Patent Application
20230190891
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 794922003200SEQLIST.TXT, date recorded: Nov. 10, 2022, size: 47,536 bytes).
The present application relates to a method for treating spinal muscular atrophy (SMA) and related disorders, comprising administering an effective amount of a component of the plasminogen activation pathway or a related compound thereof, such as plasminogen, to a subject suffering from spinal muscular atrophy (SMA) and related disorders, to repair injured nerves and improve clinical symptoms and signs.
Spinal muscular atrophy (SMA) is a disease of myasthenia and amyotrophy caused by degeneration of motor neurons in anterior horn of spinal cord. It belongs to autosomal recessive hereditary disease. The most common form of SMA is caused by mutations in the survival motor neuron (SMN) gene, and infantile SMA is the most severe form of the neurodegenerative disorder. The symptoms include muscle weakness, hypotonia, weak crying, limping or tendency to fall, difficulty in sucking or swallowing, accumulation of secretions in the lung or throat, difficulty in eating, and susceptibility to respiratory infections. Legs are often weaker than arms and fail to achieve developmental markers, such as raising head or sitting up. Generally, the earlier the onset of symptoms, the shorter the lifespan.
The progression of SMA is directly related to the degradation rate of motor neuron cells and degree of the resulting weakness. Infants with the severe forms of SMA often die of respiratory diseases due to respiratory muscle weakness. Children with milder forms of SMA survive longer, but they may need extensive medical support.
Spinal muscular atrophy is classified into 5 subtypes according to the age of onset of the patients and the severity of the disease.
Type 0 patients: generally more common in the fetus or neonate, the onset in the fetal period is manifested as decreased fetal movement, and the neonate manifests as loss of muscle reflexes, facial paralysis, atrial septal defect and joint contracture, the most serious manifestation is respiratory failure, the life expectancy of sick children is greatly shortened, and most survival time is within 6 months.
Type I patients: the infantile type, also known as Werdnig-Hoffman disease, which accounts for 50% of SMA patients, the patients present with hypotonia, poor head control, and diminished or absent tendon reflexes within 6 months after birth; severe hypotonia manifests as “frog legs” when lying down, lack of head control, inability to sit upright, weak intercostal muscles, and relatively small diaphragm muscles; patients often suffer from impaired swallowing function and respiratory failure due to respiratory muscle weakness. In absence of assisted ventilation, 92% of children with type I SMA usually die from respiratory failure before 20 months old.
Type II patients: intermediate type, accounting for about 20% of SMA patients, it usually occurs within 6-18 months after birth, patients can sit alone at some stage of development, but cannot walk independently; such patients often suffer from complications such as scoliosis, joint contractures, and ankylosis of the mandibular joint; scoliosis and intercostal muscle weakness often lead to severe lung disease, and the cognitive ability of these children is normal.
Type III patients: the juvenile type (also known as Kugelberg-Welander disease), accounts for about 30% of SMA patients, and the disease usually occurs within 18 months to 5 years after birth; the patients can walk with the help of adminicle support; unlike type II SMA, most of these patients do not have complications such as scoliosis and respiratory muscle weakness, and the cognition and life expectancy of this population are generally not affected by the disease.
Type IV patients: occurring after adolescence, the exercise capacity of the patients is gradually decreased; and those patients account for approximately 5% of the total number of SMA patients; similar to type III, but with onset in adulthood; it is generally believed that the disease occurs at the age of 30 or later.
SMA is caused by inactivating mutations or deletions of the telomere copies of a gene (SMN1) on both chromosomes, resulting in loss of function of the SMN1 gene. The 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 identical to SMN1 except for a single mutation that functions in the splicing of RNA messages. All SMA patients retain a centromeric copy of the gene (SMN2), and the number of copies of the SMN2 gene in SMA patients is generally inversely correlated with disease severity, i.e. patients with less severe SMA have more copies of SMN2. Nonetheless, SMN2 cannot fully compensate for the loss of SMN1 function due to alternative splicing of exon 7 caused by a translationally silent C to T mutation in exon 7. Thus, the majority of transcripts produced by SMN2 lack exon 7 (A7 SMN2) and encode SMN proteins that have impaired function and are rapidly degraded to truncated form. The cause of SMA is SMN1 gene deletion, and the SMN2 gene in vivo is used for compensating for function of the SMN1 gene. A difference between the SMN2 gene and the SMN1 gene is that exon 7 develops a C to T mutation. Most of the SMN2 gene with such mutation produces truncated SMN gene and SMN protein, and about 10% of full length correct SMN gene and SMN protein. Although the truncated SMN protein can also exert a function similar to that of the full-length SMN protein, it has a short half-life and is degraded rapidly.
The number of copies of SMN2 varies from person to person. In a case of SMN1 gene deletion, the number of copies of SMN2 gene in the body basically determines whether an individual suffers from type I SMA (the number of copies of SMN2 is 2), SMA type II (the number of copies of SMN2 is 3) or SMA type III (the number of copies of SMN2 is 4). Therefore, even addition of the truncated SMN gene and protein plays an important role in reducing the severity of the disease.
Clinically, SMA is usually diagnosed by clinical symptoms combined with a test for at least one copy of the SMN1 gene. In some cases, other tests such as electromyography (EMG) or muscle biopsy can also aid in the diagnosis when the SMN1 gene test shows no abnormalities. So far, the treatment of SMA has been limited to supportive care, including treatment and care for breathing, nutrition, and rehabilitation, and there are no drugs that can effectively treat the disease.
The present study found that plasminogen pathway activators such as plasminogen can significantly improve the symptoms of nerve injury in SMA subjects, improve lung function, prolong survival, promote transcription and expression of the SMN gene, and increase the level of SMN protein in brain tissue and muscle tissue, promote the expression of transcription factor such as NF-κB protein in brain tissue and muscle tissue, promote the formation of mature NGF in brain tissue, improve lung tissue injury, so as to effectively prevent and treat SMA.
In an aspect, the present application 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), which includes: administering a therapeutically effective amount of a plasminogen pathway activator to a subject with motor neuron disease such as spinal muscular atrophy (SMA), and the plasminogen pathway activator is one or more of: a component of plasminogen activation pathway, a compound directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of plasminogen activation pathway, a compound mimicking the activity of plasminogen or plasmin, a compound upregulating the expression of plasminogen or a activator of plasminogen, a plasminogen analog, a plasmin analog, a tPA or uPA analog, and an antagonist of fibrinolysis inhibitor.
In some particular embodiments, for a subject with spinal muscular atrophy (SMA), including type 0, type I, type II, type III, type IV and non-5q SMA, the plasminogen pathway activator has one or more activities selected from the group consisting of: 1. reducing or alleviating the severity of SMA; 2. delaying the onset of SMA; 3. inhibiting the progression of SMA; 4. extending the survival time of the subjects; 5. improving the quality of life of the subjects and/or improving the mental state of the subjects; 6. reducing the number of SMA-related symptoms; 7. reducing or alleviating the severity of one or more symptoms associated with SMA; 8. reducing the duration of symptoms associated with SMA; 9. preventing recurrence of symptoms associated with SMA; 10. inhibiting the development or onset of SMA symptoms; 11. inhibiting the progression of symptoms associated with SMA; 12. improving lung function; 13. improving blood oxygen saturation; 14. promoting transcription and expression of SMN gene (including: 1. promoting transcription and/or expression of the truncated SMN2 gene; or 2. promoting transcription and/or expression of the full-length SMN gene); 15. increasing the level of SMN protein in brain tissue and muscle tissue; 16. promoting expression of transcription factors, such as NF-κB protein, in brain tissue and muscle tissue; 17. promoting formation of mature NGF in brain tissue; 18. alleviating lung tissue damage; 19. increasing muscle strength; 20. reducing muscle atrophy; 21. Reducing loss of motor neurons; 22. promoting growth and development; and/or 23. improving motor function. In some particular embodiments, the plasminogen pathway activator improves muscle atrophy, increases muscle strength, and/or improves muscle tone of the subject. In some particular embodiments, the plasminogen pathway activator improves one or more of the following conditions of the subject: muscle strength, muscle tone, motor function, respiratory function, and muscle atrophy. In some particular embodiments, the plasminogen pathway activator prolongs the survival time of the subject. In some particular embodiments, the plasminogen pathway activator promotes transcription and/or expression of the SMN gene (including: 1. promoting transcription and/or expression of the truncated SMN2 gene; or 2. promoting transcription and/or expression of the full-length SMN gene). In some particular embodiments, the plasminogen pathway activator promotes recovery of the muscle function of the subject. In some particular embodiments, the plasminogen pathway activator promotes repair of neuron injury in anterior horn of spinal cord in the subjects. In some particular embodiments, the plasminogen pathway activator promotes expression of a transcription factor such as the NF-κB protein in the subject, such as expression of a transcription factor such as the NF-κB protein in brain or spinal cord tissue. In some particular embodiments, the plasminogen pathway activator promotes formation of the mature NGF in the subjects. The plasminogen pathway activator promotes formation of the mature NGF in the subjects.
In some embodiments, this application relates to a method for treating SMA, comprising administering a therapeutically effective amount of plasminogen to SMA subjects. The plasminogen has one or more of the following activities (effects): 1. promoting transcription and/or expression of the truncated SMN2 gene; and 2. promoting transcription and/or expression of the full-length SMN gene.
In some embodiments, the plasminogen pathway activator further has one or more of the following effects:
1) promoting penetration of plasminogen through the blood-brain barrier and the blood-spinal cord barrier,
2) promoting aggregation of plasminogen to brain and spinal cord tissue of SMA subjects,
3) promoting aggregation of plasminogen in an injured tissue (e.g., central nervous tissue (brain and spinal cord), lung, and muscle tissue) of SMA subjects,
4) increasing level of plasminogen in brain and spinal cord of SMA subjects,
5) increasing level of plasminogen in partially injured tissue (e.g., central nervous tissue (brain and spinal cord), lung, and muscle tissue) of SMA subjects,
6) alleviating damage to injured tissue (e.g., central nervous tissue (brain and spinal cord), lung, and muscle tissue) of SMA subjects,
7) promoting repair of inflammation of injured tissue (e.g., central nervous tissue (brain and spinal cord), lung, and muscle tissue) of SMA subjects,
8) promoting transcription of SMNΔ7 in brain and spinal cord of SMA subjects,
9) increasing level of SMN protein (including: 1. the truncated SMN2 protein; or 2. the full-length SMN protein) in brain and spinal cord of SMA subjects,
10) promoting expression of NGF in brain and spinal cord of SMA subjects, and
11) promoting growth and development of SMA subjects.
In some particular embodiments, the plasminogen pathway activator is administered in combination with one or more other medicaments and/or therapies, preferably, the therapies include cell therapy (e.g., stem cell therapy) and gene therapy, such as antisense RNA, small molecule splicing modifiers.
In some embodiments, the plasminogen pathway activator is a component of a plasminogen activation pathway.
In some embodiments, the component of the plasminogen activation pathway is selected from the group consisting of: plasminogen, recombinant human plasmin, Lys-plasminogen, Glu-plasminogen, plasmin, a variant of plasminogen and plasmin and the analog thereof comprising one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, an activator of plasminogen, tPA, and uPA. In some particular embodiments, the antagonist of a fibrinolysis inhibitor is an antagonist of PAI-1, complement C1 inhibitor, α2 anti-plasmin or an α2 macroglobulin, such as an antibody of PAI-1, complement C1 inhibitor, α2 anti-plasmin or α2 macroglobulin.
In some particular embodiments, the component of the plasminogen activation pathway is plasminogen. In some particular embodiments, the plasminogen comprises or has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 2, 6, 8, 10 or 12, and has plasminogen activity, such as proteolytic activity or lysine binding activity. In some embodiments, the plasminogen is human full-length plasminogen or a conservatively substituted variant thereof. In some embodiments, the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 2, and has lysine binding activity or proteolytic activity of plasminogen. In some embodiments, the plasminogen is a protein comprising an amino acid sequence having at 80%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO:14, and has the proteolytic activity of plasminogen. In some embodiments, the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogen, and variants thereof retaining the proteolytic activity of plasminogen. In some embodiments, the plasminogen comprises an amino acid sequence represented by SEQ ID NO: 2, 6, 8, 10 or 12, or comprises a conservatively substituted variant of the amino acid sequence represented by SEQ ID NO: 2, 6, 8, 10 or 12.
In some embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In some embodiments, the plasminogen activity is the lysine binding activity of plasminogen to a substrate molecule. In some embodiments, the plasminogen activity is the proteolytic activity of plasminogen and the lysine binding activity of plasminogen to a substrate molecule. In some embodiments, the plasminogen is a protein having an amino acid sequence with addition, deletion and/or substitution of 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, or 1 amino acid based on the sequence represented by SEQ ID NO: 2, 6, 8, 10 or 12, and having proteolytic activity and/or lysine binding activity of plasminogen. In some particular embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In some particular embodiments, the plasminogen is a protein comprising a plasminogen active fragment and having the proteolytic activity and/or lysine binding activity of plasminogen. In some embodiments, the plasminogen active fragment comprises or has a serine protease domain of plasminogen or a plasminogen protease domain. In some particular embodiments, the amino acid sequence of the plasminogen active fragment is represented by SEQ ID NO: 14. In some particular embodiments, the plasminogen is selected from the group consisting of: Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (human full-length plasminogen cleaved between amino acids 76-77), small plasminogen (containing Kringle 5 (K5) and serine protease domain), micro-plasminogen (containing serine protease domains), delta-plasminogen (containing Kringle 1 and serine protease domain), or a variant thereof retaining plasminogen activity. In some embodiments, the plasminogen is human full-length plasminogen, or a variant or fragment thereof still retaining plasminogen activity. In some embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In some embodiments, the plasminogen activity is the lysine binding activity of plasminogen to a substrate molecule. In some embodiments, the plasminogen activity is the proteolytic activity of plasminogen and the lysine binding activity of plasminogen to a substrate molecule. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or rodent, or a variant or fragment thereof still retaining the proteolytic activity and/or lysine binding activity of plasminogen. In some embodiments, the plasminogen comprises the amino acid sequence represented by SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human native plasminogen.
