方案中，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。
在一些具体实施方案中，所述纤维蛋白溶酶原激活途径的组分为纤溶酶原。在一些具体实施方案中，所述纤溶酶原包含或具有与序列2、6、8、10或12所示氨基酸序列具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，并且具有纤溶酶原活性。在一些实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水解活性。在一些实施方案中，所述纤溶酶原活性为纤溶酶原与底物分子的赖氨酸结合活性。在一些实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水解活性和纤溶酶原与底物分子的赖氨酸结合活性。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且具有纤溶酶原的蛋白水解活性和/或赖氨酸结合活性的蛋白质。在一些具体实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水解活性。在一些具体实施方案中，所述纤溶酶原为包含纤溶酶原活性片段、并且具有纤溶酶原的蛋白水解活性和/或赖氨酸结合活性的蛋白质。在一些具体实施方案中，所述纤溶酶原活性片段包含或具有纤溶酶原的丝氨酸蛋白酶结构域或称纤溶酶原蛋白酶结构域。在一些具体实施方案中，所述纤溶酶原活性片段的氨基酸序列如序列14所示。在一些具体实施方案中，所述纤溶酶原选自Glu-纤溶酶原(人全长纤溶酶原)、Lys-纤溶酶原(在第76-77位氨基酸之间切割后的人全长纤溶酶原)、小纤溶酶原(包含Kringle 5(K5)和丝氨酸蛋白酶结构域)、微纤溶 酶原(包含丝氨酸蛋白酶结构域)、delta-纤溶酶原(包含Kringle 1和丝氨酸蛋白酶结构域)或它们的保留纤溶酶原活性的变体。在一些具体实施方案中，所述纤溶酶原为人全长纤溶酶原、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，所述纤溶酶原活性为纤溶酶原的蛋白水解活性。在一些实施方案中，所述纤溶酶原活性为纤溶酶原与底物分子的赖氨酸结合活性。在一些实施方案中，所述纤溶酶原活性为纤溶酶原的蛋白水解活性和纤溶酶原与底物分子的赖氨酸结合活性。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的蛋白水解活性和/或赖氨酸结合活性的变体或片段。在一些实施方案中，所述纤溶酶原包含如序列2、6、8、10或12所示的氨基酸序列。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，所述纤溶酶原途径激活剂，例如上述纤溶酶原激活途径的组分，例如上述纤溶酶原与一种或多种其它药物和/或治疗方法联合施用。在一些实施方案中，所述纤溶酶原途径激活剂，例如纤溶酶原激活途径的组分，例如纤溶酶原通过静脉内、肌肉内、鞘内、鼻腔吸入、雾化吸入、滴鼻液或滴眼液形式给药。
在一些实施方案中，所述药物组合物、药物、制剂包含药学上可接受的载体和纤溶酶原途径激活剂，例如纤溶酶原激活途径的组分，例如纤溶酶原。在一些实施方案中，所述试剂盒和制品包含一个或多个容器，所述容器中包含所述药物组合物、药物或制剂。在一些实施方案中，所述试剂盒或制品还包含标签或使用说明书，该标签或使用说明书指示使用纤溶酶原途径激活剂，例如纤溶酶原激活途径的组分，例如纤溶酶原治疗脊髓性肌萎缩症的方法。
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容器，该容器中含有一种或多种其他药物。
在一些实施方案中，上述SMA为0型、I型、II型、III型、IV型或非5q型SMA。
本发明明确涵盖了属于本发明实施方案之间的技术特征的所有组合，并且这些组合后的技术方案在本申请中已经明确公开，就像上述技术方案 已经单独且明确公开一样。另外，本发明还明确涵盖各个实施方案及其要素的之间的组合，该组合后的技术方案在本文中明确公开。
附图简述
图1示出实施例1所述Ⅱ型SMA患者治疗前和治疗后运动神经肌电图波幅结果。结果显示，与用药前相比，患者胫神经、腓总神经动作电位波幅均有不同程度的提高。结果表明，纤溶酶原可改善II型SMA患者周围神经元传导功能，改善神经肌肉损伤。
图2示出实施例2所述患者治疗前后上下肢运动神经肌电图波幅结果。与用药前相比，患者左侧股神经、右侧尺神经、双侧腓总神经及胫神经动作电位波幅均有不同程度的提高。结果表明，纤溶酶原可改善II型SMA患者周围神经元传导功能，改善神经肌肉损伤。
图3示出实施例3所述患者治疗前后上下肢运动神经肌电图波幅结果。与用药前相比，患者双侧正中神经、胫神经、腓总神经、尺神经动作电位波幅均有不同程度的提高。结果表明，纤溶酶原可改善II型SMA患者周围神经元传导功能，改善神经肌肉损伤。
图4A-4B给予纤溶酶原后SMNΔ7 SMA小鼠生存曲线和生存时间统计结果。A为生存曲线统计结果，B为生存时间统计结果。生存曲线统计结果显示，纤溶酶原能够明显改善SMNΔ7 SMA小鼠生存曲线，统计差异显著(P＝0.029)。生存时间统计结果显示，溶媒组小鼠中位生存时间为14天，所有小鼠在第15天全部死亡；给药组中位生存时间为16天，所有小鼠在第17天全部死亡，且统计分析差异显著(P＝0.03)。说明纤溶酶原能够延长SMA模型小鼠生存时间。
图5给予纤溶酶原后SMNΔ7 SMA小鼠脊髓SMN基因qPCR检测结果。结果显示，空白对照组小鼠脊髓具有一定的SMN基因转录水平，溶媒组小鼠SMN基因转录水平低于空白对照组小鼠，给药组小鼠SMN基因转录水平明显高于溶媒组小鼠和空白对照组小鼠。该结果提示纤溶酶原能够促进SMN基因转录。
图6给予纤溶酶原后SMNΔ7 SMA小鼠脑NF-κB蛋白Western blot检测结果和光密度值定量分析结果。结果显示，空白对照组小鼠脑具有一定 量的NF-κB蛋白，溶媒组小鼠脑NF-κB蛋白水平低于空白对照组小鼠，给药组小鼠脑NF-κB蛋白水平明显高于溶媒组小鼠，且统计差异接近显著(P＝0.05)。该结果提示纤溶酶原能够促进SMNΔ7 SMA小鼠脑组织NF-κB蛋白水平增加。
图7给予纤溶酶原后SMNΔ7 SMA小鼠代表性后肢肌肉NF-κB蛋白Western blot检测结果和光密度值定量分析结果。结果显示，空白对照组小鼠肌肉具有一定量的NF-κB蛋白，溶媒组小鼠肌肉NF-κB蛋白水平低于空白对照组小鼠，给药组小鼠肌肉NF-κB蛋白水平明显高于溶媒组小鼠，且统计差异显著(*表示P<0.05)。该结果提示纤溶酶原能够促进SMNΔ7 SMA小鼠肌肉NF-κB蛋白水平增加。
图8给予纤溶酶原后SMNΔ7 SMA小鼠代表性脑SMN蛋白Western blot检测结果和光密度(OD)值定量分析结果。结果显示，空白对照组小鼠脑表达一定量的SMN蛋白，溶媒组小鼠SMN蛋白表达水平低于空白对照组小鼠，给药组小鼠SMN蛋白表达水平明显高于溶媒组小鼠。该结果提示纤溶酶原能够促进SMNΔ7 SMA小鼠脑SMN蛋白表达。
图9给予纤溶酶原后SMNΔ7 SMA小鼠代表性后肢肌肉SMN蛋白Western blot检测结果和光密度(OD)值定量分析结果。结果显示，空白对照组小鼠肌肉表达一定量的SMN蛋白，溶媒组小鼠肌肉SMN蛋白表达水平低于空白对照组小鼠，给药组小鼠肌肉SMN蛋白表达水平明显高于溶媒组小鼠。该结果提示纤溶酶原能够促进SMNΔ7 SMA小鼠肌肉SMN蛋白表达。
图10给予纤溶酶原后SMA小鼠后脑组织Western blot检测结果和NGF/Pro-NGF光密度(OD)比值定量分析结果。结果显示，空白对照组小鼠脑组织具有一定NGF/ProNGF比值，给药组小鼠脑组织NGF/ProNGF比值明显高于溶媒组小鼠，且统计差异极为显著(***表示P<0.001)。提示纤溶酶原能够促进SMA模型小鼠脑组织中ProNGF转化形成NGF，促进成熟NGF的形成。
图11给予纤溶酶原后SMA小鼠代表性后肺组织H&E染色图片结果显示，空白对照组小鼠肺组织终末细支气管上皮细胞排列整齐，清晰可辨；肺泡腔大小均匀，肺泡间隔未见增厚，血管周围未见炎细胞浸润；溶媒组 小鼠肺组织呼吸性细支气管上皮脱落，肺泡管、肺泡囊扩大，肺泡隔增宽，肺泡塌陷至结构紊乱，肺血管周围伴嗜酸性粒细胞、泡沫细胞、淋巴细胞；给药组小鼠肺组织呼吸性细支气管上皮排列有序，肺泡管、肺泡囊扩大，肺泡腔均匀扩大，但可见由单层肺泡上皮组成的肺泡壁。提示纤溶酶原能够改善SMA模型小鼠肺组织损伤。
发明详述
术语“脊髓性肌萎缩(症)”(或Spinal Muscular Atrophy，SMA)是指由两个染色体上SMN1基因的失活突变或缺失，从而导致SMN1基因功能丧失引起的疾病。SMA的症状包括肌无力、肌张力低下、哭泣无力、咳嗽无力、跛行或摔倒倾向、吮吸或吞咽困难、呼吸困难、肺或咽喉中分泌物积累、紧握的拳头和汗手、舌头颤动/振动、常常倾向一侧的头部(即使在躺下时)、倾向于弱于臂部的腿部、经常呈“蛙腿”位置的腿部、摄食困难、对呼吸道感染敏感度提高、肠/膀胱无力、低于正常的体重、不能无支撑坐立、不能行走、不能爬行、和张力减退、反射消失、以及与前hom细胞丧失相关的多发性先天性挛缩(关节挛缩)。
本申请术语“治疗脊髓性肌萎缩(SMA)”或“脊髓性肌萎缩(SMA)的治疗”包括获得以下效果中的一个或多个：1.减小或改善SMA的严重程度；2.延迟SMA的发作；3.抑制SMA的进展；4.延长受治者的存活时间；5.提高受治者的生活质量和/或改善受试者精神状态；6.减少SMA相关症状的数目；7.减小或改善与SMA相关的一种或多种症状的严重程度；8.缩短与SMA相关的症状的持续时间；9.防止与SMA相关的症状的复发；10.抑制SMA症状的发展或发作；11.抑制与SMA相关的症状的进展；12.改善肺功能；13.提高血氧饱和度；14.促进SMN基因的转录和表达；15.提高脑组织和肌肉组织中SMN蛋白水平；16.促进脑组织和肌肉组织中NF-κB蛋白的表达；17.促进脑组织成熟NGF的形成；18.减轻肺组织损伤；19.增加肌力；20.减少肌萎缩；21.减少运动神经元损失；22.促进生长、发育；和/或23.提高运动功能。
在一些实施方案中，本申请的纤维蛋白溶酶原激活途径的组分或其相关化合物，例如以上所述的纤溶酶原增强SMN基因转录和/或表达。在一 些实施方案中，本申请的纤维蛋白溶酶原激活途径的组分或其相关化合物，例如以上所述的纤溶酶原增加SMN蛋白在有此需要的人受试者中的表达。
在一些实施方案中，本申请的纤维蛋白溶酶原激活途径的组分或其相关化合物，例如纤溶酶原可以单独地或与其他药物组合地用于治疗或预防由SMN基因的失活突变或缺失引起的和/或与SMN基因功能丧失或缺陷相关的疾病。这些疾病包括但不限于脊髓性肌萎缩(SMA)。
在一些实施方案中，本申请涉及一种治疗SMN基因的失活突变或缺失引起的和/或与SMN基因功能丧失或缺陷相关的疾病，例如SMA的方法，包括给药受试者治疗有效量的纤维蛋白溶酶原激活途径的组分或其相关化合物，例如纤溶酶原。在一些实施方案中，本申请涉及治疗SMA的方法，包括给药受试者治疗有效量的纤溶酶原。
在一些实施方案中，本申请涉及治疗SMA的方法，包括给药受试者治疗有效量的纤溶酶原，所述纤溶酶原具有选自以下的一项或多项活性：1.减小或改善SMA的严重程度；2.延迟SMA的发作；3.抑制SMA的进展；4.延长受治者的存活时间；5.提高受治者的生活质量和/或改善受试者精神状态；6.减少SMA相关症状的数目；7.减小或改善与SMA相关的一种或多种症状的严重程度；8.缩短与SMA相关的症状的持续时间；9.防止与SMA相关的症状的复发；10.抑制SMA症状的发展或发作；11.抑制与SMA相关的症状的进展；12.改善肺功能；13.提高血氧饱和度；14.促进SMN基因的转录和表达；15.提高脑组织和肌肉组织中SMN蛋白水平；16.促进脑组织和肌肉组织中NF-κB蛋白的表达；17.促进脑组织成熟NGF的形成；18.减轻肺组织损伤；19.增加肌力；20.减少肌萎缩；21.减少运动神经元损失；22.促进生长、发育；和/或23.提高运动功能。
纤维蛋白溶解系统(Fibrinolytic system)也称纤溶系统，为参与纤维蛋白溶解(纤溶)过程的一系列化学物质组成的系统，主要包括纤维蛋白溶解酶原(纤溶酶原)、纤溶酶、纤溶酶原激活物、纤溶抑制剂。纤溶酶原激活物包括组织型纤溶酶原激活物(t-PA)和尿激酶型纤溶酶原激活物(u-PA)。t-PA是一种丝氨酸蛋白酶，由血管内皮细胞合成。t-PA激活纤溶酶原，此过程主要在纤维蛋白上进行；尿激酶型纤溶酶原激活物(u-PA)由 肾小管上皮细胞和血管内皮细胞产生，可以直接激活纤溶酶原而不需要纤维蛋白作为辅因子。纤溶酶原(PLG)由肝脏合成，当血液凝固时，PLG大量吸附在纤维蛋白网上，在t-PA或u-PA的作用下，被激活为纤溶酶，促使纤维蛋白溶解。纤溶酶(PL)是一种丝氨酸蛋白酶，作用如下：降解纤维蛋白和纤维蛋白原；水解多种凝血因子Ⅴ、Ⅷ、Ⅹ、Ⅶ、Ⅺ、Ⅱ等；使纤溶酶原转变为纤溶酶；水解补体等。纤溶抑制物：包括纤溶酶原激活物抑制剂(PAI)和α2抗纤溶酶(α2-AP)。PAI主要有PAI-1和PAI-2两种形式，能特异性与t-PA以1：1比例结合，从而使其失活，同时激活PLG。α2-AP由肝脏合成，与PL以1：1比例结合形成复合物，抑制PL活性；FⅩⅢ使α2-AP以共价键与纤维蛋白结合，减弱了纤维蛋白对PL作用的敏感性。体内抑制纤溶系统活性的物质：PAI-1，补体C1抑制物；α2抗纤溶酶；α2巨球蛋白。