In some particular embodiments, the plasminogen pathway activator is administered systemically or locally, e.g., by intravenous administration, intramuscular administration, intrathecal administration, nasal inhalation, aerosol inhalation, nasal or eye drops. In some embodiments, the subject is a human. In some embodiments, the subject is lack of or deficient in plasminogen. In some embodiments, the lack or deficiency is congenital, secondary and/or local. In some embodiments, the plasminogen is administered at a dose of 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg, 10-100 mg/kg (calculated by per kilogram of body weight); or at a dose of 0.0001-2000 mg/cm2, 0.001-800 mg/cm2, 0.01-600 mg/cm2, 0.1-400 mg/cm2, 1-200 mg/cm2, 1-100 mg/cm2, 10-100 mg/cm2 (calculated by per square centimeter of body surface area) every day, every two days, or every three days continuously.
In some embodiments, the above SMA is type 0, type I, type II, type III, type IV or non-5q SMA.
In one aspect, the application also relates to a pharmaceutical composition, medicament, preparation, kit, or product for treating spinal muscular atrophy (SMA), comprising the above mentioned plasminogen pathway activator, such as a component of the plasminogen activation pathway (e.g., plasminogen) as described above.
In some embodiments, the pharmaceutical composition, medicament, formulation comprises a pharmaceutically acceptable carrier and a plasminogen pathway activator, e.g., a component of the plasminogen activation pathway described above, such as plasminogen described above. In some embodiments, the kit or product comprises one or more containers containing the pharmaceutical composition, medicament or formulation. In some embodiments, the kit or product further comprises a label or instructions for use indicating the method for using a plasminogen pathway activator, e.g., a component of the plasminogen activation pathway described above, such as a method for treating spinal muscular atrophy with plasminogen described above. In some embodiments, the kit or product further comprises another one or more additional containers containing one or more other medicaments. In some embodiments, the above-mentioned SMA is type 0, type I, type II, type III, type IV or non-5q SMA.
In one aspect the present application also relates to a plasminogen pathway activator as described above, such as plasminogen as described above, for use in the treatment of spinal muscular atrophy (SMA). In some embodiments, the above-mentioned SMA is type 0, type I, type II, type III, type IV or non-5q SMA.
In one aspect, the present application also relates to use of a plasminogen pathway activator as described above, such as plasminogen as described above for treating spinal muscular atrophy (SMA). In some embodiments, the above-mentioned SMA is type 0, type I, type II, type III, type IV or non-5q SMA.
In one aspect, the present application also relates to use of a therapeutically effective amount of the above plasminogen pathway activator (e.g., a component of the plasminogen activation pathway described above, such as the plasminogen described above) in the preparation of a pharmaceutical composition, medicament, preparation, kit, or product for treating spinal muscular atrophy (SMA).
In some embodiments, the plasminogen pathway activator is selected from one or more of the following: a component of plasminogen activation pathway, a compound directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of plasminogen activation pathway, a compound mimicking the activity of plasminogen or plasmin, a compound upregulating the expression of plasminogen or an activator of plasminogen, an analog of plasminogen, an analog of plasmin, an analog of tPA or uPA, and an antagonist of fibrinolysis inhibitor.
In some particular embodiments, the component of plasminogen activation pathway is selected from the group consisting of: plasminogen, recombinant human plasmin, Lys-plasminogen, Glu-plasminogen, plasmin, a variant of plasminogen and plasmin and the analog thereof comprising one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, an activator of plasminogen, tPA and uPA. In some particular embodiments, the antagonist of the fibrinolysis inhibitor is an antagonist of PAI-1, complement C1 inhibitor, α2 antiplasmin, or α2 macroglobulin, e.g., an antibody of PAI-1, complement C1 inhibitor, α2 anti-plasmin, or α2 macroglobulin.
In some embodiments, the plasminogen pathway activator is a component of a plasminogen activation pathway.
In some embodiments, the components of the plasminogen activation pathway are selected from the group consisting of: plasminogen, recombinant human plasmin, Lys-plasminogen, Glu-plasminogen, plasmin, a variant of plasminogen and plasmin and the analog thereof comprising one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activator, tPA and uPA. In some particular embodiments, the antagonist of the fibrinolysis inhibitor is an antagonist of PAI-1, complement C1 inhibitor, α2 antiplasmin, or α2 macroglobulin, e.g., an antibody of PAI-1, complement C1 inhibitor, α2 anti-plasmin, or α2 macroglobulin.
In some particular embodiments, the component of the plasminogen activation pathway is plasminogen. In some particular embodiments, the plasminogen comprises or has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 2, 6, 8, 10 or 12, and has plasminogen activity. In some embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In some embodiments, the plasminogen activity is the lysine binding activity of plasminogen to a substrate molecule. In some embodiments, the plasminogen activity is the proteolytic activity of plasminogen and the lysine binding activity of plasminogen to a substrate molecule. In some embodiments, the plasminogen is a protein having an amino acid sequence with addition, deletion and/or substitution of 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, or 1 amino acid based on the sequence represented by SEQ ID NO: 2, 6, 8, 10 or 12, and having proteolytic activity and/or lysine binding activity of plasminogen. In some particular embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In some particular embodiments, the plasminogen is a protein comprising a plasminogen active fragment and having the proteolytic activity and/or lysine binding activity of plasminogen. In some embodiments, the plasminogen active fragment comprises or has a serine protease domain of plasminogen or a plasminogen protease domain. In some particular embodiments, the amino acid sequence of the plasminogen active fragment is represented by SEQ ID NO: 14. In some particular embodiments, the plasminogen is selected from the group consisting of: Glu-plasminogen (human full-length plasminogen), Lys-plasminogen (human full-length plasminogen cleaved between amino acids 76-77), small plasminogen (containing Kringle 5 (K5) and serine protease domain), micro-plasminogen (containing serine protease domains), delta-plasminogen (containing Kringle 1 and serine protease domain), or a variant thereof retaining plasminogen activity. In some embodiments, the plasminogen is human full-length plasminogen, or a variant or fragment thereof still retaining plasminogen activity. In some embodiments, the plasminogen activity is the proteolytic activity of plasminogen. In some embodiments, the plasminogen activity is the lysine binding activity of plasminogen to a substrate molecule. In some embodiments, the plasminogen activity is the proteolytic activity of plasminogen and the lysine binding activity of plasminogen to a substrate molecule. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or rodent, or a variant or fragment thereof still retaining the proteolytic activity and/or lysine binding activity of plasminogen. In some embodiments, the plasminogen comprises the amino acid sequence represented by SEQ ID NO: 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human native plasminogen.
In some embodiments, the plasminogen pathway activator, e.g., a component of the plasminogen activation pathway described above, such as plasminogen described above, is administered in combination with one or more other medicaments and/or therapies. In some embodiments, the plasminogen pathway activator, e.g., a component of the plasminogen activating pathway, such as plasminogen, is administered by intravenous administration, intramuscular administration, intrathecal administration, nasal inhalation, aerosol inhalation, nasal or eye drops.
In some embodiments, the pharmaceutical composition, medicament, formulation comprises a pharmaceutically acceptable carrier and a plasminogen pathway activator, e.g., a component of the plasminogen activating pathway, such as plasminogen. In some embodiments, the kit or product comprises one or more containers containing the pharmaceutical composition, medicament or formulation. In some embodiments, the kit or product further comprises a label or instructions for use indicating the method for using a plasminogen pathway activator, e.g., a component of the plasminogen activation pathway, such as plasminogen to treat spinal muscular atrophy.
In some embodiments, the kit or the product further comprises one or more additional containers in containing one or more other medicaments.
In some embodiments, the above mentioned SMA is type 0, type I, type II, type III, type IV or non-5q SMA.
The present application explicitly encompasses all the combinations of the technical features belonging to the embodiments of the present application, and these combined technical solutions have been explicitly disclosed in this application, just as the separately and explicitly disclosed above technical solutions. In addition, the present application also explicitly encompasses the combinations of each embodiment and its elements, and the combined technical solutions are explicitly disclosed herein.
The term “spinal muscular atrophy” (SMA) refers to a disease caused by inactivating mutations or deletions of the SMN1 gene on both chromosomes, resulting in loss of function of the SMN1 gene. Symptoms of SMA include muscle weakness, hypotonia, weak crying, weak coughing, limping or tendency to fall, difficulty in sucking or swallowing, difficulty in breathing, accumulation of secretions in the lungs or throat, clenched fists and sweaty hands, tongue fluttering/vibration, head often tilted to one side (even when lying down), legs tending to be weaker than arms, legs often in “frog legs” position, difficulty in feeding, increased susceptibility to respiratory infections, bowel/bladder weakness, below 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 anterior horn cells.
The term “treating spinal muscular atrophy (SMA)” or “treatment of spinal muscular atrophy (SMA)” herein includes obtaining one or more of the following effects: 1. reducing or alleviating the severity of SMA; 2. delaying the onset of SMA; 3. inhibiting the progression of SMA; 4. extending the survival time of the subject; 5. improving the quality of life of the subject and/or improving the mental state of the subject; 6. reduce the number of SMA-related symptoms; 7. reducing or alleviating the severity of one or more symptoms associated with SMA; 8. reducing the duration of symptoms associated with SMA; 9. preventing recurrence of symptoms associated with SMA; 10. inhibiting the development or onset of SMA symptoms; 11. inhibiting the progression of symptoms associated with SMA; 12. improving lung function; 13. improving blood oxygen saturation; 14. promoting the transcription and expression of SMN gene (including: 1. promoting the transcription and/or expression of the truncated SMN2 gene; or 2. promoting the transcription and/or expression of the full-length SMN gene); 15. increasing the level of SMN protein (including: 1. the truncated SMN2 protein; or 2. the full-length SMN protein) in brain tissue and muscle tissue; 16. promoting the expression of NF-κB protein in brain tissue and muscle tissue; 17. promoting the formation of mature NGF in brain tissue; 18. alleviating lung tissue injury; 19. increasing muscle strength; 20. reducing muscle atrophy; 21. reducing loss of motor neurons; 22. promoting growth and development; and/or 23. improving motor function. In some embodiments, a component of the plasminogen activation pathway of the present application or a related compound thereof, such as plasminogen described above, enhances SMN gene transcription and/or expression. In some embodiments, a component of the plasminogen activation pathway of the present application, or a related compound thereof, such as the plasminogen described above, increases the expression of SMN protein in a human subject in need thereof.
In some embodiments, a component of the plasminogen activation pathway of the present application, or a related compound thereof, such as plasminogen, may be used alone or in combination with other medicaments to treat or prevent diseases caused by inactivating mutations or deletions in the SMN gene or diseases associated with loss or deficiency of SMN gene function. These diseases include, but are not limited to, spinal muscular atrophy (SMA).
In some embodiments, the present application relates to a method for treating a disease, such as SMA, caused by an inactivating mutation or deletion of SMN gene and/or associated with a loss or deficiency of SMN gene function, comprising administering to a subject a therapeutically effective amount of a component of the plasminogen activation pathway or a related compound, such as plasminogen. In some embodiments, the present application relates to a method for treating SMA, comprising administering to a subject a therapeutically effective amount of plasminogen.
In some embodiments, the present application relates to a method for treating SMA, comprising administering to a subject a therapeutically effective amount of plasminogen, wherein the plasminogen has one or more activities selected from the group consisting of: 1. reducing or alleviating the severity of SMA; 2. delaying the onset of SMA; 3. inhibiting the progression of SMA; 4. extending the survival time of the subject; 5. improving the quality of life of the subject and/or improving the mental state of the subject; 6. reducing the number of SMA-related symptoms; 7. reducing or alleviating the severity of one or more symptoms associated with SMA; 8. reducing the duration of symptoms associated with SMA; 9. preventing recurrence of symptoms associated with SMA; 10. inhibiting the development or onset of SMA symptoms; 11. inhibiting the progression of symptoms associated with SMA; 12. improving lung function; 13. improving blood oxygen saturation; 14. promoting the transcription and expression of SMN gene (including: 1. promoting the transcription and/or expression of the truncated SMN2 gene; or 2. promoting the transcription and/or expression of the full-length SMN gene); 15. increasing the level of SMN protein (including: 1. the truncated SMN2 protein; or 2. the full-length SMN protein) in brain tissue and muscle tissue; 16. promoting the expression of NF-κB protein in brain tissue and muscle tissue; 17. promoting the formation of mature NGF in brain tissue; 18. alleviating lung tissue injury; 19. increasing muscle strength; 20. reducing muscle atrophy; 21. reducing loss of motor neurons; 22. promoting growth and development; and/or 23. improving motor function.
In some embodiments, this application relates to a method for treating SMA, comprising administering a therapeutically effective amount of plasminogen to SMA subjects. The plasminogen has one or more of the following activities (effects): 1. promoting transcription and/or expression of the truncated SMN2 gene; and 2. promoting transcription and/or expression of the full-length SMN gene.