本发明“纤维蛋白溶酶原途径激活剂”或“纤溶酶原途径激活剂”术语涵盖纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
本发明的术语“纤维蛋白溶酶原激活途径的组分”或“纤溶酶原激活途径的组分”涵盖：
1.纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、微纤溶酶原(micro-plasminogen)、delta-纤溶酶原；它们的变体或类似物；
2.纤维蛋白溶酶以及它们的变体或类似物；和
3.纤维蛋白溶酶原激活剂，例如tPA和uPA以及包含一个或多个tPA或uPA的结构域(如一个或多个kringle结构域和蛋白水解结构域)的tPA或uPA变体和类似物。
所述的“纤溶抑制剂的拮抗剂”术语涵盖PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的拮抗剂，例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抗体。
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”涵盖包含或具有与序列2、6、8、10或12所示氨基酸序列具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且具有纤溶酶原的蛋白水解活性和/或赖氨酸结合活性的蛋白质。例如本发明的“纤溶酶原变体”可以是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原的蛋白水解活性和/或赖氨酸结合活性的蛋白质。具体地，本发明纤溶酶原变体包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式，以及通过保守性氨基酸取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸获得的这些蛋白质的突变变体。
本发明的纤溶酶原可以为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原的蛋白水解活性和/或赖氨酸结合活性的变体，例如序列2、6、8、10或12所示的纤溶酶原，例如序列2所示的人天然纤溶酶原。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“类似物”包括分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用的化合物。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和“类似物”涵盖包含一个或多个结构域(例如一个或多个kringle结构域和蛋白水 解结构域)的纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和“类似物”。例如，纤维蛋白溶酶原的“变体”和“类似物”涵盖包含一个或多个纤溶酶原结构域(例如一个或多个kringle(k)结构域和蛋白水解结构域(或称丝氨酸蛋白酶结构域，或称纤溶酶原蛋白酶结构域)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结构域和蛋白水解结构域)的纤维蛋白溶酶“变体”和“类似物”，例如小纤维蛋白溶酶(mini-plasmin)和δ-纤维蛋白溶酶(delta-plasmin)。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的“变体”或“类似物”是否分别具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的活性，或者是否分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用可以通过本领域已知方法进行检测，例如，通过基于酶谱法(enzymography)、ELISA(酶联免疫吸附测定)和FACS(荧光激活细胞分选方法)通过激活的纤维蛋白溶酶活性水平来衡量，例如可以参照选自如下文献中记载的方法测量：Ny，A.，Leonardsson，G.，Hagglund，A.C，Hagglof，P.，Ploplis，V.A.，Carmeliet，P.and Ny，T.(1999).Ovulation inplasminogen-deficient mice.Endocrinology 140，5030-5035；Silverstein RL,Leung LL,Harpel PC,Nachman RL(November 1984)."Complex formation of platelet thrombospondin with plasminogen.Modulation of activation by tissue activator".J.Clin.Invest.74(5):1625–33；Gravanis I,Tsirka SE(February2008)."Tissue-type plasminogen activator as a therapeutic target in stroke".Expert Opinion on Therapeutic Targets.12(2):159–70；Geiger M,Huber K,Wojta J,Stingl L,Espana F,Griffin JH,Binder BR(Aug 1989)."Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo".Blood.74(2):722–8.
在本发明的一些实施方案中，本发明的“纤维蛋白溶酶原激活途径的组分”为纤溶酶原，选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶 原活性和/或赖氨酸结合活性的保守突变变体或其片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性和/或赖氨酸结合活性的保守突变变体或其片段。在一些实施方案中，所述纤溶酶原的氨基酸序列包含或具有如序列2、6、8、10或12所示的氨基酸序列。在一些实施方案中，所述纤溶酶原是人全长纤溶酶原。在一些实施方案中，所述纤溶酶原是如序列2所示的人全长纤溶酶原。
“能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物”指能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的任何化合物，例如tPA、uPA、链激酶、沙芦普酶、阿替普酶、瑞替普酶、替奈普酶、阿尼普酶、孟替普酶、拉诺替普酶、帕米普酶、葡激酶。
本发明“纤溶抑制剂的拮抗剂”为拮抗、减弱、封闭、阻止纤溶抑制剂作用的化合物。所述纤溶抑制剂例如PAI-1、补体C1抑制物、α2抗纤溶酶和α2巨球蛋白。所述拮抗剂例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抗体，或阻断或下调例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白表达的反义RNA或小RNA，或占据PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的结合位点但无PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白功能的化合物”，或封闭PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的结合结构域和/或活性结构域的化合物。
纤溶酶是纤溶酶原激活系统(PA系统)的关键组分。它是一种广谱的蛋白酶，能够水解细胞外基质(ECM)的几个组分，包括纤维蛋白、明胶、纤连蛋白、层粘连蛋白和蛋白聚糖。此外，纤溶酶能将一些金属蛋白酶前体(pro-MMPs)激活形成具有活性的金属蛋白酶(MMPs)。因此纤溶酶被认为是胞外蛋白水解作用的一个重要的上游调节物。纤溶酶是由纤溶酶原通过两种生理性的PAs：组织型纤溶酶原激活剂(tPA)或尿激酶型纤溶酶原激活剂(uPA)蛋白水解形成的。由于纤溶酶原在血浆和其他体液中相对水平较高，传统上认为PA系统的调节主要通过PAs的合成和活性水平实现。PA系统组分的合成受不同因素严格调节，如激素、生长因子和细胞 因子。此外，还存在纤溶酶和PAs的特定生理抑制剂。纤溶酶的主要抑制剂是α2-抗纤溶酶(α2-antiplasmin)。PAs的活性同时被uPA和tPA的纤溶酶原激活剂抑制剂-1(PAI-1)抑制以及主要抑制uPA的溶酶原激活剂抑制剂-2(PAI-2)调节。某些细胞表面具有直接水解活性的uPA特异性细胞表面受体(uPAR)。
纤溶酶原是一个单链糖蛋白，由791个氨基酸组成，分子量约为92kDa。纤溶酶原主要在肝脏合成，大量存在于胞外液中。血浆中纤溶酶原含量约为2μM。因此纤溶酶原是组织和体液中蛋白质水解活性的一个巨大的潜在来源。纤溶酶原存在两种分子形式：谷氨酸-纤溶酶原(Glu-plasminogen)和赖氨酸-纤溶酶原(Lys-plasminogen)。天然分泌和未裂解形式的纤溶酶原具有一个氨基末端(N-末端)谷氨酸，因此被称为谷氨酸-纤溶酶原。然而，在纤溶酶存在时，谷氨酸-纤溶酶原在Lys76-Lys77处水解成为赖氨酸-纤溶酶原。与谷氨酸-纤溶酶原相比，赖氨酸-纤溶酶原与纤维蛋白具有更高的亲和力，并可以更高的速率被PAs激活。这两种形式的纤溶酶原的Arg560-Val561肽键可被uPA或tPA切割，导致二硫键连接的双链蛋白酶纤溶酶的形成。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringles，羧基末端部分包含蛋白酶结构域。一些kringles含有介导纤溶酶原与纤维蛋白及其抑制剂α2-AP特异性相互作用的赖氨酸结合位点。最新发现一个纤溶酶原为38kDa的片段，其中包括kringles1-4，是血管生成的有效抑制剂。这个片段被命名为血管抑素，可通过几个蛋白酶水解纤溶酶原产生。
纤溶酶的主要底物是纤维蛋白，纤维蛋白的溶解是预防病理性血栓形成的关键。纤溶酶还具有对ECM几个组分的底物特异性，包括层粘连蛋白、纤连蛋白、蛋白聚糖和明胶，表明纤溶酶在ECM重建中也起着重要作用。间接地，纤溶酶还可以通过转化某些蛋白酶前体为活性蛋白酶来降解ECM的其他组分，包括MMP-1，MMP-2，MMP-3和MMP-9。因此，有人提出，纤溶酶可能是细胞外蛋白水解的一个重要的上游调节器。此外，纤溶酶具有激活某些潜在形式的生长因子的能力。在体外，纤溶酶还能水解补体系统的组分并释放趋化补体片段。
“纤溶酶”是存在于血液中的一种非常重要的酶，能将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体。
“纤溶酶原”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源纤溶酶原氨基酸序列(序列4)计算由810个氨基酸组成，分子量约为90kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的纤溶酶原包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基Cys103-Cys181，Kringle2包括残基Glu184-Cys262，Kringle3包括残基Cys275-Cys352，Kringle4包括残基Cys377-Cys454，Kringle5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。Delta-纤溶酶原(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶(结构)域(也可称为蛋白水解结构域，或称纤溶酶原蛋白酶结构域)，有文献报道了delta-纤溶酶原的氨基酸序列(序列8)，编码该氨基酸序列的cDNA序列如序列7。小纤溶酶原(Mini-plasminogen)由Kringle5和丝氨酸蛋白酶域组成，有文献报道其包括残基Val443-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，其氨基酸序列如序列10所示，编码该氨基酸序列的cDNA序列如序列9所示。而微纤溶酶原(Micro-plasminogen)仅含有丝氨酸蛋白酶结构域，有文献报道其氨基酸序列包括残基Ala543-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，也有专利文献CN102154253A报道其序列包括残基Lys531-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨 基酸)，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”、“纤维蛋白溶解酶原”可互换使用，含义相同。
在本申请中，所述纤溶酶原“缺乏”的含义或活性为受试者体内纤溶酶原的含量比正常人低，低至足以影响所述受试者的正常生理功能；所述纤溶酶原“缺失”的含义或活性为受试者体内纤溶酶原的含量显著低于正常人，甚至活性或表达极微，只有通过外源提供才能维持正常生理功能。
本领域技术人员可以理解，本发明纤溶酶原的所有技术方案适用于纤溶酶，因此，本发明描述的技术方案涵盖了纤溶酶原和纤溶酶。在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时，在纤溶酶原激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性纤溶酶。具有活性的纤溶酶可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中纤溶酶原的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为纤溶酶原激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”在本申请中包括1)在纤溶酶原蛋白中，能够与底物中的靶序列结合的活性片段，也称为赖氨酸结合片段，例如包含Kringle 1、Kringle 2、Kringle 3、Kringle 4和/或Kringle 5的片段(所述纤溶酶原结构参见Aisina R B,Mukhametova L I.Structure and function of plasminogen/plasmin system[J].Russian Journal of Bioorganic Chemistry,2014,40(6):590-605所述)；2)在纤溶酶原蛋白中发挥蛋白水解功能的活性片段，例如包含序列14所示的纤溶酶原活性(蛋白水解功能)的片段；3)在纤溶酶原蛋白中，既具有与底物中的靶序列结合活性(赖氨酸结合活性)又具有纤溶酶原活性(蛋白水解功能)的片段。在本申请的一些实施方案中，所述纤溶酶原为包含序列14所示的纤溶酶原活性片段的蛋白质。在本申请的一些实施方案中，所述纤溶酶原为包含Kringle 1、Kringle 2、 Kringle 3、Kringle 4和/或Kringle 5的赖氨酸结合片段的蛋白质。在一些实施方案中，本申请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。在一些实施方案中，本申请的纤溶酶原包括Kringle 1、Kringle 2、Kringle 3、Kringle 4和/或Kringle 5、或与Kringle 1、Kringle 2、Kringle 3、Kringle 4或Kringle 5具有至少80％、90％、95％、96％、97％、98％、99％同源性并且仍然具有赖氨酸结合活性的蛋白质。
目前，对于血液中纤溶酶原及其活性测定方法包括：对组织纤溶酶原激活剂活性的检测(t-PAA)、血浆组织纤溶酶原激活剂抗原的检测(t-PAAg)、对血浆组织纤溶酶原活性的检测(plgA)、血浆组织纤溶酶原抗原的检测(plgAg)、血浆组织纤溶酶原激活剂抑制物活性的检测、血浆组织纤溶酶原激活剂抑制物抗原的检测、血浆纤维蛋白溶酶-抗纤维蛋白溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤溶酶原活性成正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤溶酶原包括人的天然纤溶酶原，还包括来源于不同物种的、具有纤溶酶原活性的纤溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性，碱性，疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸，蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性 可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤溶酶原是指从其天然环境分离和/或回收的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原会纯化(1)至大于90％、大于95％、或大于98％的纯度(按重量计)，如通过Lowry法所确定的，例如超过99％(按重量计)，(2)至足以通过使用旋转杯序列分析仪获得N端或内部氨基酸序列的至少15个残基的程度，或(3)至同质性，该同质性是通过使用考马斯蓝或银染在还原性或非还原性条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定的。分离的纤溶酶原也包括通过生物工程技术从重组细胞制备，并通过至少一个纯化步骤分离的纤溶酶原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指任何长度的氨基酸的聚合形式，其可以包括遗传编码的和非遗传编码的氨基酸，化学或生物化学修饰的或衍生化的氨基酸，和具有经修饰的肽主链的多肽。该术语包括融合蛋白，包括但不限于具有异源氨基酸序列的融合蛋白，具有异源和同源前导序列(具有或没有N端甲硫氨酸残基)的融合物；等等。
关于参照多肽序列的“氨基酸序列同一性百分数(％)”定义为在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式实现，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员能决定用于比对序列的适宜参数，包括对所比较序列全长实现最大对比需要的任何算法。然而，为了本发明的目的，氨基酸序列同一性百分数值是使用序列比较计算机程序ALIGN-2产生的。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相 对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：
分数X/Y乘100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基的数目，且其中Y是B中的氨基酸残基的总数。应当领会，在氨基酸序列A的长度与氨基酸序列B的长度不相等的情况下，A相对于B的％氨基酸序列同一性会不等于B相对于A的％氨基酸序列同一性。除非另有明确说明，本文中使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
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。
术语疾病状态的“治疗”包括抑制或阻止所述疾病状态或其临床症状的发展，或减轻所述疾病状态或症状，使得所述疾病状态或其临床症状暂时或永久性的退去。
术语“肌力”指肢体作随意运动时肌肉收缩的力量或肌肉主动运动时的力量。根据肌力的情况，通常将肌力分为以下0--5级：0级，完全瘫痪，测不到肌肉收缩；1级，仅测到肌肉收缩，但不能产生动作；2级，肢体能在床上平行移动，但不能抵抗自身重力，即不能抬离床面；3级，肢体可以克服地心引力，能抬离床面，但不能抵抗阻力；4级，肢体能做对抗外界阻力的运动，但不完全；5级，肌力正常。
术语“肌张力”指肌肉静止松弛状态下的紧张度。肌张力是维持身体各种姿势以及正常运动的基础。肌张力表现为多种形式，例如人在静卧休息时，身体各部肌肉所具有的张力称静止性肌张力。躯体站立时，虽不见肌肉显著收缩，但躯体前后肌肉亦保持一定张力，以维持站立姿势和身体稳定，称为姿势性肌张力。肌肉在运动过程中的张力，称为运动性肌张 力，是保证肌肉运动连续、平滑(无颤抖、抽搐、痉挛)的重要因素。在病理状况下，肌张力增高或者减低，影响人体正常的姿势或运动。
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.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)是可以用于克隆纤溶酶原编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(enterobacteriaceae)，诸如沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物种。在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子系统，色氨酸(trp)启动子系统，beta-内酰胺酶启动子系统，或来自噬菌体λ的启动子系统。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
在微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达并生成本发明的纤溶酶原(例如编码纤溶酶原的多核苷酸)。参见Winnacker,From Genes to Clones,VCH Publishers,N.Y.,N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起点，启动子和增强子(Queen等,Immunol.Rev.89:49(1986))，以及必需的加工信息位点，诸如核糖体结合位点，RNA剪接位点，多聚腺苷酸化位点，和转录终止子序列。合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛乳头瘤病毒、巨细胞病毒等衍生的启动子。参见Co等,J.Immunol.148:1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀，亲和柱，柱层析，高效液相层析(HPLC)，凝胶电泳等来纯化本发明所述的纤溶酶原。该纤溶酶原是基本上纯的，例如至少约80％至85％纯的，至少约85％至90％纯的，至少约90％至95％纯的，或98％至99％纯的 或更纯的，例如不含污染物，所述污染物如细胞碎片，除纤溶酶原以外的大分子，等等。
药物配制剂
可以通过将具有所需纯度的纤溶酶原与可选的药用载体，赋形剂，或稳定剂(Remington's Pharmaceutical Sciences,16版，Osol,A.ed.(1980))混合形成冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。例如，抗高血压的药物，抗心律失常的药物，治疗糖尿病的药物等。
本发明的纤溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送系统(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980)。
用于体内给药的本发明的纤溶酶原必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤溶酶原可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J. Biomed.Mater.Res.,15:167-277(1981)；Langer,Chem.Tech.,12:98-105(1982))或聚(乙烯醇)，聚交酯(美国专利3773919,EP 58,481)，L-谷氨酸与γ乙基-L-谷氨酸的共聚物(Sidman，等，Biopolymers 22:547(1983))，不可降解的乙烯－乙烯乙酸酯(ethylene-vinyl acetate)(Langer，等，出处同上)，或可降解的乳酸-羟基乙酸共聚物如Lupron DepotTM(由乳酸-羟基乙酸共聚物和亮氨酰脯氨酸(leuprolide)乙酸酯组成的可注射的微球体)，以及聚D-(-)-3-羟丁酸。聚合物如乙烯-乙酸乙烯酯和乳酸-羟基乙酸能持续释放分子100天以上，而一些水凝胶释放蛋白的时间却较短。可以根据相关机理来设计使蛋白稳定的合理策略。例如，如果发现凝聚的机理是通过硫代二硫键互换而形成分子间S-S键，则可通过修饰巯基残基、从酸性溶液中冻干、控制湿度、采用合适的添加剂、和开发特定的聚合物基质组合物来实现稳定。
给药和剂量
可以通过不同方式，例如通过静脉内，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，其包含可用于治疗由糖尿病引起的心血管病及其相关病症的本发明纤溶酶原或纤溶酶。所述制品优选包括一个容器，标签或包装插页。适当的容器有瓶子，小瓶，注射器等。容器可由各种材料如玻璃或塑料制成。所述容器含有组合物，所述组合物可有效治疗本发明的疾病或病症并具有无菌入口(例如所述容器可为静脉内溶液包或小瓶，其含有可被皮下注射针穿透的塞子的)。所述组合物中至少一种活性剂为纤溶酶原/纤溶酶。所述容器上或所附的标签说明所述组合物用于治疗本发明所述由糖尿病引起的心血管病及其相关病症。所述制品可进一步包含含有可药用缓冲液的第二容器，诸如磷酸盐缓冲的盐水，林格氏溶液以及葡萄糖溶液。其可进一步包含从商业和使用者角度来看所需的其它物质，包括其它缓冲液，稀释剂，过滤物，针和注射器。此外，所述制品包含带有使用说明的包装插页，包括例如指示所述组合物的使用者将纤溶酶原组合物以及治疗伴随的疾病的其它药物给药患者。
实施例
以下所有实施例中使用的人纤溶酶原来自捐赠者血浆，基于文献[1-3]所描述的方法并进行工艺优化，从人捐赠者血浆纯化获得，其中人Lys-纤维蛋白溶酶原(Lys-纤溶酶原)和Glu-纤维蛋白溶酶原(Glu-纤溶酶原)>98％。
以下实施例1-7中所有患者均签署知情同意书，自愿接受上述纯化自人血浆的纤溶酶原的治疗，并获得医院伦理委员会批准。根据病情严重程度和病程不同，调节用法用量。给药方式为雾化吸入或静脉注射。雾化吸入、静脉注射的药品浓度均为5mg/ml，以生理盐水为溶媒。