In some embodiments, the plasminogen further has one or more of the following effects or activities:
1) promoting penetration of plasminogen through the blood-brain barrier and the blood-spinal cord barrier,
2) promoting aggregation of plasminogen to brain and spinal cord tissue of SMA subjects,
3) promoting aggregation of plasminogen in an injured tissue (e.g., central nervous tissue (brain and spinal cord), lung, and muscle tissue) of SMA subjects,
4) increasing level of plasminogen in brain and spinal cord of SMA subjects,
5) increasing level of plasminogen in partially injured tissue (e.g., central nervous tissue (brain and spinal cord), lung, and muscle tissue) of SMA subjects,
6) alleviating damage to injured tissue (e.g., central nervous tissue (brain and spinal cord), lung, and muscle tissue) of SMA subjects,
7) promoting repair of inflammation of injured tissue (e.g., central nervous tissue (brain and spinal cord), lung, and muscle tissue) of SMA subjects,
8) promoting transcription of SMNΔ7 in brain and spinal cord of SMA subjects,
9) increasing level of SMN protein (including: 1. the truncated SMN2 protein; or 2. the full-length SMN protein) in brain and spinal cord of SMA subjects,
10) promoting expression of NGF in brain and spinal cord of SMA subjects, and
11) promoting growth and development of SMA subjects.
Fibrinolytic system is a system consisting of a series of chemical substances involved in the process of fibrinolysis, mainly including plasminogen, plasmin, plasminogen activator, and fibrinolysis inhibitor. Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA is a serine protease that is synthesized by vascular endothelial cells. t-PA activates plasminogen, which is mainly carried out on fibrin; urokinase-type plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells, and may directly activate plasminogen without the need for fibrin as a cofactor. Plasminogen (PLG) is synthesized by liver. When blood coagulates, a large amount of PLG is adsorbed on the fibrin network, and under the action of t-PA or u-PA it is activated into plasmin to promote fibrinolysis. Plasmin (PL) is a serine protease whose functions are as follows: degrading fibrin and fibrinogen; hydrolyzing various coagulation factors V, VIII, X, VII, XI, and II, etc.; converting plasminogen into plasmin; hydrolyzing complement, etc. Fibrinolysis inhibitors: including plasminogen activator inhibitor (PAI) and α2 antiplasmin (α2-AP). PAI mainly has two forms, PAI-1 and PAI-2, which may specifically bind to t-PA in a ratio of 1:1, thereby inactivating it and activating PLG at the same time. α2-AP is synthesized by liver, and binds to PL in a ratio of 1:1 to form a complex to inhibit the activity of PL; FXIII makes α2-AP covalently bound to fibrin, reducing the sensitivity of fibrin to PL. Substances that inhibit the activity of the fibrinolytic system in vivo: PAI-1, complement C1 inhibitor; α2 antiplasmin; α2 macroglobulin.
The term “plasminogen pathway activator” of the present application encompasses components of the plasminogen activation pathway, compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway, compounds mimicking the activity of plasminogen or plasmin, compounds up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolysis inhibitors.
The term “component of the plasminogen activation pathway” according to the present application encompasses:
1. plasminogen, Lys-plasminogen, Glu-plasminogen, micro-plasminogen, delta-plasminogen; variants or analogs thereof,
2. plasmin and a variant or an analog thereof, and
3. plasminogen activators, such as tPA and uPA, and tPA or uPA variants and analogs comprising one or more domains of tPA or uPA, such as one or more kringle domains and proteolytic domains.
The term “antagonist of fibrinolysis inhibitor” encompasses antagonists of PAI-1, complement C1 inhibitor, α2 antiplasmin or α2 macroglobulin, such as an antibody of PAI-1, complement C1 inhibitor, α2 antiplasmin or α2 macroglobulin.
“Variants” of the above plasminogen, plasmin, tPA and uPA include all naturally occurring human genetic variants as well as other mammalian forms of these proteins, as well as a protein obtained by addition, deletion and/or substitution of such as 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, or 1 amino acid, and still retaining the activity of plasminogen, plasmin, tPA or uPA. For example, “variants!” of plasminogen, plasmin, tPA and uPA include mutational variants of these proteins obtained by substitution of such as 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, or 1 conservative amino acid.
A “plasminogen variant” of the application encompasses a protein comprising or having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence of SEQ ID NO: 2, 6, 8, 10 or 12, and retaining the proteolytic and/or lysine-binding activity of plasminogen. For example, a “plasminogen variant” according to the present application may be a protein obtained by addition, deletion and/or substitution of 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, or 1 amino acid on the basis of SEQ ID NO: 2, 6, 8, 10 or 12, and still retaining the proteolytic and/or lysine-binding activity of plasminogen. Particularly, the plasminogen variants according to the present application include all naturally occurring human genetic variants as well as other mammalian forms of these proteins, as well as mutational variants of these proteins obtained by substitution of such as 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, or 1 conservative amino acid.
The plasminogen according to the present application may be a human plasminogen ortholog from a primate or rodent, or a variant thereof still retaining the proteolytic and/or lysine-binding activity of plasminogen, for example, a plasminogen represented by SEQ ID NO: 2, 6, 8, 10 or 12, such as a human natural plasminogen represented by SEQ ID NO: 2.
The “analogs” of the above plasminogen, plasmin, tPA, and uPA include compounds that respectively provide substantially similar effect to plasminogen, plasmin, tPA, or uPA.
The “variants” and “analogs” of above plasminogen, plasmin, tPA and uPA encompass “variants” and “analogs” of plasminogen, plasmin, tPA and uPA comprising one or more domains (e.g., one or more kringle domains and proteolytic domains). For example, “variants” and “analogs” of plasminogen encompass “variants” and “analogs” of plasminogen comprising one or more plasminogen domains (e.g., one or more kringle (K) domains and proteolytic domains, or referred to as serine protease domain, or plasminogen protease domain), such as mini-plasminogen. “Variants” and “analogs” of plasmin encompass “variants” and “analogs” of plasmin comprising one or more plasmin domains (e.g., one or more kringle domains and proteolytic domains), such as mini-plasmin, and delta-plasmin.
Whether a “variant” or “analog” of the above plasminogen, plasmin, tPA or uPA respectively has the activity of plasminogen, plasmin, tPA or uPA, or whether the “variant” or “analog” provides substantially similar effect to plasminogen, plasmin, tPA or uPA, may be detected by methods known in the art, for example, it is measured by the level of activated plasmin activity based on enzymography, ELISA (enzyme-linked immunosorbent assay), and FACS (fluorescence-activated cell sorting method), for example, it is detected by referring to a method selected from the following documents: Ny, A., Leonardsson, G., Hagglund, A. C, Hagglof, P., Ploplis, V. A., Carmeliet, P. and Ny, T. (1999). Ovulation inplasminogen-deficient mice. Endocrinology 140, 5030-5035; Silverstein R L, Leung L L, Harpel P C, Nachman R L (November 1984). “Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator”. J. Clin. Invest. 74(5):1625-33; Gravanis I, Tsirka SE (February 2008). “Tissue-type plasminogen activator as a therapeutic target in stroke”. Expert Opinion on Therapeutic Targets. 12(2):159-70; Geiger M, Huber K, Wojta J, Stingl L, Espana F, Griffin J H, Binder B R (August 1989). “Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo”. Blood. 74(2):722-8.
In some embodiments of the present application, the “component of plasminogen activation pathway” according to the present application is a plasminogen selected from the group consisting of: Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogen, or variants thereof retaining plasminogen activity. In some embodiments, the plasminogen is natural or synthetic human plasminogen, or a conservative mutant variant or fragment thereof still retaining plasminogen activity and/or lysine binding activity. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or rodent or a conservative mutant variant or fragment thereof still retaining plasminogen activity and/or lysine binding activity. In some embodiments, the amino acid sequence of the plasminogen comprises or has an amino acid sequence represented by SEQ ID NO: 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is a human full length plasminogen. In some embodiments, the plasminogen is a human full length plasminogen represented by SEQ ID NO: 2.
“A compound capable of directly activating plasminogen, or indirectly activating plasminogen by activating an upstream component of plasminogen activation pathway”, refers to any compound capable of directly activating plasminogen, or indirectly activating plasminogen by activating an upstream component of plasminogen activation pathway, such as tPA, uPA, streptokinase, saruplase, alteplase, reteplase, tenecteplase, anistreplase, monteplase, lanoteplase, pamiteplase, staphylokinase.
The “antagonist of a fibrinolysis inhibitor” according to the present application is a compound that antagonizes, weakens, blocks, or prevents the action of a fibrinolysis inhibitor. Such fibrinolysis inhibitors are e.g., PAI-1, complement C1 inhibitor, α2 antiplasmin, and α2 macroglobulin. Such an antagonist is: e.g., an antibody of PAI-1, complement C1 inhibitor, α2 antiplasmin, or α2 macroglobulin; or an antisense RNA or small RNA blocking or downregulating the expression of such as PAI-1, complement C1 inhibitor, α2 antiplasmin or α2 macroglobulin; or a compound occupying the binding site of PAI-1, complement C1 inhibitor, α2 antiplasmin, or α2 macroglobulin but without the function of PAI-1, complement C1 inhibitor, α2 antiplasmin, or α2 macroglobulin; or a compound blocking the binding domains and/or active domains of PAI-1, complement C1 inhibitor, α2 antiplasmin, or α2 macroglobulin.
Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease capable of hydrolyzing several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans. In addition, plasmin may activate some metalloproteinase precursors (pro-MMPs) to form active metalloproteinases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis. Plasmin is formed by proteolysis of plasminogen by two physiological PAs: tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). Due to the relatively high levels of plasminogen in plasma and other body fluids, it has traditionally been thought that the regulation of the PA system is mainly achieved through the synthesis and activity levels of PAs. The synthesis of components of the PA system is strictly regulated by different factors, such as hormone, growth factor and cytokine. In addition, there are specific physiological inhibitors of plasmin and PAs. The main inhibitor of plasmin is α2-antiplasmin. The activity of PAs is inhibited by plasminogen activator inhibitor-1 (PAI-1) of both uPA and tPA, and regulated by plasminogen activator inhibitor-2 (PAI-2) which mainly inhibits uPA. Certain cell surfaces have uPA-specific cell surface receptors (uPARs) with direct hydrolytic activity.
Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. Plasminogen is mainly synthesized in liver, and is abundantly present in the extracellular fluid. The content of plasminogen in plasma is approximately 2 μM. Plasminogen is thus a huge potential source of proteolytic activity in tissues and body fluids. Plasminogen exists in two molecular forms: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). The naturally secreted and uncleaved form of plasminogen has an amino-terminal (N-terminal) glutamate, and is therefore referred to as glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed at Lys76-Lys77 into lysine-plasminogen. Compared with glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin, and may be activated by PAs at a higher rate. The Arg560-Val561 peptide bond of these two forms of plasminogen may be cleaved by either uPA or tPA, resulting in the formation of a two-chain protease plasmin linked by disulfide. The amino-terminal part of plasminogen comprises five homologous tri-cycles, i.e., so-called kringles, and the carboxy-terminal part comprises the protease domain. Some kringles comprise lysine-binding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor α2-AP. A recently found plasminogen is a 38 kDa fragment, including kringles1-4, and it is a potent inhibitor of angiogenesis. This fragment is named as angiostatin, and is produced by the hydrolysis of plasminogen by several proteases.
The main substrate of plasmin is fibrin, and the dissolution of fibrin is the key to preventing pathological thrombosis. Plasmin also has substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycans, and gelatin, indicating that plasmin also plays an important role in ECM remodeling. Indirectly, plasmin may also degrade other components of the ECM, including MMP-1, MMP-2, MMP-3 and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been proposed that plasmin may be an important upstream regulator of extracellular proteolysis. In addition, plasmin has the ability to activate certain latent forms of growth factors. In vitro, plasmin also hydrolyzes components of the complement system, and releases chemotactic complement fragments.
“Plasmin” is a very important enzyme present in the blood that hydrolyzes fibrin clots to fibrin degradation products and D-dimers.
“Plasminogen” is the zymogen form of plasmin. According to the sequence in swiss-prot, it consists of 810 amino acids calculated by the natural human plasminogen amino acid sequence (SEQ ID NO: 4) containing the signal peptide, and the molecular weight is about 90 kD, and it is a glycoprotein mainly synthesized in liver and capable of circulating in blood, the cDNA sequence encoding this amino acid sequence is represented by SEQ ID NO: 3. Full-length plasminogen contains seven domains: a C-terminal serine protease domain, an N-terminal Pan Apple (PAp) domain, and five Kringle domains (Kringle1-5). Referring to the sequence in swiss-prot, its signal peptide comprises residues Met1-Gly19, PAp comprises residues Glu20-Val98, Kringle1 comprises residues Cys103-Cys181, Kringle2 comprises residues Glu184-Cys262, Kringle3 comprises residues Cys275-Cys352, Kringle4 comprises residues Cys377-Cys454, and Kringle5 comprises residues Cys481-Cys560. According to NCBI data, the serine protease domain comprises residues Val581-Arg804.