实施例1 II型SMA
患者，女，38个月。出生10个月时无法爬行，双腿无力，1岁以后运动能力几乎没有提高。1岁9个月基因检测确诊为SMA，肌力二级，下肢不能持重，开始服用沙丁胺醇、甲钴胺、辅酶Q10，2周岁时已经退化到不能爬，只能短时间坐立。2岁2个月时开始间充质干细胞治疗，平衡力有所好转，不容易生病，但是仍然无法站立。2岁11个月开始穴位注射鼠神经生长因子，平衡力有所好转，下肢开始有支撑力，肌力测试达3级。
用药情况
静脉注射；剂量：100-200mg/次；频率：每隔1天一次、每隔2天一次或每隔3天一次；2周为一个疗程；每个疗程间隔2-3周。共治疗6个疗程。
Hammersmith运动功能评分量表(Expanded Hammersmith Functional Motor Scale,HFMSE)专门用以评估II型和III型SMA患者运动功能，反映疾病严重性。它相较于基线的变化而定义，评估患儿运动功能的变化情况，分值越高，运动功能越好[4-6]。
神经肌电图检查是运动神经元病主要诊断和鉴别手段，复合肌肉动作电位波幅反映神经元轴突损伤。SMA是一种运动神经元退行性疾病，运动神经元大量死亡，出现肌无力和复合肌肉动作电位波幅降低甚至无法检测出[7]。
药效情况
HFMSE评分：用药前20分，第1个疗程治疗后评分为21分。第2疗程治疗前评分为23分，治疗后评分为24分。第6个疗程治疗后评分为25分。
运动功能评价：治疗前患者不能无辅助站立，用药2个疗程后患者能够辅助站立，用药3个疗程后患者实现辅助行走，并且患者的头控能力明显改善。随着治疗的进行，患者的运动功能进一步改善，包括辅助站立时间延长和辅助行走距离增加等。
肌电图：较用药前，用药后双侧胫神经和腓总神经及股神经动作电位波幅明显升高(图1)。
此外，用药后患者的精神状态明显改善，活力增加。治疗过程中未见药物相关的副作用。
以上结果说明纤溶酶原可提高Ⅱ型SMA患者HFMSE评分，改善患者运动功能，改善患者的神经肌肉功能和精神状态。
实施例2 II型SMA患者
患者，男，30个月。出生后，无异常。9个月时，无法独站，无法自主翻身，双手偶尔有轻微颤抖，无法抓握，无法抬头。10个月时基因检测确诊为II型SMA患者。1-2岁时，开始在康复中心做康复，一周一次，可以保持独坐，双手可以抓取轻物，小腿缓慢伸展，辅助下可完成翻身动作。2岁2个月时，静脉输注骨髓间充质干细胞三次，每次两个单位，改善并不明显。2岁4个月时，注射了两次神经干细胞，每次两个单位，注射后改善比较明显。现状：可以独坐，独坐时，身体可以向两侧稍微倾斜，用双手支撑身体，但双臂无法举起。头部控制有所改善，可以来回快速摇头，在家长辅助四点支撑的情况下保持抬头状态。腿部力量增加，可以坐在座椅上小腿来回踢踹。可以斜靠家长身上保持站立，但需轻微辅助膝盖。
用药情况
第一次：静脉注射，50mg；第二次：静脉注射，50mg；第三次和第四次：静脉注射，100mg。用药频率为每隔2天一次或每隔3天一次，共用药2周。
药效情况
用药前HFMSE评分表为2分，第四次用药后HFMSE评分为8分，患者可独坐双手举起。肌电图结果显示，较用药前，用药后左侧股神经、右侧尺神经、腓总神经及胫神经动作电位波幅增加(图2)。
以上结果说明纤溶酶原可提高II型SMA患者HFMSE评分，改善患者运动功能，改善神经肌肉功能，且治疗过程中未见药物相关的副作用。
实施例3 II型SMA患者
患者，女，24个月。1岁时可扶站，不会四点支撑爬。16个月时肌电图显示神经源性受损，基因检测确诊为II型SMA患者。现状：独坐不稳， 躺着自己坐不起来，能扶站，靠站，扶走，状态时好时坏，双手举起困难。
用药情况
静脉注射；剂量：50mg-100mg每次；频率：每隔1天一次或每隔3天一次；2周为一个疗程，每个疗程间隔3-4周，共治疗8个疗程。
药效情况
HFMSE评分：用药前为23分，第1个疗程用药后为24分。患者第1个疗程和第2个疗程之间间隔了大约两个月。第2个疗程治疗前为23分，治疗后为24分。第8个疗程治疗后评分为28分。
运动功能：用药前患者不能将手举过头顶。治疗两个疗程后，患者实现无辅助站立和辅助行走，并且能够将手举过头顶。经过持续用药，患者的运动功能进一步改善，独坐、扶站、扶走平稳且时间逐渐延长。
肌电图：治疗后双侧正中神经、胫神经、腓总神经、尺神经的动作电位波幅均有不同程度增加(图3)。
此外，治疗过程中未见药物相关的副作用。
以上结果说明纤溶酶原可提高II型SMA患者HFMSE评分，改善患者运动功能，改善神经肌肉功能。
实施例4 Ⅱ型SMA患者
患者，男，43个月。12个月大时检查肌电图显示神经源损害，14个月大做基因检测确诊为II型SMA。到24个月大时已经退化到不能独坐，只能靠坐。29个月开始吃沙丁胺醇，甲钴胺，辅酶q10，运动功能逐渐轻微改善。36个月开始用间充质干细胞治疗，共静脉注射过5针间充质干细胞，注射间充质干细胞后患者整体状态有明显进步，精神状态好转，不容易累，不容易生病，但是仍然不能站立。38个月时穴位注射鼠神经生长因子，未见明显效果。
用药情况
第一次：静脉注射，50mg；第二次：静脉注射，50mg；第三次：静脉注射，100mg；第四次：静脉注射，150mg，每次间隔3天，用药2周。
药效情况
患者用药后右臂功能有所改善，不需借助左手力量可以自行抬起90度，但HFMSE评分没有提高。治疗过程中未见药物相关的副作用。
实施例5 II型SMA患者
患者，男，26个月。10个月时，基因检测确定为SMA，SMN2(运动神经元存活基因2)拷贝数是3。12个月时，失去翻身能力，胳膊无力支撑身体，只能完全趴在床上，手指不能自主用力。13个月时，开始注射脐带间充质干细胞，注射干细胞后，腿部力量增大，呼吸有所改善，睡眠质量有所提高。16个月时，开始服用中药。在间充质干细胞、中药和康复训练综合治疗后，患者能独坐并自己掌握平衡，但不能左右转身或左右捡玩具。能自主连续翻身。腿部力量增加明显，呼吸有所改善，声音变大，漏斗胸改善明显，肋骨外翻迹象好转，胳膊和手的力量有所增加，但还不能抓着大人的手被拽起来。胳膊抬高有所退化，之前胳膊能自己抬高到头顶位置，但在下述治疗时只能到脸部位置。
用药情况
静脉注射两周，隔天1次，第一周用药4次，剂量分别为10mg，20mg，30mg，40mg；第二周用药4次，剂量分别为50mg，50mg，100mg，100mg。
药效情况
第一周第二次用药后，小腿和脚踝力量有所增强。两周用药结束9天后，手和胳膊力量增加，手抓物品力量增加50％。且治疗过程中未见药物相关的副作用。
以上结果说明纤溶酶原可改善II型SMA患者运动功能。
实施例6 I型SMA患者
患者，男，11月龄，6个月时基因检测确诊为I型SMA；医生确诊时告知此类病患者生存周期平均在2周岁，家属未采取任何治疗措施。症状：头支撑无力；上肢双臂无力、无法抬起，双手摆动少、抓握无力、中指无力；下肢无力、摆动少，脚趾可动；无法独坐，不可自主翻身，吮吸 力小、吞咽困难，全天24小时进行血氧监测，血氧饱和度为92-97％，呼吸时胸部起伏弱。
用药方法
雾化吸入(2-3次/天)+静脉推注(3天一次)，治疗周期为十个疗程(2周为1个疗程)。每个疗程间隔2周。雾化吸入剂量为5-10mg，静脉注射剂量为50-200mg。
以CHOP INTEND评分表(费城儿童医院婴儿神经肌肉疾病测试量表)评估该Ⅰ型SMA患者运动功能改善情况，评分高说明运动功能越好[8]。
治疗效果
生存状态：患者接受10个疗程治疗后，患者年龄大于25个月，超过了I型SMA患者自然史研究显示的大多数患者10个月的存活时间[9]。此外，本研究发现用药SMA后患者精神状态和生长发育(身高、体重、胸围)状况明显好转，且相较于同年龄未用纤溶酶原治疗的患者，用纤溶酶原治疗患者肌肉萎缩情况也明显改善，SMA典型的胸廓塌陷体征明显改善。此外，研究中观察到用药纤溶酶原后患者睡眠状态也明显改善。
CHOP INTEND评分：治疗前CHOP INTEND评分为30分，用药5个疗程后评分增加到50分。由于一些原因第5个和第6个疗程间间隔大约2个月，评分降至36分，第6个疗程用药后评分为44分。第6个疗程和第7个疗程间隔大约2个月。第7个疗程用药前为44分，用药后为45分。第10个疗程治疗后，评分为46分。
运动功能：第1个疗程治疗后患者可独坐，头部支撑约为30秒。第4个疗程后独坐时间延长，约为30秒。随着治疗的进行，患者的运动功能持续改善，手抓握有力，手臂可自主轻微抬起，脚活动、摆动频率增加。
吞咽功能：用药后患者吃饭和饮水时呛咳的次数减少，并且说话功能良好。
呼吸功能：第1个疗程用药第2天患者的血氧饱和度即达到97-98％，偶尔为95-96％。用药2个疗程后，患者呼吸力量增强。10个疗程后，患者已经大于25个月，在无通气支持的条件下，患者仍保持良好的呼吸状态。 I型SMA自然史研究显示，在没有通气支持的条件下，20个月时的存活率仅有8％[10]。
以上结果说明纤溶酶原可提高I型SMA患者CHOP INTEND评分，改善患者运动功能，可实现无辅助支撑坐立30秒和头支撑30秒运动的里程碑式改善；还可改善患者的吞咽功能和说话能力；改善患者胸廓塌陷体征，改善肺部功能，升高血氧饱和度；并可改善患者精神状态和睡眠。
实施例7 非5q型SMA患者
患者，女，40月龄(3岁4个月)。6个月大时发病，1.5岁(18月龄)时经基因检测确诊为非5q型SMA，因肺感染、排痰困难堵塞呼吸道导致呼吸无力，由间断使用呼吸机后逐渐自主呼吸衰竭无法脱机，使用呼吸机约1.5年。语言功能丧失，面瘫，无法活动，肌力0级。症状表现：使用呼吸机至今，每日使用排痰器、吸痰器、吸氧、雾化(每日2次)，鼻饲进食。肌电图结果显示，双侧上下肢运动神经元严重损伤，未见动作电位。
用药方案
第一疗程(2周)：雾化吸入，10mg/次，3次/天，合并50-100mg静脉注射给药，3天一次。
第一疗程结束后月2个月进行第二疗程。
第二至第四疗程(每个疗程中间间隔2周)：静脉注射，每隔2天一次，剂量为150mg-250mg。
治疗效果
第一疗程：双手悬挂晃动时间增长，幅度增大，力度提高，左上臂可以在辅助托起情况下自主向内移动。下肢辅助屈膝立起持续30分钟，面部表情增多，可眨眼，嘴可自主抽动。
第二疗程：偶尔可吞咽汤水，睡眠改善
第三疗程：排便正常，头可左右晃动，有辅助支撑可抬头维持几秒。手腕略有力，指尖可循环转动，左臂可自主晃动，幅度更大。血氧维持在97％，不再输氧。
第四疗程：左臂协调性更好，右臂动作幅度小，但是摆动频率快。双下肢肌肉柔软不僵硬，面部表情增多，可自主排便。
通过治疗发现患者的精神状态明显改善，呼吸能力明显加强，对通气支持需求逐渐减少。
结果表明，纤溶酶原可改善非5q型SMA患者运动功能，包括肢体运动力度、幅度、范围增加，丰富患者的面部表情；改善患者肺部功能和呼吸功能，减少输氧，升高血氧饱和度；改善患者的吞咽功能；改善患者睡眠质量。
以下实施例8-15为对动物模型的用药研究，所述纤溶酶原仍为上述从人捐赠者血浆提纯的纤溶酶原蛋白。
FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1MsdTg(SMN2*delta7)4299Ahmb/J基因突变小鼠(以下简称SMNΔ7 SMA小鼠)具有SMN1基因纯合突变并且表达人的SMN2基因，该小鼠临床及病理表现类似于人类SMA。种鼠于美国Jackson实验室购买(谱系号：005025)。
实施例8 纤溶酶原延长SMA模型小鼠生存时间
SMNΔ7 SMA小鼠出生称量体重，根据体重随机分为溶媒组(6只)和给药组(5只)。小鼠出生3天后开始给药，溶媒组小鼠腹腔每天注射溶媒6ml/kg，给药组小鼠按照60mg/kg腹腔每天注射给予纤溶酶原。记录小鼠的存活情况。
生存曲线统计结果显示，纤溶酶原能够明显改善SMNΔ7 SMA小鼠生存曲线，统计差异显著(P＝0.029)。生存时间统计结果显示，溶媒组小鼠中位生存时间为14天，所有小鼠在第15天全部死亡；给药组中位生存时间为16天，所有小鼠在第17天全部死亡，且统计分析差异显著(P＝0.03)。说明纤溶酶原能够延长SMA模型小鼠生存时间，见图4A和4B。
实施例9 纤溶酶原促进SMA模型小鼠脊髓SMN基因转录
取出生3天的SMNΔ7 SMA小鼠2只，一只为溶媒组小鼠，每天上、下午各腹腔注射一次给予6μl牛血清白蛋白溶液(5mg/ml)；一只为给药组小鼠，按照30μg/6μl每天上、下午各腹腔注射一次给予纤溶酶原；取野 生型(FVB)小鼠一只做为空白对照组，每天上、下午各腹腔注射一次给予6μl牛血清白蛋白溶液(5mg/ml)。连续用药9天。处死后取材脊髓，进行所有SMN基因转录产物qPCR检测，正向引物为F:GCGGCGGCAGTGGTGGCGGC(序列15)；反向引物为R:AGTAGATCGGACAGATTTTGCT(序列16)。
结果显示，空白对照组小鼠脊髓具有一定的SMN基因转录水平，溶媒组小鼠SMN基因转录水平低于空白对照组小鼠，给药组小鼠SMN基因转录水平明显高于溶媒组小鼠和空白对照组小鼠(图5)。该结果提示纤溶酶原能够促进SMN基因转录。
实施例10 纤溶酶原促进SMA模型小鼠脑NF-κB水平增加
取出生3天的SMNΔ7 SMA小鼠7只，4只为溶媒组小鼠，前9天每天上、下午各腹腔注射一次给予6μl牛血清白蛋白溶液(5mg/ml)，第10天开始，每天腹腔注射一次给予6μl牛血清白蛋白溶液(10mg/ml)；3只为给药组小鼠，前9天按照30μg/6μl每天上、下午各腹腔注射一次给予纤溶酶原，第10天开始，，每天按照60μg/6μl腹腔注射一次纤溶酶原；取野生型小鼠4只做为空白对照组，前9天每天上、下午各腹腔注射一次给予6μl牛血清白蛋白溶液(5mg/ml)，第10天开始，每天腹腔注射一次给予6μl牛血清白蛋白溶液(10mg/ml)。第12天，处死小鼠取材脑组织，制备脑组织匀浆，进行NF-κB蛋白的Western blot检测。根据SDS-PAGE凝胶制备试剂盒(Solarbio，P1320)的配胶说明制备10％凝胶。各组样品分别与4×上样缓冲液(TaKaRa，e2139)以体积比为3:1混匀后，100℃加热5min，冷却后离心2min，然后取20μL上样。电泳条件为30V跑胶30min，然后100V电泳至胶底。电泳结束后剥取凝胶转移到活化的PVDF膜(GE，A29433753)上，电转条件为15V，2.5h。转移后的PVDF膜浸泡在封闭液(5％脱脂乳液)中于4℃冰箱中封闭过夜，TBST(0.01M Tris-NaCl，pH7.6缓冲液)洗4次后，加入兔抗小鼠NF-κB抗体(Cell Signaling Technology,8242)室温孵育3h，TBST洗4次后，加入山羊抗兔IgG(HRP)抗体(Abcam，ab6721)二抗室温孵育1h，TBST洗4次后，将PVDF膜放于干净成像板上，加入Immobilon Western HRP Substrate(MILLIPORE， WBKLS0100)显色，在生物分子成像仪下拍照并用Image J获取每个条带的光密度值，进行定量分析。
核因子κB(nuclear factor kappa-B,NF-κB)是一类关键性的核转录因子。NF-κB家族成员主要包括RelA(p65)，c-Rel，RelB，NF-κB1(p50蛋白及其前体p105)和NF-κB2(p52蛋白及其前体p100)，各成员可形成同源或异源二聚体而行使功能。在哺乳动物细胞中最常见的是p65与p50结合形成p65/p50二聚体。在未受刺激的细胞中，NF-κB转录因子与抑制性IκB(Inhibitor of kappa B)蛋白结合在一起，因而被滞留在细胞质中。上游信号的刺激导致IκB蛋白在IKK(IκB kinase)的作用下被磷酸化修饰，继而被泛素连接酶复合体识别，促使IκB蛋白以蛋白酶体依赖的方式被降解，NF-κB从而被释放出来，进入细胞核并启动靶基因的表达[11]。在几乎所有的动物细胞中都能发现NF-κB，它们参与细胞对外界刺激的响应，在细胞的炎症反应、免疫应答等过程起关键性作用。NF-κB也与突触的可塑性、记忆有关[12]。
结果显示，空白对照组小鼠脑具有一定量的NF-κB蛋白，溶媒组小鼠脑NF-κB蛋白水平低于空白对照组小鼠，给药组小鼠脑NF-κB蛋白水平明显高于溶媒组小鼠，且统计差异接近显著(P＝0.05)(图6)。该结果提示纤溶酶原能够促进SMNΔ7 SMA小鼠脑组织NF-κB蛋白水平增加。
实施例11 纤溶酶原促进SMA模型小鼠肌肉NF-κB水平增加
从上述实施例10的处死小鼠取材肌肉，按照上述实施例10所述方法进行NF-κB蛋白的Western blot检测。
结果显示，空白对照组小鼠肌肉具有一定量的NF-κB蛋白，溶媒组小鼠肌肉NF-κB蛋白水平低于空白对照组小鼠，给药组小鼠肌肉NF-κB蛋白水平明显高于溶媒组小鼠，且统计差异显著(*表示P<0.05)(图7)。该结果提示纤溶酶原能够促进SMNΔ7 SMA小鼠肌肉NF-κB蛋白水平增加。
实施例12 纤溶酶原促进SMA模型小鼠脑组织SMN蛋白水平增加
取出生3天的SMNΔ7 SMA小鼠2只，1只为溶媒组小鼠，每天上、下午各腹腔注射一次给予6μl牛血清白蛋白溶液(5mg/ml)；1只为给药组小 鼠，按照30μg/6μl每天上、下午各腹腔注射一次给予纤溶酶原；取野生型(FVB)小鼠2只做为空白对照组，每天上、下午各腹腔注射一次给予6μl牛血清白蛋白溶液(5mg/ml)。用药9天后，处死小鼠取材脑组织，制备脑组织匀浆，进行SMN蛋白的Western blot检测。根据SDS-PAGE凝胶配胶说明制备12％凝胶。各组样品分别与4×上样缓冲液(TaKaRa，e2139)以体积比为3:1混匀后，100℃加热5min，冷却后离心2min，然后取20μL上样。电泳条件为30V跑胶30min，然后100V电泳至胶底。电泳结束后剥取凝胶转移到活化的PVDF膜(GE，A29433753)上，电转条件为15V，2.5h。转移后的PVDF膜浸泡在封闭液(5％脱脂乳液)中于4℃冰箱中封闭过夜，TBST(0.01M Tris-NaCl，pH7.6缓冲液)洗4次后，加入兔抗小鼠SMN抗体(Proteintech，11708-1-AP)室温孵育3h，TBST洗4次后，加入山羊抗兔IgG(HRP)抗体(Abcam，ab6721)二抗室温孵育1h，TBST洗4次后，将PVDF膜放于干净成像板上，加入Immobilon Western HRP Substrate(MILLIPORE，WBKLS0100)显色，在生物分子成像仪下拍照并用Image J软件获取每个条带的光密度值，进行定量分析。
结果显示，空白对照组小鼠脑表达一定量的SMN蛋白，溶媒组小鼠SMN蛋白表达水平低于空白对照组小鼠，给药组小鼠SMN蛋白表达水平明显高于溶媒组小鼠(图8)。该结果提示纤溶酶原能够促进SMNΔ7 SMA小鼠脑SMN蛋白表达。
实施例13 纤溶酶原促进SMA模型小鼠肌肉SMN蛋白水平增加
从实施例12所述处死小鼠取材后肢肌肉组织，制备组织匀浆，进行SMN蛋白的Western blot检测，检测方法如实施例12所述。
结果显示，空白对照组小鼠肌肉表达一定量的SMN蛋白，溶媒组小鼠肌肉SMN蛋白表达水平低于空白对照组小鼠，给药组小鼠肌肉SMN蛋白表达水平明显高于溶媒组小鼠(图9)。该结果提示纤溶酶原能够促进SMNΔ7 SMA小鼠肌肉SMN蛋白表达。
实施例14 纤溶酶原促进SMA模型小鼠脑组织成熟NGF形成
从实施例12的处死小鼠取材脑组织，制备组织匀浆，进行NGF蛋白的Western blot检测。根据SDS-PAGE凝胶配胶说明制备12％凝胶。各组样品分别与4×上样缓冲液(TaKaRa，e2139)以体积比为3:1混匀后，100℃加热5min，冷却后离心2min，然后取20μL上样。电泳条件为30V跑30min，然后100V电泳至胶底。电泳结束后剥取凝胶转移到活化的PVDF膜(GE，A29433753)上，电转条件为15V，2.5h。转移后的PVDF膜浸泡在封闭液(5％脱脂乳液)中于4℃冰箱中封闭过夜，TBST(0.01M Tris-NaCl，pH7.6缓冲液)洗4次后，加入兔抗小鼠NGF抗体室温孵育3h，TBST洗4次后，加入山羊抗兔IgG(HRP)抗体(Abcam，ab6721)二抗室温孵育1h，TBST洗4次后，将PVDF膜放于干净成像板上，加入Immobilon Western HRP Substrate(MILLIPORE，WBKLS0100)显色，在生物分子成像仪下拍照并用Image J软件获取每个条带的光密度值进行定量分析。
神经生长因子(Nerve growth factor,NGF)是神经营养因子家族中的重要成员。在体内以前体形式合成，包括信号肽、前导肽和成熟肽。研究报道神经生长因子NGF前体(ProNGF)与其裂解形成NGF发挥相反的作用。ProNGF能够促进神经细胞凋亡。成熟的NGF参与调节神经细胞的生长、发育、分化、生存和损伤后再修复等过程，对中枢及周围神经元功能性表达也有重要调控作用[12]。NGF/ProNGF比值＝NGF光密度(OD)/ProNGF光密度(OD)值。
结果显示，空白对照组小鼠脑组织具有一定NGF/ProNGF比值，给药组小鼠脑组织NGF/ProNGF比值明显高于溶媒组小鼠，且统计差异极为显著(***表示P<0.001)(图10)。提示纤溶酶原能够促进SMA模型小鼠ProNGF转化形成NGF，促进成熟NGF的形成。
实施例15 纤溶酶原改善SMA模型小鼠肺组织损伤
从实施例12的处死小鼠取材肺组织，在10％中性福尔马林固定液中固定24小时。固定后的肺组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为5μm，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，切片在显微镜下200倍下观察。
结果显示，空白对照组小鼠肺组织终末细支气管上皮细胞排列整齐，清晰可辨；肺泡腔大小均匀，肺泡间隔未见增厚，血管周围未见炎细胞浸润；溶媒组小鼠肺组织呼吸性细支气管上皮脱落，肺泡管、肺泡囊扩大，肺泡隔增宽，肺泡塌陷至结构紊乱，肺血管周围伴嗜酸性粒细胞、泡沫细胞、淋巴细胞；给药组小鼠肺组织呼吸性细支气管上皮排列有序，肺泡管、肺泡囊扩大，肺泡腔均匀扩大，但可见由单层肺泡上皮组成的肺泡壁(图11)。提示纤溶酶原能够改善SMA模型小鼠肺组织损伤。
参考文献
[1]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.
[2]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.
[3]HAGAN JJ,ABLONDI FB,DE RENZO EC.Purification and biochemical properties of human plasminogen.J Biol Chem.1960Apr；235:1005-10.
[4]Main M,Kairon H,Mercuri E,Muntoni F.The Hammersmith functional motor scale for children with spinal muscular atrophy:a scale to test ability and monitor progress in children with limited ambulation.Eur J Paediatr Neurol.2003；7(4):155–9.
[5]O’Hagen JM,Glanzman AM,McDermott MP,Ryan PA,Flickinger J,Quigley J,Riley S,Sanborn E,Irvine C,Martens WB,et al.An expanded version of the Hammersmith Functional Motor Scale for SMA II and III patients.Neuromuscul Disord.2007；17(9-10):693–7.
[6]Mercuri E,Messina S,Battini R,Berardinelli A,Boffi P,Bono R,Bruno C,Carboni N,Cini C,Colitto F,et al.Reliability of the Hammersmith functional motor scale for spinal muscular atrophy in a multicentric study.Neuromuscul Disord.2006；16(2):93–8.
[7]Ke11y JJ,Thibodeau L,Andros PA.Use of electrophysiological tests to measure disease progression in ALS therape utic trials[J].Muscle Nerv e,1990,13(3):471.
[8]Glanzman AM,Mazzone E,Main M,Pelliccioni M,Wood J,Swoboda KJ,et al.The Children’s Hospital of Philadelphia Infant Test of Neuromuscular Disorders(CHOP INTEND):test development and reliability.Neuromuscul Disord 2010；20:155-161.
[9]Sithara Ramdas,Laurent Servais.New treatments in spinal muscular atrophy:an overview of currently available data.Expert Opin Pharmacother.2020 Feb；21(3):307-315.
[10]Linda P.Lowes,Lindsay N.Alfano,W.David et al.Impact of Age and Motor Function in a Phase 1/2A Study of Infants with SMA Type 1 Receiving Single-Dose Gene Replacement Therapy.Pediatr Neurol.2019 Sep；98:39-45.
[11]Lenardo M J,Baltimore D.NF-κB:A pleiotropic mediator of inducible and tissue-specific gene control[J].Cell,1989,58(2):227-229.
[12]Aloe L,Rocco M L,Bianchi P,et al.Nerve growth factor:from the early discoveries to the potential clinical use[J].Journal of Translational Medicine,2012,10(1).