Glu-plasminogen is a natural full-length plasminogen, consisting of 791 amino acids (without a signal peptide of 19 amino acids); the cDNA sequence encoding this amino acid sequence is represented by SEQ ID NO: 1, and the amino acid sequence is represented by SEQ ID NO: 2. In vivo, there is also a Lys-plasminogen produced by the hydrolysis of the peptide bond between amino acids 76 and 77 of Glu-plasminogen, as represented by SEQ ID NO: 6; and the cDNA sequence encoding this amino acid sequence is represented by SEQ ID NO: 5. Delta-plasminogen (6-plasminogen) is a fragment of full-length plasminogen that lacks the Kringle2-Kringle5 structure, and only contains Kringle1 and a serine protease domain (also known as a protease domain (PD)). The amino acid sequence of delta-plasminogen (SEQ ID NO: 8) is reported in a literature, and the cDNA sequence encoding this amino acid sequence is represented by SEQ ID NO: 7. Mini-plasminogen consists of Kringle5 and a serine protease domain, and it is reported that it comprises residues Val443-Asn791 (with the Glu residue of the Glu-plasminogen sequence without the signal peptide as the starting amino acid), the amino acid sequence of the mini-plasminogen is represented by SEQ ID NO: 10, and the cDNA sequence encoding this amino acid sequence is represented by SEQ ID NO: 9. While micro-plasminogen comprises only a serine protease domain, and it is reported that its amino acid sequence comprises residues Ala543-Asn791 (with the Glu residue of the Glu-plasminogen sequence without the signal peptide as the starting amino acid); additionally, it is disclosed in patent document CN102154253A that its sequence comprises residues Lys531-Asn791 (with the Glu residue of the Glu-plasminogen sequence without the signal peptide as the starting amino acid); the sequence of micro-plasminogen of the present application refers to the patent document CN102154253A, the amino acid sequence of micro-plasminogen is represented by SEQ ID NO: 12, and the cDNA sequence encoding this amino acid sequence is represented by SEQ ID NO: 11. The structure of the full-length plasminogen has also been described in the article of Aisina et al. (Aisina R B, Mukhametova L I. Structure and function of plasminogen/plasmin system [J]. Russian Journal of Bioorganic Chemistry, 2014, 40 (6): 590-605). In this article, Aisina et al. describe that plasminogen includes Kringles 1, 2, 3, 4, and 5 and a serine protease domain (also known as a protease domain (PD)), wherein the Kringles are responsible for the binding of plasminogen to ligands with low-molecular-weight and high-molecular-weight (i.e., the lysine binding activity), which enables plasminogen to be transformed into a more open conformation and is more easily activated; the protease domain (PD) is the residues Val562 to Asn791, tPA and uPA specifically cleave the activation bond between Arg561 and Val562 of plasminogen, so that plasminogen be transformed to plasmin, therefore, the protease domain (PD) is the region providing the proteolytic activity of plasminogen.
In the present application, “plasmin” and “fibrinolytic enzyme” may be used interchangeably with the same meaning; and “plasminogen” and “fibrinolytic zymogen” may be used interchangeably with the same meaning.
In the present application, “lack” of plasminogen or plasminogen activity means that the content of plasminogen in a subject is lower than that of a normal person, and is sufficiently low to affect the normal physiological function of the subject; “deficiency” of plasminogen or plasminogen activity means that the content of plasminogen in a subject is significantly lower than that of a normal person, and even the activity or expression is extremely low, and the normal physiological function may only be maintained by external supply of plasminogen.
Those skilled in the art may understand that, all technical solutions of plasminogen according to the present application are applicable to plasmin, thus the technical solutions described in the present application encompass plasminogen and plasmin. During circulation, plasminogen is present in a closed, inactive conformation, but when bound to a thrombus or cell surface, it is converted into active plasmin with an open conformation after being mediated by plasminogen activator (PA). Active plasmin may further hydrolyze the fibrin clot into degradation products of fibrin and D-dimers, thereby dissolving the thrombus. The PAp domain of plasminogen comprises an important determinant for maintaining plasminogen in an inactive closed conformation, while the KR domain may bind to a lysine residue present on a receptor and substrate. A variety of enzymes are known to act as plasminogen activators, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hageman factor) etc.
“Plasminogen active fragment” in this application includes 1) in a plasminogen protein, an active fragment capable of binding to a target sequence in a substrate, also known as lysine-binding fragment, such as a fragment comprising Kringle 1, Kringle 2, Kringle 3, Kringle 4 and/or Kringle 5 (for the structure of plasminogen see, Aisina R B, Mukhametova L I. Structure and function of plasminogen/plasmin system[J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6):590-605); 2) an active fragment exerting proteolytic function in plasminogen protein, such as a fragment comprising the plasminogen activity represented by SEQ ID NO: 14; 3) a fragment of plasminogen protein, which has both binding activity to a target sequence in a substrate (lysine binding activity) and plasminogen activity (proteolytic function). In some embodiments of the present application, the plasminogen is a protein comprising the active fragment of plasminogen represented by SEQ ID NO: 14. In some embodiments of the present application, the plasminogen is a protein with the lysine-binding fragment comprising Kringle 1, Kringle 2, Kringle 3, Kringle 4, and/or Kringle 5. In some embodiments, the plasminogen active fragment of the present application comprises SEQ ID NO: 14, or a protein with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% homology with SEQ ID NO: 14. Therefore, the plasminogen of the present application comprises a protein having the plasminogen active fragment and still retaining the plasminogen activity. In some embodiments, the plasminogen of the present application comprises Kringle 1, Kringle 2, Kringle 3, Kringle 4 and/or Kringle 5, or a protein having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology with Kringle 1, Kringle 2, Kringle 3, Kringle 4 or Kringle 5, and still retaining lysine binding activity.
At present, the methods for measuring plasminogen and its activity in blood comprise: detection of tissue plasminogen activator activity (t-PAA), 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 the activity of plasma tissue plasminogen activator inhibitor, detection of the antigen of plasma tissue plasminogen activator inhibitor, and detection of plasma plasmin-antiplasmin complex (PAP); wherein the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and a chromogenic substrate to the plasma to be detected, the PLG in the plasma to be detected is converted into PLM under the action of SK, and PLM acts on the chromogenic substrate; subsequently, the detection by spectrophotometer indicates that the increase in absorbance is proportional to plasminogen activity. In addition, the plasminogen activity in blood may also be detected by immunochemical method, gel electrophoresis, immunoturbidimetry, and radioimmunodiffusion, etc.
“Ortholog or orthologs” refer to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs and vertical homologs; particularly it refers to proteins or genes evolved from the same ancestral gene in different species. The plasminogen according to the present application includes human natural plasminogen, and also includes plasminogen ortholog or orthologs derived from different species and having plasminogen activity.
A “conservative substitution variant” refers to a variant in which a given amino acid residue is altered without changing the overall conformation and function of the protein or enzyme, including but not limited to those variants in which the amino acid(s) in the amino acid sequence of the parent protein are replaced by amino acid(s) with similar properties (e.g., acidic, basic, hydrophobic, etc.). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic basic amino acids and are interchangeable. Similarly, isoleucine is a hydrophobic amino acid, and may be replaced by leucine, methionine or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may differ; for example, 70% to 99% similarity (identity) based on the MEGALIGN algorithm. “Conservative substitution variants” also include polypeptides or enzymes having more than 60%, preferably more than 75%, more preferably more than 85%, or even most preferably more than 90% amino acid sequence identity determined by BLAST or FASTA algorithm, and having the same or substantially similar properties or functions as the natural or parent protein or enzyme.
“Isolated” plasminogen refers to a plasminogen protein isolated and/or recovered from its natural environment. In some embodiments, the plasminogen will be purified: (1) to more than 90%, more than 95%, or more than 98% purity (by weight), as determined by Lowry's method, e.g., more 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 spinning cup sequence analyzer, or (3) to homogeneity as determined by using Coomassie blue or silver staining through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions. Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.
The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms include fusion proteins including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without N-terminal methionine residues); and the like.
“Percent (%) of amino acid sequence identity” with respect to a reference polypeptide sequence is defined as, after introducing gaps as necessary to achieve maximum percent sequence identity, and no conservative substitutions are considered as part of the sequence identity, the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence. Alignment for purposes of determining percent amino acid sequence identity may be accomplished in a variety of ways within the technical scope in the art, e.g., by publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art may determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences to be compared. However, for the purpose of the present application, the values of percent amino acid sequence identity are generated by using the computer program ALIGN-2 for sequence comparison.
Where ALIGN-2 is used to compare amino acid sequences, the percentage (%) of amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or may be expressed as a given amino acid sequence A having a certain percentage (%) of amino acid sequence identity relative to, with or with respective to a given amino acid sequence B) is calculated as follows:
Fraction X/Y×100%
wherein X is the number of amino acid residues scored as identical matches during the alignment of sequences A and B by the sequence alignment program ALIGN-2, and wherein Y is the total number of amino acid residues in sequence B. It should be appreciated that, where the length of amino acid sequence A is not equal to that of amino acid sequence B, the percentage (%) of amino acid sequence identity of A with respect to B will not equal to the percentage (%) of amino acid sequence identity of B with respect to A. Unless expressly stated otherwise, all the values of percentage (%) of amino acid sequence identity used herein are obtained by using the ALIGN-2 computer program as described in the preceding paragraph.
The terms “individual”, “subject” and “patient” are used interchangeably herein to refer to mammals including, but not limited to, murine (rat, mouse), non-human primate, human, canine, feline, hoofed animals (e.g., horses, cattle, sheep, pigs, goats), etc.
A “therapeutically effective amount” or “effective amount” refers to an amount of plasminogen sufficient to prevent and/or treat a disease when administrated to a mammal or other subject for treating the disease. A “therapeutically effective amount” will vary depending on the component of plasminogen in use, the severity of the disease and/or symptoms thereof in the subject to be treated, as well as the age, weight, and the like.
The term “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, resulting in temporary or permanent regression of the disease state or its clinical symptoms.
The term “muscle strength” refers to the strength of muscle contraction during voluntary movement of a limb or the strength of a muscle during active movement. According to the situation of muscle strength, muscle strength is usually divided into the following 0-5 grades: grade 0, complete paralysis, no muscle contraction can be measured; grade 1, only muscle contraction can be measured, but no movement; grade 2, the limbs can move horizontally on the bed, but cannot resist their own gravity, i.e., they cannot be lifted off the bed surface; grade 3, the limbs can overcome gravity and can be lifted off the bed surface, but cannot resist resistance; grade 4, the limbs can do movement against external resistance, but incomplete; grade 5, normal muscle strength.
The term “muscle tone” refers to the tension of a muscle in its resting, relaxed state. Muscle tone is the basis for maintaining various postures and normal movements of the body. Muscle tone manifests itself in various forms. For example, when a person is lying down and resting, the tension of the muscles of various parts of the body is called resting muscle tone. When the body is standing, although the muscles are not significantly contracted, the muscles in the front and rear of the body also maintain a certain tension to maintain the standing posture and body stability, which is called postural muscle tone. The tension of muscles during exercise, called exercise muscle tone, is an important factor to ensure continuous and smooth muscle movement (without tremors, twitches, and spasms). In pathological conditions, muscle tone increases or decreases, affecting the normal posture or movement of human body.
Plasminogen may be isolated from nature, and purified for further therapeutic use, or it may be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is synthesized chemically, the synthesis may be carried out via liquid phase or solid phase. Solid-phase polypeptide synthesis (SPPS) (in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by the sequential addition of the retaining amino acids in the sequence) is a suitable method for chemical synthesis of plasminogen. Various forms of SPPS, such as Fmoc and Boc, may be used to synthesize plasminogen. Techniques for solid-phase synthesis are described in Barany and Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85:2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10, and Camarero J A et al. 2005, Protein Pept Lett. 12:723-8. Briefly, small insoluble porous beads are treated with functional units on which peptide chains are constructed; after repeated cycles of coupling/deprotection, the attached solid-phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected to reveal new N-terminal amines that may be attached to other amino acids. The peptide remains immobilized on the solid phase, subsequently it is cleaved off.
Plasminogen according to the present application may be produced by standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector to be operably linked to regulatory sequences in the expression vector. The regulatory sequences for expression include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Expression regulation may be a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell (e.g., COS or CHO cell). 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.
A suitable expression vector is typically replicated in a host organism as an episome or as an integrated part of the host chromosomal DNA. Typically, an expression vector contains a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance marker) to facilitate the detection of those cells transformed with desired exogenous DNA sequence.
Escherichia coli is an example of a prokaryotic host cell that may be used to clone a plasminogen-encoding polynucleotide. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors may also be generated, which typically contain an expression control sequence (e.g., origin of replication) that are compatible with the host cell. In addition, there are many well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from bacteriophage lambda. A promoter will typically control the expression, optionally in case of an operator gene sequence, and have ribosome binding site sequence, etc., to initiate and complete transcription and translation.
Other microorganisms, such as yeast, may also be used for expression. Yeast (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells, and as required a suitable vector has an expression control sequence (e.g., promoter), origin of replication, termination sequence, etc. A typical promoter comprises 3-phosphoglycerate kinase and other saccharolytic enzymes. Particularly, inducible yeast promoters include promoters from ethanol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) may also be used to express and produce the plasminogen of the application (e.g., polynucleotides encoding plasminogen). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for use in these cells may comprise expression control sequences such as origin of replication, promoter and enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary sites for processing information such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol. 148:1149 (1992).
Once synthesized (chemically or recombinantly), the plasminogen of the present application may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like. The plasminogen is substantially pure, e.g., at least about 80-85% pure, at least about 85-90% pure, at least about 90-95% pure, or 98-99% pure or purer, e.g., free of contaminants such as cellular debris, macromolecules other than the plasminogen, and the like.
Medicament Formulation
A therapeutic formulation may be prepared by mixing the plasminogen of desired purity with an optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), to form a lyophilized formulation or an aqueous solution. An acceptable carrier, excipient, or stabilizer is non-toxic to a recipient at the employed dosage and concentration, including buffers such as phosphate, citrate and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexanediamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parahydroxybenzoate such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; carbohydrates such as sucrose, mannitol, fucose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and/or nonionic surfactants such as TWEN™, PLURONICS™ or polyethylene glycol (PEG).
The formulations according to the present application may also contain more than one active compound as required for the particular condition to be treated, preferably those compounds are complementary in activity and do not have side effects with each other. For example, antihypertensive medicaments, antiarrhythmic medicaments, diabetes medicaments, etc.
The plasminogen according to the present application may be encapsulated in microcapsules prepared by techniques such as coacervation or interfacial polymerization, for example, the plasminogen may be placed in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
The plasminogen according to the present application for in vivo administration must be sterile. This may be easily achieved by filtration through sterilizing filters before or after lyophilization and reformulation.