Claims (15)

  1. 一种治疗脊髓性肌萎缩症(SMA)的方法，包括给药患脊髓性肌萎缩症(SMA)的受试者治疗有效量的纤溶酶原途径激活剂。
  2. 权利要求1的方法，其中所述纤溶酶原途径激活剂促进SMN基因的转录和/或表达。
  3. 权利要求1或2的方法，其中所述纤溶酶原途径激活剂改善受试者的肌力。
  4. 权利要求1-3任一项的方法，其中所述纤溶酶原途径激活剂延长受试者生存期。
  5. 权利要求1-4任一项的方法，其中所述纤溶酶原途径激活剂改善受试者的肌张力。
  6. 权利要求1-5任一项的方法，其中所述纤溶酶原途径激活剂促进受试者
    Figure PCTCN2020129461-appb-100001
    蛋白的表达。
  7. 权利要求1-6任一项的方法，其中所述纤溶酶原途径激活剂促进受试者生长发育。
  8. 权利要求1-7任一项的方法，其中所述纤溶酶原途径激活剂与一种或多种其它药物或治疗方法联合施用。
  9. 权利要求1-8任一项的方法，其中所述纤溶酶原途径激活剂通过静脉内、肌肉内、鞘内、鼻腔吸入、雾化吸入、滴鼻液或滴眼液形式给药。
  10. 权利要求1-9任一项的方法，其中所述纤溶酶原途径激活剂为纤溶酶原激活途径的组分。
  11. 权利要求1-10任一项的方法，所述纤溶酶原途径激活剂为纤溶酶原。
  12. 权利要求11的方法，其中所述纤溶酶原包含与序列2、6、8、10或12所示氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，并且具有纤溶酶原活性。
  13. 权利要求11的方法，所述纤溶酶原为包含纤溶酶原活性片段、并且具有纤溶酶原活性的蛋白质。
  14. 权利要求11的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活 性的变体。
  15. 权利要求11的方法，所述纤溶酶原包含序列2、6、8、10或12所示氨基酸序列。
PCT/CN2020/129461 2020-05-11 2020-11-17 一种治疗脊髓性肌萎缩症的方法和药物 WO2021227417A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA3182911A CA3182911A1 (en) 2020-05-11 2020-11-17 Method and drug for treating spinal muscular atrophy
KR1020227041422A KR20230005298A (ko) 2020-05-11 2020-11-17 척수성 근위축증의 치료 방법 및 약물
CN202080099318.3A CN115697385A (zh) 2020-05-11 2020-11-17 一种治疗脊髓性肌萎缩症的方法和药物
US17/924,617 US20230181699A1 (en) 2020-05-11 2020-11-17 Method and drug for treating spinal muscular atrophy
JP2022567202A JP2023525257A (ja) 2020-05-11 2020-11-17 脊髄性筋萎縮症を治療する方法及び薬剤
EP20935975.1A EP4140498A4 (en) 2020-05-11 2020-11-17 METHOD AND MEDICINE FOR THE TREATMENT OF SPINAL AMYOTROPHY