The plasminogen according to the present application may be prepared as a sustained-release formulation. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers which have a certain shape and contain glycoprotein, for example, membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981); Langer, Chem. Tech., 12:98-105 (1982)), or poly(vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman, et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al., supra), or degradable lactic acid-glycolic acid copolymers such as Lupron Depot™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid may release molecules continuously for more than 100 days, while some hydrogels release proteins for shorter period of time. Rational strategies to stabilize proteins may be devised based on the relevant mechanisms. For example, if the mechanism of condensation is found to form intermolecular S—S bond through thiodisulfide interchange, then stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, using suitable additives, and developing specific polymer matrix composition.
Administration of the pharmaceutical composition according to the present application may be accomplished by different means, e.g., intravenous administration, intraperitoneal administration, subcutaneous administration, intracranial administration, intrathecal administration, intra-arterial administration (e.g., via the carotid artery), and intramuscular administration.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, etc.
Dosing regimens will be determined by medical personnel based on various clinical factors. As is well known in the medical field, the dosage for any patient depends on a variety of factors, including the patient's size, body surface area, age, the particular compound to be administrated, sex, number and route of administration, general health, and other concomitantly administrated medicaments. The dosage range of the pharmaceutical composition comprising the plasminogen according to the present application may be about 0.0001-2000 mg/kg, or about 0.001-500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 10 mg/kg, 50 mg/kg, etc.) body weight of the subject per day. For example, the dose may be 1 mg/kg body weight, or 50 mg/kg body weight, or in the range of 1-50 mg/kg, or at least 1 mg/kg. Dosages above or below this exemplary range are also contemplated, especially in view of the factors set forth above. Intermediate doses within the above ranges are also included within the scope of the present application. Subjects may be administrated such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary dosage schedule includes 1-10 mg/kg on consecutive days. Real-time evaluation of therapeutic efficacy and safety is required during the administration of the medicament of the present application.
One embodiment of the present application relates to a product or kit comprising plasminogen or plasmin of the present application useful in treating cardiovascular disease caused by diabetes and related disorders. The product preferably comprises a container, a label or package insert. Suitable containers are bottles, vials, syringes, etc. The container may be made of various materials such as glass or plastic. The container contains a composition which is effective for treatment of the disease or condition according to the present application and has a sterile access port (e.g., the container may be an intravenous solution pack or vial containing a stopper penetrable by a hypodermic needle). 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 treating cardiovascular disease caused by diabetes and related disorders mentioned in the present application. The product may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and glucose solution. It may further contain other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes. In addition, the product comprises a package insert with instructions for use, including, for example, instructing the user of the composition to administrate the composition comprising plasminogen to the patient along with other medicaments for treatment of concomitant diseases.
Plasminogen used in the following examples is human plasminogen and was obtained from the plasma of a donor, and was purified from the plasma of the human donor based on the method described in the References [1-3] with process optimization, wherein the human Lys-plasminogen and Glu-plasminogen >98%.
All patients in the following Examples 1-11 signed an informed consent, voluntarily received the above-mentioned treatment of plasminogen purified from human plasma, and obtained the approval of the hospital ethics committee. The administration regimen and dosage are adjusted according to the severity and course of the disease. The mode of administration is aerosol inhalation or intravenous injection. The drug concentration of aerosol inhalation and intravenous injection is 5 mg/ml, and normal saline is used as vehicle.
Female patient, 38 months old. At 10 months old, she was unable to crawl, her legs were weak, and her motor ability barely improved after 1 year of age. At the age of 1 year and 9 months, she was diagnosed with SMA by genetic testing, muscle strength was grade 2, and her lower limbs could not carry weight. Then she started taking salbutamol, methylcobalamin, and coenzyme Q10. At the age of 2 years, her motor ability further degenerated so that she could not climb, and could only sit and stand for a short time. At the age of 2 years and 2 months, she started mesenchymal stem cell therapy, and the balance ability was improved and not easy to get sick, but she still could not stand. At the age of 2 years and 11 months, she started acupoint injection of the mouse nerve growth factor, then the balance ability was improved, the lower limbs began to have supporting strength, and the measured muscle strength reached grade 3.
Intravenous injection; dosage: 100-200 mg/time; frequency: once every other day, once every other 2 days, or once every other 3 days; 2 weeks as a course of treatment; 2-3 weeks between adjacent courses of treatment. A total of 6 courses of treatment were performed.
The Expanded Hammersmith Functional Motor Scale (HFMSE) is designed to assess motor function in patients with type II and type III SMA, reflecting the disease severity. The functional motor scale is defined as change from baseline to assess changes in motor function in children; the higher the scores, the better the motor function [4-6].
Nerve electromyography (EMG) is the main diagnosis and identification means for motor neuron disease, and the amplitude of compound muscle action potential reflects the neuronal axon injury. SMA is a degenerative motor neuron disease with massive motor neuron death, exhibiting muscle weakness and reduced or even undetectable compound muscle action potential amplitudes [7].
HFMSE scores: the score before treatment was 20 points, and the score after the first course of treatment was 21 points. The score before the second course of treatment was 23 points, and the score after treatment was 24 points. The score after the sixth course of treatment was 25 points (see
Motor function evaluation: before treatment, the patient could not stand without assistance. After 2 courses of treatment, the patient was able to stand with assistance. After 3 courses of treatment, the patient achieved assisted walking, and the patient's head control ability was significantly improved. With the progress of treatment, the patient's motor function was further improved, including prolonged standing time with assistance and increased walking distance with assistance.
EMG: the action potential amplitudes of bilateral tibial nerves, common peroneal nerve and femoral nerve were significantly increased after treatment, as compared with those before treatment (
In addition, the patient's mental state was significantly improved and the vitality increased after the treatment. There were no drug-related side effects during the treatment.
The above results indicate that, plasminogen can improve the HFMSE score of a patient with type II SMA, and improve the motor function, neuromuscular function, and mental state of the patient.
Male patient, 30 months old. There was no abnormality after birth. At the age of nine months, he could not stand independently and turn over on his own; his hands occasionally trembled slightly, could not grasp and raise his head. At the age of ten months, the patient was diagnosed with type II SMA by genetic testing. At the age of 1-2 years, he started to rehabilitate in the rehabilitation center, once a week; then he could keep sitting alone, grab light objects with both hands, stretch his calf slowly, and complete turning over with assistance. At the age of 2 years and 2 months, intravenous infusion of bone marrow mesenchymal stem cells was administered three times, two units each time; and the improvement was not significant. At the age of 2 years and 4 months, the neural stem cells were injected twice, two units each time; and the improvement was obvious after the injection. Status: he can sit independently, when sitting alone, the body can lean slightly to the sides and support the body with both hands, but the arms cannot be raised. Head control has been improved, and he can quickly shake head back and forth, and keep his head up by four-point support with the parent's assistance. The strength of the legs is increased, and the calf can be kicked back and forth while sitting on the seat. He may lean on the parent to maintain a standing position with slight knee assistance.
The first time: intravenous injection, 50 mg; the second time: intravenous injection, 50 mg; the third and fourth time: intravenous injection, 100 mg. The administration frequency is once every other 2 days, or once every other 3 days for 2 weeks.
The HFMSE score was 2 points before the treatment, and the HFMSE score was 8 points after the fourth treatment (see
The above results indicate that, plasminogen can improve the HFMSE score of a patient with type II SMA, improve the motor function, and improve the neuromuscular function of the patient, and there are no drug-related side effects during the treatment.
Female patient, 24 months old. At the age of 1 year, she could stand with support, was unable to climb on four-point support. At 16 months old, the electromyography showed neurogenic injury, and the patient was diagnosed with type II SMA by genetic testing. Status: Unstable sitting alone, unable to sit up while lying down, able to stand up with handrail, stand up by leaning, and walk with handrail; sometimes the status was in good condition, and sometimes in bad condition, she was difficult to lift both hands.
Intravenous injection; dosage: 50 mg-100 mg each time; frequency: once every other day, or once every other 3 days; 2 weeks is a course of treatment, and 3-4 weeks between adjacent courses of treatment, with a total of 8 courses of treatment.
HFMSE scores: the score before treatment was 23 points, and the score after the first course of treatment was 24 points. There was an interval of approximately two months between the patient's first and second courses of treatment. The score before the second course of treatment was 23 points, and the score after the treatment was 24 points. The score was 28 points after the eighth course of treatment (see
Motor function evaluation: before treatment, the patient could not raise the hands above head. After 2 courses of treatment, the patient achieved standing without assistance and walking with assistance, and he was able to raise his hands above his head. After continuous administration, the patient's motor function was further improved, and was capable of sitting independently, standing with handrail, and stably walking with handrail; and the time for these activities was gradually extended.
EMG: after treatment, the action potential amplitudes of bilateral median nerve, tibial nerve, common peroneal nerve, and ulnar nerve all increased to varying degrees (
In addition, no drug-related side effect was seen during the treatment.
The above results indicate that, plasminogen can improve the HFMSE score, motor function and neuromuscular function of a patient with type II SMA.
Male patient, 43 months old. At the age of 12 months, the electromyography detection showed neurogenic injury, and at the age of 14 months he was diagnosed with type II SMA by genetic testing. By the age of 24 months, he degenerated to a status that he could not sit independently, and could only sit by leaning. At the age of 29 months, he started taking salbutamol, methylcobalamin, and coenzyme q10, and his motor function was gradually and slightly improved. At the age of 36 months, he started treatment with mesenchymal stem cells, and totally received 5 injections of mesenchymal stem cells intravenously. After the injection of mesenchymal stem cells, the patient's overall state was improved significantly, his mental state was improved, he was not easy to get tired and get sick, but he was still not able to stand. At the age of 38 months, acupoint injection of mouse nerve growth factor was performed, but there was no obvious effect.
The first time: intravenous injection, 50 mg; the second time: intravenous injection, 50 mg; the third time: intravenous injection, 100 mg; the fourth time: intravenous injection, 150 mg, the administration was performed every other 3 days, for 2 weeks.
The patient's right arm function was improved after treatment, and the patient could lift his right arm to 90 degrees without help of the left hand, but the HFMSE score was not improved. There were no drug-related side effects during the treatment.
Male patient, 26 months old. At the age of 10 months, he was diagnosed with SMA by genetic testing, and the copy number of SMN2 (survival motor neuron gene 2) was 3. At the age of 12 months, he lost the ability of turning over, his arms were unable to support his body, he could only lie on the bed completely, and his fingers could not exert force on their own. At the age of 13 months, he started the injection of umbilical cord mesenchymal stem cells. After the injection of stem cells, the strength of the legs was increased, the breathing was improved, and the quality of sleep was also improved. At the age of 16 months, he started taking Chinese medicine. After the comprehensive treatment of mesenchymal stem cells, traditional Chinese medicine and rehabilitation training, the patient could sit independently and keep the balance by himself, but he was unable to turn left or right or pick up toys on his left or right. He was able to roll over independently, and the leg strength was increased significantly, breathing was improved, and voice became louder, the pectus excavatum was improved significantly, rib valgus signs were improved, and strength of the arms and hands was increased, but still could not be pulled up by grasping an adult's hand. The arm lift was degenerated in some way, and the arm could be raised to the top of the head by itself before, but only to the face when the following treatment was started.
Intravenous injection for two weeks, once every other day, 4 times administration in the first week with the doses of 10 mg, 20 mg, 30 mg, and 40 mg respectively; 4 times administration in the second week with the doses of 50 mg, 50 mg, 100 mg, and 100 mg respectively.
Calf and ankle strength was increased after the second administration in the first week. Nine days after the two-week treatment, strength of the hands and arms was increased, and the grasping power was increased by 50%. There were no drug-related side effects during the treatment.
The above results indicate that plasminogen can improve motor function in a patient with type II SMA.
Male patient, 11 months old. He was diagnosed with type I SMA by genetic testing at 6 months old; the doctor informed that the average life cycle of patients with this type of disease was 2 years old at the time of diagnosis, and the family members did not take any treatment measures. Symptoms: weak head support; weak upper limbs and arms and inability to lift, less hand swing, weak grasping, weak middle finger; weak lower limbs, less swinging, movable toes; unable to sit independently, unable to turn over independently, weak sucking, difficulty in swallowing; blood oxygen monitoring was performed 24 hours a day, blood oxygen saturation was 92-97%, and the chest rose and fell weakly when breathing.
The drug was administered by aerosol inhalation (2 or 3 times a day) and intravenous injection (once every three days) for a total of 16 courses of treatment (2 weeks as a course of treatment). Adjacent courses of treatment were separated by 2 weeks. The aerosol inhalation dose was 5-10 mg, and the intravenous injection dose was 50-200 mg, started from 50 mg and gradually increased to 200 mg.
The CHOP INTEND scale (Children's Hospital of Philadelphia Test Scale for Infant Neuromuscular Diseases) was used to evaluate the improvement of motor function in patients with type I SMA. A higher score indicates better motor function [8].
Survival status: after 16 courses of treatment, the patient was older than 30 months, exceeding the 10-month survival time for most patients shown by natural history studies of patients with type I SMA [9]. In addition, this study found that the mental state and growth and development (height, weight, chest circumference) of a patient with SMA were significantly improved, and compared with a patient of the same age who was not treated with plasminogen, muscle atrophy in a patient treated with plasminogen was also improved significantly (see
CHOP INTEND scores: CHOP INTEND score was 30 points before treatment, and the score increased to 50 points after 5 courses of treatment. For some reason, the 5th and 6th courses were separated by about 2 months, and the score dropped to 36 points, and the score was 44 points after the 6th course of treatment. The 6th and 7th courses were separated by about 2 months. The score was 44 points before the 7th course of treatment, and 45 points after the treatment. During the tenth to sixteenth courses of treatment, the score fluctuated between 44 points and 46 points (see
Motor function: after the first course of treatment, the patient was able to sit independently with head support for about 30 seconds. After the 4th course of treatment, the time of sitting independently was prolonged, about 30 seconds. With progress of the treatment, the patient's motor function continued to improve, the hand grasped strongly, the arm could be lifted slightly and voluntarily, and the foot movement and swing frequency were increased.