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNPCT/CN2020/089631 2020-05-11
CN2020089631 2020-05-11

Publications (1)

Publication Number Publication Date
WO2021227417A1 true WO2021227417A1 (zh) 2021-11-18

Family

ID=78525998

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2020/129461 WO2021227417A1 (zh) 2020-05-11 2020-11-17 一种治疗脊髓性肌萎缩症的方法和药物
PCT/CN2021/093039 WO2021228086A1 (zh) 2020-05-11 2021-05-11 一种治疗脊髓性肌萎缩症的方法和药物

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/093039 WO2021228086A1 (zh) 2020-05-11 2021-05-11 一种治疗脊髓性肌萎缩症的方法和药物

Country Status (8)

Country Link
US (2) US20230181699A1 (zh)
EP (2) EP4140498A4 (zh)
JP (2) JP2023525257A (zh)
KR (2) KR20230005298A (zh)
CN (2) CN115697385A (zh)
CA (2) CA3182911A1 (zh)
TW (2) TW202142256A (zh)
WO (2) WO2021227417A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3182911A1 (en) * 2020-05-11 2021-11-18 Talengen International Limited Method and drug for treating spinal muscular atrophy

Citations (5)

* 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
EP0058481A1 (en) 1981-02-16 1982-08-25 Zeneca Limited Continuous release pharmaceutical compositions
CN101384256A (zh) * 2006-02-27 2009-03-11 惠氏公司 用于治疗肌肉病状的pai-1抑制剂
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
CN108210917A (zh) * 2016-12-15 2018-06-29 深圳瑞健生命科学研究院有限公司 一种预防和治疗肥胖症的方法和药物

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020165147A1 (en) * 1996-10-11 2002-11-07 Manuel Yepes Brain-associated inhibitor of tissue-type plasminogen activator
US6372473B1 (en) * 1997-05-28 2002-04-16 Human Genome Sciences, Inc. Tissue plasminogen activator-like protease
US20030219431A1 (en) * 2002-05-24 2003-11-27 Empire Pharmaceuticals, Inc. Treatment of neuronal and neurological damage associated with spinal cord injury
ES2534744T3 (es) * 2004-04-22 2015-04-28 Grifols Therapeutics Inc. Plasmina modificada de forma recombinante
CA2707266C (en) * 2007-11-29 2013-12-31 Talecris Biotherapeutics, Inc. Recombinantly modified plasmin
WO2009146178A1 (en) * 2008-04-15 2009-12-03 President And Fellows Of Harvard College Angiogenin and amyotrophic lateral sclerosis
JP6118314B2 (ja) * 2011-05-04 2017-04-19 ライゼン・ファーマシューティカルズ・エスアー タンパク質キナーゼのモジュレーターとしての新規化合物
US20140212405A1 (en) * 2013-01-28 2014-07-31 2294719 Ontario Limited Fibrinolytic/Proteolytic Treatment of Myofacial and Neuropathic Pain and Related Conditions
ES2961967T3 (es) * 2015-12-18 2024-03-14 Talengen Int Ltd Plasminógeno para su uso en el tratamiento de la angiocardiopatía diabética
CA3182911A1 (en) * 2020-05-11 2021-11-18 Talengen International Limited Method and drug for treating spinal muscular atrophy

Patent Citations (5)

* 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
EP0058481A1 (en) 1981-02-16 1982-08-25 Zeneca Limited Continuous release pharmaceutical compositions
CN101384256A (zh) * 2006-02-27 2009-03-11 惠氏公司 用于治疗肌肉病状的pai-1抑制剂
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
CN108210917A (zh) * 2016-12-15 2018-06-29 深圳瑞健生命科学研究院有限公司 一种预防和治疗肥胖症的方法和药物

Non-Patent Citations (39)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1980
AISINA R BMUKHAMETOVA L I: "Structure and function of plasminogen/plasmin system[J", RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY, vol. 40, no. 6, 2014, pages 590 - 605, XP055713150, DOI: 10.1134/S1068162014060028
ALOE LROCCO M LBIANCHI P ET AL.: "Nerve growth factor: from the early discoveries to the potential clinical use[J", JOURNAL OF TRANSLATIONAL MEDICINE, vol. 10, no. 1, 2012, XP021134204, DOI: 10.1186/1479-5876-10-239
BARANY AND SOLID-PHASE PEPTIDE SYNTHESIS, pages 3 - 284
C. L. LORSON, H. RINDT, M. SHABABI: "Spinal muscular atrophy: mechanisms and therapeutic strategies", HUMAN MOLECULAR GENETICS, vol. 19, no. R1, 15 April 2010 (2010-04-15), GB , pages R111 - R118, XP055340572, ISSN: 0964-6906, DOI: 10.1093/hmg/ddq147 *
CAMARERO JA ET AL., PROTEIN PEPT LETT., vol. 12, 2005, pages 723 - 8
CO ET AL., J. IMMUNOL., vol. 148, 1992, pages 1149
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
GLANZMAN AMMAZZONE EMAIN MPELLICCIONI MWOOD JSWOBODA KJ ET AL.: "The Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND): test development and reliability", NEUROMUSCUL DISORD, vol. 20, 2010, pages 155 - 161, XP026965893
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
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
KELLY JJTHIBODEAU LANDROS PA: "Use of electrophysiological tests to measure disease progression in ALS therape utic trials[J", MUSCLE NERV E, vol. 13, no. 3, 1990, pages 471
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
LANGER ET AL., J. BIOMED. MATER. RES., vol. 15, 1981, pages 167 - 277
LANGER, CHEM. TECH., vol. 12, 1982, pages 98 - 105
LENARDO M JBALTIMORE D.: "NF-xB: A pleiotropic mediator of inducible and tissue-specific gene control[J", CELL, vol. 58, no. 2, 1989, pages 227 - 229, XP024244616, DOI: 10.1016/0092-8674(89)90833-7
LINDA P. LOWESLINDSAY N. ALFANOW. DAVID ET AL.: "Impact of Age and Motor Function in a Phase 1/2A Study of Infants with SMA Type 1 Receiving Single-Dose Gene Replacement Therapy", PEDIATR NEUROL., vol. 98, September 2019 (2019-09-01), pages 39 - 45, XP055622028, DOI: 10.1016/j.pediatrneurol.2019.05.005
MAIN MKAIRON HMERCURI EMUNTONI F: "The Hammersmith functional motor scale for children with spinal muscular atrophy: a scale to test ability and monitor progress in children with limited ambulation", EUR J PAEDIATR NEUROL., vol. 7, no. 4, 2003, pages 155 - 9
MERCURI EMESSINA SBATTINI RBERARDINELLI ABOFFI PBONO RBRUNO CCARBONI NCINI CCOLITTO F ET AL.: "Reliability of the Hammersmith functional motor scale for spinal muscular atrophy in a multicentric study", NEUROMUSCUL DISORD., vol. 16, no. 2, 2006, pages 93 - 8, XP025107866, DOI: 10.1016/j.nmd.2005.11.010
MERRIFIELD ET AL., J. AM. CHEM. SOC., vol. 85, 1963, pages 2149 - 2156
NADERI JASMIN, BERNREUTHER CHRISTIAN, GRABINSKI NICOLE, PUTMAN CHARLES T., HENKEL BIRGIT, BELL GORDON, GLATZEL MARKUS, SULTAN KARI: "Plasminogen Activator Inhibitor Type 1 Up-Regulation Is Associated with Skeletal Muscle Atrophy and Associated Fibrosis", THE AMERICAN JOURNAL OF PATHOLOGY, vol. 175, no. 2, 1 August 2009 (2009-08-01), US , pages 763 - 771, XP055866382, ISSN: 0002-9440, DOI: 10.2353/ajpath.2009.081009 *
NY, A.LEONARDSSON, G.HAGGLUND, A.CHAGGLOF, PPLOPLIS, V.A.CARMELIET, P.NY, T.: "Ovulation inplasminogen-deficient mice", ENDOCRINOLOGY, vol. 140, 1999, pages 5030 - 5035
O'HAGEN JMGLANZMAN AMMCDERMOTT MPRYAN PAFLICKINGER JQUIGLEY JRILEY SSANBOM EIRVINE CMARTENS WB ET AL.: "An expanded version of the Hammersmith Functional Motor Scale for SMA II and III patients", NEUROMUSCUL DISORD., vol. 17, no. 9-10, 2007, pages 693 - 7, XP022325966, DOI: 10.1016/j.nmd.2007.05.009
QUEEN ET AL., IMMUNOL. REV., vol. 89, 1986, pages 49
See also references of EP4140498A4
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 - 3 3
SITHARA RAMDASLAURENT SERVAIS: "New treatments in spinal muscular atrophy: an overview of currently available data", EXPERT OPIN PHARMACOTHER., vol. 21, no. 3, February 2020 (2020-02-01), pages 307 - 315, XP055891962, DOI: 10.1080/14656566.2019.1704732
SPECIAL METHODS IN PEPTIDE SYNTHESIS
SPROULE DOUGLAS M., KAUFMANN PETRA: "Therapeutic developments in spinal muscular atrophy", THERAPEUTIC ADVANCES IN NEUROLOGICAL DISORDERS MAY 2010, vol. 3, no. 3, 1 May 2010 (2010-05-01), pages 173 - 185, XP055866388, ISSN: 1756-2864, DOI: 10.1177/1756285610369026 *
STEWART ET AL.: "Solid Phase Peptide Synthesis", 1984, PIERCE CHEM. CO.
SUGARMAN EANAGAN NZHU HAKMAEV VRZHOU ZROHLFS EMFLYNN KHENDRICKSON BCSCHOLL TSIRKO-OSADSA DA: "Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy: clinical laboratory analysis of >72,400 specimens", EUR J HUM GENET., vol. 20, no. 1, January 2012 (2012-01-01), pages 27 - 32
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
TALBOTTIZZANO, GENE THER, vol. 24, no. 9, 2017, pages 529 - 533
THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY., vol. 2
VERHAART IECROBERTSON AWILSON IJAARTSMA-RUS ACAMERON SJONES CCCOOK SFLOCHMULLER H: "Prevalence, incidence and carrier frequency of 5q-linked spinal muscular atrophy - a literature review", ORPHANET J RARE DIS., vol. 12, no. 1, 4 July 2017 (2017-07-04), pages 124
WINNACKER: "From Genes to Clones", 1987, VCH PUBLISHERS
ZOU TIE, LING CHANGCHUN, XIAO YAO, TAO XIANMEI, MA DUAN, CHEN ZU-LIN, STRICKLAND SIDNEY, SONG HOUYAN: "Exogenous Tissue Plasminogen Activator Enhances Peripheral Nerve Regeneration and Functional Recovery After Injury In Mice : ", JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY, vol. 65, no. 1, 1 January 2006 (2006-01-01), NEW YORK, NY. , pages 78 - 86, XP055866385, ISSN: 0022-3069, DOI: 10.1097/01.jnen.0000195942.25163.f5 *

Also Published As

Publication number Publication date
KR20230005298A (ko) 2023-01-09
TW202142256A (zh) 2021-11-16
TWI811675B (zh) 2023-08-11
JP2023525256A (ja) 2023-06-15
CA3182911A1 (en) 2021-11-18
US20230190891A1 (en) 2023-06-22
WO2021228086A1 (zh) 2021-11-18
EP4140498A4 (en) 2023-11-01
KR20230004752A (ko) 2023-01-06
JP2023525257A (ja) 2023-06-15
EP4144364A4 (en) 2023-11-22
CN115697385A (zh) 2023-02-03
CA3183106A1 (en) 2021-11-18
CN115551535A (zh) 2022-12-30
TW202200192A (zh) 2022-01-01
EP4144364A1 (en) 2023-03-08
US20230181699A1 (en) 2023-06-15
EP4140498A1 (en) 2023-03-01

Similar Documents

Publication Publication Date Title
TWI714862B (zh) 一種調控glp-1/glp-1r的方法和藥物
CN116669757A (zh) 一种提高bdnf水平的方法和药物
WO2021143906A1 (zh) 一种治疗神经损伤及其相关病症的方法
WO2021227417A1 (zh) 一种治疗脊髓性肌萎缩症的方法和药物
WO2021190558A1 (zh) 一种治疗阿尔茨海默病的方法和药物
TWI787732B (zh) 一種預防和治療多發性硬化症的方法和藥物
TWI741597B (zh) 一種治療肌萎縮側索硬化的方法和藥物
WO2021190563A1 (zh) 一种治疗亨廷顿病的方法和药物
WO2021190561A1 (zh) 一种治疗帕金森病的方法和药物
WO2020228681A1 (zh) 一种治疗肌萎缩侧索硬化的方法和药物
TW202408566A (zh) 一種治療神經損傷及其相關病症的方法
TW202228766A (zh) 一種治療神經損傷及其相關病症的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20935975

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022567202

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 3182911

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 20227041422

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020935975

Country of ref document: EP

Effective date: 20221125

NENP Non-entry into the national phase

Ref country code: DE