Swallowing function: after administration, the number of times of choking when eating and drinking was reduced, and the speaking function was good.
Respiratory function: On day 2 of the first course of treatment, the patient's blood oxygen saturation reached 97-98%, occasionally 95-96%. After 2 courses of treatment, the patient's breathing strength increased. After 16 courses of treatment, the patient was more than 30 months old, and the patient still maintained a good breathing state without ventilatory support. The natural history study of type I SMA showed that without ventilatory support, the survival rate at 20 months old was only 8% [10].
The above results indicate that, plasminogen can improve the CHOP INTEND score and motor function of a patient with type I SMA, and can achieve a milestone improvement of sitting without support for 30 seconds and head support for 30 seconds; it can also improve patients' swallowing function and speaking ability, improve lung function, increase blood oxygen saturation, and improve the patient's mental state and sleep.
Female patient, 23 months old. The symptoms showed up at 26 days of age, then she was diagnosed with type I SMA by genetic testing. Conditions before treatment: she was unable to sit up without assistance, and the limbs had no antigravity motion. She was able to move the fingers of the left hand, the joints of the right hand, the forefinger within a wide range, and the other four fingers slightly; the ankle joint of the left lower limb was movable, the knee joint of the right lower limb could move slightly, but couldn't be uplifted; was able to shake head slightly from side to side; and the CHOP INTEND score was 4 points. She was unable to breath spontaneously and dependent on a ventilator for 24 h, with the blood oxygen saturation of 95-100% during the day and 94-97% at night. The patient completely lost the swallowing function, and needed gastrostomy feeding. The amplitude of action potential of bilateral median nerve, ulnar nerve, common peroneal nerve and tibial nerve decreased (80-100%) due to extensive neurogenic injury. Suck sputum 40-50 times a day, and the sputum was bluish white and slightly sticky.
The drug was administered by intravenous injection at a dose of 50 mg once every other day for 3 consecutive days.
Motor function: after 2 days of intravenous injection of plasminogen, the CHOP INTEND score was increased from 4 points to 5 points, increased by 1 point (
Respiratory function: before administration, the patient needed 40 to 50 sputum suctions every day, and sputum was bluish white and slightly sticky; on day 3 of aerosol inhalation, the number of sputum suctions was decreased to about 30 times; on day 9 of administration, the patient could cough up a small amount of mucus; on day 13 of administration, the volume of sputum was decreased by 10%, but the stickiness did not change; although the number of sputum suctions was slightly increased in the first few days after drug withdrawal, on day 12 of drug withdrawal, the volume of sputum was decreased by 10%, the number of sputum suctions was further decreased; and on day 11, the patient could spontaneously control the drool.
Female patient, 29 months old. The symptoms showed up at 5 months of age, then she was diagnosed with type I SMA by genetic testing. Conditions before treatment: the patient was able to sit independently for 10-15 seconds when in good condition occasionally, but was unstable; was able to turn from side to side, but was unable to roll over independently; was able to raise head for 1 min when sitting without assistance, and for 3 to 5 min when sitting by leaning; was able to raise her head upright for 40 s and shake head when picked up; could raise upper limbs 10 cm when sitting; had weak grasping strength, could grasp spoons and paintbrushes; was unable to raise lower limbs, and was able to bend knees with assistance and swing calves left and right within a small range for 2 min. The CHOP INTEND score was 31 points. The patient had alternating thoracic and abdominal breathing (⅓ of thoracic breathing, and ⅔ of abdominal breathing). The patient had weak swallowing function, took 1 h to eat, could eat granular food only, and sometimes choked when drinking. The patient had intermittent mild tongue flutter; poor sleep, crying.
The drug was administered by intravenous injection at a dose of 50-100 mg/day, 50 mg on day 1, gradually increased to 100 mg, once every other day for 14 consecutive days.
Motor function: after 9 days of administration of plasminogen, the patient achieved independent sitting without assistance for 10 s. After 14 days of administration, the CHOP INTEND score was increased from 31 points to 40 points, increased by 9 points (
Male patient, 10 months old. He was diagnosed with type I SMA by genetic testing after the onset of symptoms at 42 days of age. Conditions before treatment: the patient did not have head control ability, was unable to raise head upright and swing head; was unable to sit independently or sit by leaning; was unable to roll over; and was able to raise forearms when bending elbows; his hands could grasp but were powerless; and his lower limbs and feet were immobile. The CHOP INTEND score was 31 points. The patient did not have swallowing ability, and needed nasal feeding. The patient had a lot of sputum and saliva, and needed about 10 deep sputum suctions and about 20 oral sputum and saliva suctions every day.
The drug was administered by aerosol inhalation twice a day, 5 mg each time. The drug was also administered by intravenous injection at a dose of 50-100 mg/day, 50 mg on day 1, gradually increased to 100 mg, once every other day.
Motor function: the patient could not move his arm when lying on his back with the arm flat, but on day 7 of administration of plasminogen, the patient could turn arms inside and outside spontaneously; and on day 10 of administration, the patient could bend knees and stand upright for about 2 min. After 14 days of administration, the CHOP INTEND score was increased from 31 points to 40 points, increased by 9 points (
Respiratory function: the patient used a ventilator 24 hours before administration at a ventilator pressure of 15; after 14 days of administration, the ventilator pressure was reduced to 13, and the patient could occasionally wean off the ventilator and breath smoothly.
Female patient, 12 months old. The patient was diagnosed with type I SMA by genetic testing after the onset of symptoms at 3 months of age. Conditions before treatment: the patient did not have head control ability, could not raise head upright and shake head; could not sit independently and sit by leaning; could not roll over; arms and legs: only fingers, wrists, and toes were mobile. The CHOP INTEND score was 0 points. The patient was dependent on a ventilator at night. The patient did not have swallowing ability, and needed gastric tube feeding. The patient needed about 10 sputum suctions per day.
The drug was administered by intravenous injection, 50-100 mg each time, 50 mg on day 1, gradually increased to 100 mg, once every other day for 14 days.
Motor function: on day 11 of administration of plasminogen, the patient could turn upper limbs outward 450 when upper limbs were flat, could stand for more than 2 min with bent knees, and cloud slightly nod. After 8 days of administration, the CHOP INTEND score was increased from 0 point to 8 points, increased by 8 points (
Female patient, 40-month-old (3 years and 4 month). The onset of the disease was at 6 months of age, and was diagnosed with non-5q SMA at the age of 1.5 years (18 months) by genetic testing, specifically spinal muscular atrophy with respiratory distress type 1 (SMARD1), caused by mutations in related IGHMBP2-encoding genes on chromosome 11. Due to lung infection and difficulty in expelling sputum to block the respiratory tract, resulting in weakness of breathing, and gradually spontaneous respiratory failure after intermittent use of the ventilator, unable to breath without ventilator, using ventilator for about 1.5 years; loss of language function, facial paralysis, immobility, muscle strength grade 0. Symptoms: since the use of the ventilator, daily use of expectoration machines, sputum suction devices, oxygen inhalation, atomization (twice a day), and nasal feeding. The electromyography results showed that, the motor neurons of both upper and lower limbs were severely injured, and no action potential was observed.
The first course of treatment (2 weeks): aerosol inhalation, 10 mg/time, 3 times/day, combined with 50-100 mg intravenous injection, once every 3 days.
The second course of treatment was carried out 2 months after completion of the first course of treatment.
The second to fourth courses (2 weeks between adjacent courses): intravenous injection, once every other 2 days, the dose was 150 mg-250 mg.
The first course of treatment: the time of hands hanging and shaking was increased, the amplitude and the strength were increased, and the left upper arm was able to move inwardly with the assistance of support. The lower extremity was able to bend and stand up for 30 minutes with assistance, the facial expressions were increased, the eyes could blink, and the mouth could twitch voluntarily.
The second course of treatment: the patient could occasionally swallow soup, and her sleep quality was improved.
The third course of treatment: defecation was normal, the head could be shaken from side to side, and the head could be held for a few seconds with auxiliary support. The wrist was slightly strong, the fingertips could be rotated cyclically, and the left arm could be swayed autonomously with larger amplitude. Blood oxygen was maintained at 97% and no oxygen was given.
The fourth course of treatment: the coordination of the left arm was better, and the movement range of the right arm was small, but the swing frequency was fast. The muscles of the lower limbs were soft and not stiff, the facial expressions were increased, and she was able to defecate voluntarily.
During process of the treatment, the mental state of the patient was significantly improved, the respiratory ability was significantly improved, and the need for ventilatory support was gradually reduced.
The results show that, plasminogen can improve the motor function of a patients with non-5q SMA, including increasing in the strength, amplitude and range of limb movements, and enriching the patient's facial expressions; improving the patient's lung function and respiratory function, reducing oxygen infusion, and increasing blood oxygen saturation; improving the patient's swallowing function; improving the patient's sleep quality.
The following examples 12-31 are administration studies on animal models, and the plasminogen used in the following examples is still the above-mentioned plasminogen protein purified from human donor plasma.
SMNΔ7 SMA mice used in the following examples are FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1MsdTg(SMN2*delta7) 4299Ahmb/J gene mutant mice (SMNΔ7 SMA mice for short) having SMN1 gene homozygous mutation, and expressing human SMN2 gene. The clinical and pathological manifestations of the mice are similar to human SMA. The breeding mice were purchased from Jackson Laboratory in the United States (pedigree number: 005025).
SMNΔ7 SMA mice were weighed at birth, and randomly divided into vehicle group (6 mice) and plasminogen group (5 mice) according to body weight. The mice were given plasminogen after 3 days of birth. The mice in the vehicle group were intraperitoneally injected with 6 ml/kg of vehicle every day, and the mice in the plasminogen group were intraperitoneally injected with 60 mg/kg of plasminogen every day. The survival of the mice was recorded.
The statistical results of survival curve showed that, plasminogen can significantly improve the survival curve of SMNΔ7 SMA mice, and the statistical difference is significant (P=0.029). The statistical results of survival time showed that, the median survival time of the mice in the vehicle group is 14 days, and all mice died on day 15; the median survival time of the mice in the plasminogen group is 16 days, and all the mice died on day 17, and the difference of statistical analysis is significant (P=0.03), indicating that plasminogen can prolong the survival time of SMA model mice, as shown in
Two 3-day-old SMNΔ7 SMA mice were taken, one mouse was in the vehicle group and was given 6 μl of bovine serum albumin solution (5 mg/ml) by intraperitoneal injection once in the morning and once in the afternoon each day; another mouse was in the plasminogen group and was given plasminogen by intraperitoneal injection (as per 30 μg/6 μl) once in the morning and once in the afternoon each day. One wild-type (FVB) mouse was taken as blank control group, and 6 μl of bovine serum albumin solution (5 mg/ml) was given by intraperitoneal injection once in the morning and once in the afternoon each day. The administration was performed for 9 consecutive days. The spinal cord was harvested after sacrificing the mice, and all SMN gene transcripts were detected by qPCR. The forward primer was F: GCGGCGGCAGTGGTGGCGGC (SEQ ID NO: 15); the reverse primer was R: AGTAGATCGGACAGATTTTGCT (SEQ ID NO: 16).
The results show that, the spinal cord of the mice in the blank control group has a certain level of SMN gene transcription, the level of SMN gene transcription in the mice of the vehicle group was lower than that in the mice of the blank control group, and the level of SMN gene transcription in the mice of the plasminogen group was significantly higher than that in the mice of the vehicle group and the blank control group (
Seven 3-day-old SMNΔ7 SMA mice were taken. 4 mice were in the vehicle group, for the first 9 days, 6 μl of bovine serum albumin solution (5 mg/ml) was given by intraperitoneal injection once in the morning and once in the afternoon each day; starting from day 10, 6 μl of bovine serum albumin solution (10 mg/ml) was given by intraperitoneal injection once every day. 3 mice were in the plasminogen group, for the first 9 days, plasminogen was given by intraperitoneal injection (as per 30 μg/6 μl) once in morning and once in the afternoon each day; starting from day 10, and plasminogen was injected as per 60 μg/6 μl by intraperitoneal injection once a day. 4 wild-type mice were taken as blank control group, for the first 9 days, 6 μl of bovine serum albumin solution (5 mg/ml) was administered by intraperitoneal injection once in the morning and once in the afternoon; starting from day 10, 6 μl of bovine serum albumin solution (10 mg/ml) was administered by intraperitoneal injection once a day. On day 12, the mice were sacrificed to collect brain tissue, and the brain tissue homogenate was prepared for Western blot detection of NF-κB protein. 10% gels were prepared according to the dispensing instructions of the SDS-PAGE gel preparation kit (Solarbio, P1320). Samples in each group were taken to respectively mix well with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heating at 100° C. for 5 min, cooling and centrifuging for 2 min, and then 20 μL of the mixture was taken for loading. The electrophoresis conditions: running at 30V for 30 min, and then running to the bottom of the gel at 100V. After electrophoresis, the gel was stripped and transferred to an activated PVDF membrane (GE, A29433753), and the electrotransfer conditions were 15V for 2.5h. The transferred PVDF membrane was immersed in blocking solution (5% skim milk) and blocked overnight in a 4° C. refrigerator. After washing 4 times with TBST (0.01M Tris-NaCl, pH 7.6 buffer), rabbit anti-mouse NF-κB antibody (Cell Signaling Technology, 8242) was added to incubate at room temperature for 3 h; after washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abeam, ab6721) secondary antibody was added to incubate at room temperature for 1 h, washing 4 times with TBST, then placing the PVDF membrane on a clean imaging plate and adding Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographing under a biomolecular imager, and then Image J software was used to obtain the optical density value of each band for quantitative analysis.
Nuclear factor kappa-B (NF-κB) is a key nuclear transcription factor. NF-κB family members mainly include RelA (p65), c-Rel, RelB, NF-κB1 (p50 protein and its precursor p105) and NF-κB2 (p52 protein and its precursor p100), each member can form homologous or heterologous dimers to function. Most commonly in mammalian cells, p65 binds to p50 to form a p65/p50 dimer. In unstimulated cells, the NF-κB transcription factor binds to the inhibitory IκB (inhibitor of kappa B) protein and is thus retained in the cytoplasm. The stimulation of upstream signals leads to the phosphorylation of IκB protein under the action of IKK (IκB kinase), which is then recognized by the ubiquitin ligase complex, thereby promoting the degradation of IκB protein in a proteasome-dependent manner, and then the NF-κB is released to enter the nucleus and initiate the expression of target genes [11]. NF-κB can be found in almost all animal cells, and they are involved in the response of cells to external stimuli, and play a key role in cellular inflammatory response, immune response and other processes. NF-κB is also related to synaptic plasticity and memory [12].
The results show that, the brains of the mice in the blank control group have a certain amount of NF-κB protein, the level of NF-κB protein in the brains of the mice in the vehicle group is lower than that of the mice in the blank control group, and the level of NF-κB protein in the brains of the mice in the plasminogen group is significantly higher than that of the mice in the vehicle group, and the statistical difference is close to significant (P=0.05) (
Muscles were obtained from the sacrificed mice in Example 14 above, and Western blot detection of NF-κB protein was performed according to the method described in Example 14 above.
The results show that, the muscles of mice in the blank control group have a certain amount of NF-κB protein, the level of NF-κB protein in the muscles of mice in the vehicle group is lower than that of the mice in the blank control group, and the level of NF-κB protein in the muscles of mice in the plasminogen group is significantly higher than that of the mice in the vehicle group, and the statistical difference is significant (* indicates P<0.05) (
Two 3-day-old SMNΔ7 SMA mice were taken, one mouse was in vehicle group and was given 6 μl bovine serum albumin solution (5 mg/ml) by intraperitoneal injection once in the morning and once in the afternoon each day; another mouse was in plasminogen group and was given plasminogen by intraperitoneal injection (as per 30 μg/6 μl) once in the morning and once in the afternoon each day. 2 wild-type (FVB) mice were taken as the blank control group, and 6 μl of bovine serum albumin solution (5 mg/ml) was given to each mouse by intraperitoneal injection once in the morning and once in the afternoon each day. After 9 days of administration, the mice were sacrificed to collect brain tissue, and the brain tissue homogenate was prepared for Western blot detection of SMN protein. 12% gels were prepared according to the dispensing instructions of SDS-PAGE gel. Samples in each group were taken to respectively mix well with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heating at 100° C. for 5 min, cooling and centrifuging for 2 min, and then 20 μL of the mixture was taken for loading. The electrophoresis conditions: running at 30V for 30 min, and then running to the bottom of the gel at 100V. After electrophoresis, the gel was stripped and transferred to an activated PVDF membrane (GE, A29433753), and the electrotransfer conditions were 15V for 2.5h. The transferred PVDF membrane was immersed in blocking solution (5% skim milk) and blocked overnight in a 4° C. refrigerator. After washing 4 times with TBST (0.01M Tris-NaCl, pH 7.6 buffer), rabbit anti-mouse SMN antibody (Proteintech, 11708-1-AP) was added to incubate at room temperature for 3 h; after washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abeam, ab6721) secondary antibody was added to incubate at room temperature for 1 h, washing 4 times with TBST, then placing the PVDF membrane on a clean imaging plate and adding Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographing under a biomolecular imager, and then Image J software was used to obtain the optical density value of each band for quantitative analysis.
The results show that, the brain of the mice in the blank control group expresses a certain amount of SMN protein, the expression level of SMN protein in the mice in the vehicle group is lower than that in the mice in the blank control group, and the expression level of SMN protein in the mice in the plasminogen group is significantly higher than that in the mice in the vehicle group (
The hindlimb muscle tissue was collected from the sacrificed mice described in Example 16, and the tissue homogenate was prepared for Western blot detection of SMN protein. The detection method was as described in Example 16.
The results show that, the muscles of the mice in the blank control group express a certain amount of SMN protein, the expression level of SMN protein in the muscles of the mice in the vehicle group is lower than that of the mice in the blank control group, and the expression level of SMN protein in the muscles of the mice in the plasminogen group is significantly higher than that of the mice in the vehicle group (
Seven 3-day-old SMNΔ7 SMA mice were taken and randomly divided into two groups, 4 mice in the vehicle group, and 3 mice in the plasminogen group. The mice in the vehicle group were intraperitoneally injected with bovine serum albumin solution (5 mg/mL) at a dose of 6 μL/g once in the morning and once in the afternoon every day for the first 10 days, and were intraperitoneally injected with bovine serum albumin solution (10 mg/mL) at a dose of 6 μL/g every day after 10 days of administration. The mice in the plasminogen group were intraperitoneally injected with plasminogen (5 mg/mL) at a dose of 30 mg/kg once in the morning and once in the afternoon every day for the first 10 days, and were intraperitoneally injected with plasminogen (10 mg/mL) at a dose of 60 mg/kg every day after 10 days of administration. 4 wild-type mice were taken as mice in the blank control group, and were intraperitoneally injected with bovine serum albumin solution (5 mg/mL) at a dose of 6 μL/g once in the morning and once in the afternoon every day for the first 10 days, then they were intraperitoneally injected with bovine serum albumin solution (10 mg/mL) at a dose of 6 μL/g every day after 10 days of administration. On day 11 of administration, the mice were killed, and brain tissue were taken out and used for Western blot assays of the NGF protein. 12% gels were prepared according to the dispensing instructions of SDS-PAGE gel. Samples in each group were taken to respectively mix well with 4× loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heating at 100° C. for 5 min, cooling and centrifuging for 2 min, and then 20 μL of the mixture was taken for loading. The electrophoresis conditions: running at 30V for 30 min, and then running to the bottom of the gel at 100V. After electrophoresis, the gel was stripped and transferred to an activated PVDF membrane (GE, A29433753), and the electrotransfer conditions were 15V for 2.5h. The transferred PVDF membrane was immersed in blocking solution (5% skim milk) and blocked overnight in a 4° C. refrigerator. After washing 4 times with TBST (0.01M Tris-NaCl, pH 7.6 buffer), rabbit anti-mouse NGF antibody (Abcam, ab52918) was added to incubate at room temperature for 3 h; after washing 4 times with TBST, goat anti-rabbit IgG (HRP) antibody (Abeam, ab6721) secondary antibody was added to incubate at room temperature for 1 h, washing 4 times with TBST, then placing the PVDF membrane on a clean imaging plate and adding Immobilon Western HRP Substrate (MILLIPORE, WBKLS0100) for color development, photographing under a biomolecular imager, and then Image J software was used to obtain the optical density value of each band for quantitative analysis.
Nerve growth factor (NGF) is an important member of the neurotrophic factor family, and it is synthesized in vivo in precursor form, including signal peptide, leader peptide, and mature peptide. Studies have reported that the precursor of nerve growth factor NGF (ProNGF) plays an opposite role relative to NGF which is formed by cleavage of ProNGF. ProNGF may promote neuronal apoptosis, while mature NGF is involved in regulating the growth, development, differentiation, survival and repair of nerve cells after injury, and also plays an important role in regulating the functional expression of central and peripheral neurons [12]. NGF/ProNGF ratio=NGF optical density (OD) value/ProNGF optical density (OD) value.
The results show that, the brain tissue of the mice in the blank control group has a certain ratio of NGF/ProNGF, and the ratio of NGF/ProNGF in the brain tissue of the mice in the plasminogen group is significantly higher than that of the mice in the vehicle group, and the statistical difference is extremely significant (*** indicates P<0.001) (
Eight 3-day-old SMNΔ7 SMA mice were taken and randomly divided into two groups, 4 mice in the vehicle group, and 4 mice in the plasminogen group. 7 wild-type mice from the same litter were taken as mice in the blank control group. The mice in the vehicle group and the blank control group were intraperitoneally injected with 3 ml/kg of vehicle once in the morning and once in the afternoon every day for the first 3 days, and were intraperitoneally injected with 6 ml/kg of vehicle once a day after 3 days of administration. The mice in the plasminogen group were intraperitoneally injected with plasminogen (10 mg/ml) at the dose of 30 mg/kg once in the morning and once in the afternoon every day for the first 3 days, and were intraperitoneally injected with plasminogen (10 mg/mL) at the dose of 60 mg/kg once a day after 3 days of administration. After 9 days of administration, the mice were sacrificed, lung tissue were taken out and fixed in 10% neutral formalin fixing solutions for 24 hours. The fixed lung tissue was dehydrated with ethanol gradient, cleared with xylene, and embedded in paraffin. The tissue was cut into slices with thickness of 5 μm, the slices were dewaxed, rehydrated, stained with hematoxylin and eosin (H&E staining), differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia water, dehydrated with ethanol gradient, sealed, and observed under a microscope at 200× magnification.
The results show that, the terminal bronchiolar epithelial cells of the lung tissue of the mice in the blank control group are neatly arranged and clearly distinguishable; the alveolar cavities are uniform in size, the alveolar septum is not thickened, and there is no inflammatory cell infiltration around the blood vessels; as for the lung tissue of the mice in the vehicle group, the respiratory bronchiolar epithelium is fallen off, the alveolar ducts and alveolar sacs are enlarged, the alveolar septum is widened, the alveoli collapse to structural disorder, and there are eosinophils, foam cells, and lymphocytes around the pulmonary blood vessels; the respiratory bronchiolar epithelium of the mice in the plasminogen group are arranged in an orderly manner, the alveolar ducts and alveolar sacs are enlarged, and the alveolar cavities are evenly enlarged, but the alveolar wall composed of a single layer of alveolar epithelium is visible (
Lung tissue of the sacrificed mice of Example 19 were taken out and fixed in 10% neutral formalin fixing solutions for 24 h. The fixed lung tissue was dehydrated with ethanol gradient, cleared with xylene, and embedded in paraffin. The tissue was cut into slices with thickness of 4 μm, and the slices were dewaxed, rehydrated, and washed once with water. The tissue was circled using a PAP pen, incubated in 3% hydrogen peroxide for 15 min, and washed twice with 0.01 M PBS for 5 min each time. The slices were blocked in 5% normal goat serum (Vector laboratories, Inc., USA) for 30 min, the goat serum was removed after the blocking was completed, a rabbit anti-mouse F4/80 antibody (Abcam, ab100790) was added dropwise, the slices were incubated at 4° C. for overnight and washed twice with 0.01 M PBS for 5 min each time. A goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added, and the slices were incubated at room temperature for 1 h and washed twice with 0.01 M PBS for 5 min each time. The slices were developed using a DAB kit (Vector laboratories, Inc., USA), washed three times with water, restained with hematoxylin for 30 s, and rinsed with running water for 5 min. The slices were dehydrated with ethanol gradient, cleared with xylene, sealed by neutral gum, and observed under an optical microscope at 200× magnification.
F4/80 is a highly glycosylated G-protein-coupled receptor and is an identification marker for murine macrophages.
The results show that there is a certain level of F4/80-positive cells in the lung tissue of the mice in the blank control group (
Spinal cord tissue of the sacrificed mice of Example 19 were taken out and used for PCR amplification of SMNΔ7 mRNA, and the amplified samples were detected using 1.5% agarose gel. The primers were: 5′-GCTGATGCTTTGGGAAGTATGTTA-3′ (SEQ ID NO: 17) and 5′-ATTCCAGATCTGTC TGATCG-3′ (SEQ ID NO: 18).
It has been reported that improvement of the mRNA transcription of SMNΔ7 can promote increase of the functional SMN protein, so as to improve the course of the disease and delay the progression of the disease [13].
The study results of the present example show that the SMNΔ7 transcription level in the spinal cord of the mice in the plasminogen group is significantly higher than that of the mice in the vehicle group, and the P value is 0.002 (
Muscle tissue of the sacrificed mice of Example 19 were taken out and fixed in 10% neutral formalin fixing solution for 24 h. The fixed muscle tissue was dehydrated with ethanol gradient, cleared with xylene, and embedded in paraffin. The tissue was cut into slices with thickness of 5 μm, the slices were dewaxed, rehydrated, stained with hematoxylin and eosin (H&E staining), differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia water, dehydrated with ethanol gradient, sealed, and observed under a microscope at 200× magnification.
The results show that in the muscle tissue of the mice in the blank control group (
Brain tissue of the sacrificed mice of Example 19 were taken out and fixed in 10% neutral formalin fixing solutions for 24 h. The fixed brain tissue was dehydrated with ethanol gradient, cleared with xylene, and embedded in paraffin. The tissue was cut into slices with thickness of 5 μm, the slices were dewaxed, rehydrated, stained with hematoxylin and eosin (H&E staining), differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia water, dehydrated with ethanol gradient, sealed, and observed under a microscope at 200× magnification.
The results show that in the mice in the blank control group (
Eight 3-day-old or 7-day-old SMNΔ7 SMA mice and 4 wild-type mice of the same age were taken, the SMNΔ7 SMA mice were randomly divided into vehicle group and plasminogen group, 4 mice per group, and the 4 wild-type mice were used as blank control group. The mice in the plasminogen group were intraperitoneally injected with plasminogen (10 mg/ml) at a dose of 60 mg/kg/day, the mice in the vehicle group and the blank control group were intraperitoneally injected with vehicle at a dose of 6 ml/kg/day, and the administration was performed until 11 days after birth. On day 11 after birth, all mice were sacrificed and dissected 2 hours after administration, a partial lung tissue was taken out, homogenized, and detected in accordance with instructions of Human Plasminogen ELISA Kit (Producer: AssayMax, EP1200-1). The concentration of each sample was calibrated using a human plasminogen working standard as an internal standard, and the amount of plasminogen in total protein per unit of each sample was calculated by dividing the calibrated concentration by the total protein concentration and conducted statistical analysis.
The results show that the plasminogen level in the lung tissue of the mice in the blank control group is 1.22±1.12 ng/mg; the plasminogen level in the lung tissue of the mice in the vehicle group is 2.36±0.87 ng/mg, which is about 1.9 times that of the mice in the blank control group; and the plasminogen level in the lung tissue of the mice of the plasminogen group is 96.94±20.49 ng/mg, which is about 41 times that of the mice in the vehicle group (
Muscle tissue of the sacrificed mice of Example 24 were taken out, homogenized, and detected in accordance with instructions of the Human Plasminogen ELISA Kit (Producer: AssayMax, EP1200-1). The concentration of each sample was calibrated using a human plasminogen working standard as an internal standard, and the amount of plasminogen in total protein per unit of each sample was calculated by dividing the calibrated concentration by the total protein concentration and conducted statistical analysis.
The results show that the plasminogen level in the muscle tissue of the mice in the blank control group is 5.95±3.84 ng/mg; The plasminogen level in the muscle tissue of the mice in the vehicle group is 15.11±10.38 ng/mg, which is about 2.5 times that of the mice in the blank control group; and the plasminogen level in the muscle tissue of the mice in the plasminogen group is 901.30±398.51 ng/mg, which is about 60 times that of the mice in the vehicle group (
Brain tissue of the sacrificed mice of Example 24 were taken out, homogenized, and detected in accordance with instructions of the Human Plasminogen ELISA Kit (Producer: AssayMax, EP1200-1). The concentration of each sample was calibrated using a human plasminogen working standard as an internal standard, and the amount of plasminogen in total protein per unit of each sample was calculated by dividing the calibrated concentration by the total protein concentration and conducted statistical analysis.
The results show that the plasminogen level in the brain tissue of the wild-type mouse in the blank control group is 1.18±1.54 ng/mg; the plasminogen level in the brain tissue of the mice in the vehicle group is 1.49±1.59 ng/mg, which is not significantly different from that of the mice in the vehicle group; the plasminogen level in the brain tissue of the SMA transgenic mouse in the plasminogen group is 12.09±5.32 ng/mg, which is about 8 times that of the mice in the vehicle group (
Eight 3-day-old or 7-day-old SMNΔ7 SMA mice, 4 wild-type mice of the same age, and 3 SMA heterozygous mice of the same age were taken, the SMNΔ7 SMA mice were randomly divided into the vehicle group and the plasminogen group, 4 mice per group, the 4 wild-type mice were used as mice in the blank control group, and the 3 SMA heterozygous mice were used as mice in the normal administration control group. The mice in the plasminogen group and the normal administration control group were intraperitoneally injected with plasminogen (10 mg/ml) at a dose of 60 mg/kg/day, the mice in the vehicle group and the blank control group were intraperitoneally injected with the vehicle at a dose of 6 ml/kg/day, and the administration was performed until 11 days after birth. On day 11 after birth, all the mice were sacrificed and dissected 2 hours after administration, a partial spinal cord tissue was taken out, homogenized, and used for an ELISA assay of plasminogen.
The results show that the plasminogen level in the spinal cord of the wild-type mouse in the blank control group is 8.51±9.51 ng/mg. The plasminogen level in the spinal cord of the SMA transgenic mouse in the vehicle group is 19.95±4.06 ng/mg, which is slighter higher than that of the mice in the blank control group. The plasminogen level in the spinal cord of the mice in the normal administration control group is 13.03±7.51 ng/mg. The plasminogen level in the spinal cord of the SMA transgenic mouse in the plasminogen group is 62.33±17.37 ng/mg, which is about 3 times that of the mice in the vehicle group, the statistical analysis P value between the plasminogen group and the vehicle group is 0.029. The plasminogen level in the spinal cord of the SMA transgenic mouse in the plasminogen group is about 4.8 times that of the mice in the normal administration control group, and the statistical analysis P value between the plasminogen group and the normal administration control group is 0.026 (
9 SMA heterozygous mice (purchased from Jackson Laboratory, 005025) were taken and randomly divided into two groups according to the body weight, 3 mice in the blank control group and 6 mice in the model group. All the mice were anesthetized with Zoletil 50, the mice in the model group were instilled with lipopolysaccharide (LPS) solution (2.5 mg/ml) via the trachea at a dose of 5 mg/kg for model construction, and the mice in the blank control group were instilled with normal saline via the trachea at a dose of 2 ml/kg. 2 hours after model construction, all mice in the model group were randomly divided into two groups according to the body weight, 3 mice in the vehicle group and 3 mice in the plasminogen group. After grouping, the mice started administration, the mice in the plasminogen group were injected with plasminogen via the tail vein at a dose of 50 mg/kg, and the mice in the vehicle group and the blank control group were injected with the vehicle via the tail vein at a dose of 5 ml/kg. 2 hours after administration, all the mice were sacrificed and dissected, and spinal cord tissue were taken out, homogenized, and detected in accordance with instructions of Human Plasminogen ELISA Kit (Producer: AssayMax, EP1200-1). The concentration of each sample was calibrated using a human plasminogen working standard as an internal standard, and the amount of plasminogen in total protein per unit of each sample was calculated by dividing the calibrated concentration by the total protein concentration and conducted statistical analysis.
The results show that the plasminogen level in the spinal cord homogenate of the mice in the blank control group is 2.70±0.74 ng/mg; the plasminogen level in the spinal cord homogenate of the mice in the vehicle group after instillation of LPS via the trachea is 3.17±1.51 ng/mg, which is not significantly different from that of the mice in the blank control group; the plasminogen level in the spinal cord homogenate of the mice in the plasminogen group after supplementation of plasminogen at a dose 2.5 times the physiological dose is 121.16±44.68 ng/mg, which is about 38.2 times that of the mice in the vehicle group (
Spinal cord tissue of the sacrificed mice of Example 28 were taken out, homogenized, and used for enzyme-substrate kinetic assays of plasminogen. Standard solutions, blanks, and samples at seven different concentrations were added in sequence to ELISA plate (producer: NUNC, 446469) at amount of 85 μL/well, 15 μL of mixture of 20 mM S-2251 solution (producer: Chromogenix, 82033239) and 100 ng/NL uPA solution (mixed in volume ratio of 2:1 right before use) was then added to each well, and incubated at 37° C. Starting from 0 min of the reaction, the absorbance A405 was read using a multifunctional microplate reader every 5 min until 90 min of the reaction. Straight-line fitting was performed on each reaction based on the time and the absorbance to obtain a straight line with the slope being the reaction rate (ΔA405/min) of the standard/sample. Finally, the potency of the samples tested was calculated by using the potency value of the standard and ΔA405/min for making the standard curves. The activity of plasminogen in total protein per unit of each sample was calculated.
The results show that the level of plasminogen in the spinal cord tissue of the mice in the blank control group is 0.00011±4.51×10−5 U/mg; the level of plasminogen in the spinal cord of the mice in the vehicle group after instillation of LPS via the trachea is 0.00010±9.72×10−6 U/mg, which is not significantly different from that of the mice in the blank control group; and the plasminogen level in the spinal cord of the mice in the plasminogen group after supplementation of plasminogen at a dose of 2.5 times the physiological dose is 0.00034±1.04×10−4 U/mg, and the statistical analysis P value is 0.058 (
Lung tissue of the sacrificed mice of Example 28 were taken out, homogenized, and detected in accordance with instructions of Human Plasminogen ELISA Kit (producer: AssayMax, EP1200-1). The concentration of each sample was calibrated using a human plasminogen working standard as an internal standard, and the amount of plasminogen in total protein per unit of each sample was calculated by dividing the calibrated concentration by the total protein concentration and conducted statistical analysis.
The results show that the plasminogen level in the lung tissue of the mice in the blank control group is 1.49±0.47 ng/mg; the plasminogen level in the lung tissue of the mice in the vehicle group after instillation of LPS via the trachea is 3.67±1.01 ng/mg, which is about 2.5 times that of the mice in the blank control group; and the plasminogen level in the lung tissue of the mice in the plasminogen group after supplementation of plasminogen at a dose 2.5 times the physiological dose is 562.68±102.85 ng/mg, which is about 153 times that of the mice in the vehicle group (
Lung tissue of the sacrificed mice of Example 28 were taken out, homogenized, and used for enzyme-substrate kinetic assay of plasminogen. Standard solutions, blanks, and samples at seven different concentrations were added in sequence to ELISA plate (producer: NUNC, 446469) at amount of 85 μL/well, 15 μL of mixture of 20 mM S-2251 solution (producer: Chromogenix, 82033239) and 100 ng/μL uPA solution (mixed in volume ratio of 2:1 right before use) was added to each well, and incubated at 37° C. Starting from 0 min of the reaction, the absorbance A405 was read using multifunctional microplate reader every 5 min until 90 min of the reaction. Straight-line fitting was performed on each reaction based on the time and the absorbance to obtain straight line with the slope being reaction rate (ΔA405/min) of the standard/sample. Finally, the potency of the samples tested was calculated using the potency value of the standard and ΔA405/min as standard curve. The activity of plasminogen in total protein per unit of each sample was calculated.
The results show that the plasminogen level in the lung tissue of the mice in the blank control group is 0.00022±1.31×10−5 U/mg; after the SMA heterozygous mice develop LPS-induced acute pneumonia, the plasminogen level in the lung tissue of the mice in the vehicle group is increased to 0.00033±3.70×10−5 U/mg; and 2 hours after administration of plasminogen at a dose 2.5 times the physiological dose, the plasminogen level in the lung tissue of the mice in the plasminogen group is increased to 0.0023±1.78×104 U/mg, which is about 7 times that of the mice in the vehicle group (
Spinal cord tissue of the sacrificed mice of Example 28 were taken out and fixed in 10% neutral formalin fixing solutions for 24 h. The fixed spinal cord tissue was dehydrated with ethanol gradient, cleared with xylene, and embedded in paraffin. The tissue was cut into slices with thickness of 4 μm, and the slices were dewaxed, rehydrated, and washed once with water. The tissue was circled with a PAP pen, incubated in 3% hydrogen peroxide for 15 min, and washed twice with 0.01 M PBS for 5 min each time. The slices were blocked in 5% normal goat serum (Vector laboratories, Inc., USA) for 30 min, the goat serum was removed after the blocking was completed, rabbit anti-mouse NGF antibody (Abcam, ab52918) was added dropwise, and the slices were incubated at 4° C. overnight and washed twice with 0.01 M PBS for 5 min each time. Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was added, and the slices were incubated at the room temperature for 1 h and washed twice with 0.01 M PBS for 5 min each time. The slices were developed using DAB kit (Vector laboratories, Inc., USA), washed three times with water, restained with hematoxylin for 30 s, and rinsed with running water for 5 min. The slices were dehydrated with ethanol gradient, cleared with xylene, sealed by neutral gum, and observed under an optical microscope at 200× magnification.
The results show that a certain level of NGF is expressed in the spinal cord of the mice in the blank control group (
Eight 3-day-old SMNΔ7 SMA mutant mice were taken and randomly divided into two groups, 4 mice in the vehicle group and 4 mice in the plasminogen group. 7 wild-type mice from the same litter were used as mice in the blank control group. The mice in the vehicle group and the blank control group were intraperitoneally injected with the vehicle at a dose of 3 ml/kg once in the morning and once in the afternoon every day for the first 3 days, and were intraperitoneally injected with the vehicle at a dose of 6 ml/kg once a day after 3 days of administration. The mice in the plasminogen group were intraperitoneally injected with plasminogen (10 mg/ml) at a dose of 30 mg/kg once in the morning and once in the afternoon every day for the first 3 days, and were intraperitoneally injected with plasminogen (10 mg/ml) at a dose of 60 mg/kg once a day after 3 days of administration. After 9 days of administration, i.e., PD11, the mice were sacrificed, and spinal cord tissue were taken out and used for qPCR assays of SMN mRNA.
The results show that the level of full-length SMN mRNA in the spinal cord tissue of the mice in the plasminogen group is significantly higher than that of the mice in the vehicle group, and a statistical difference is significant (** indicates P<0.01) (
Righting reflex test was performed on mice of Example 33 on day 8 and day 10 after birth to assess the motor function of the mice.
Righting reflex test is used for assessing the motor ability of young mice. The specific procedure was: the mice were placed on their back in a supine position, the time spent on spontaneous righting reflex of the mice was recorded, the maximum testing time was 30 s, each mouse was tested three times, and the average value was calculated [14].
The results show that the righting reflex time of the mice in the vehicle group is significantly longer than that of the mice in the blank control group, which indicates that the motor ability of the mice in the vehicle group is significantly weaker; and after the SMA mice are administrated with plasminogen, the motor ability is improved and the righting reflex time is reduced comparing to the mice in the vehicle group. Although the P value is close to 0.05, it is not statistically significant due to the number problem (
Tube test was performed on mice of Example 33 on day 10 after birth, i.e., PD10, to assess the motor function of the mice.
Tube test is used for assessing the muscle strength, weakness, and fatigue of the proximal hindlimbs, and neogenesis situation of old mince. In addition, it can also be used for assessing general neuromuscular function, and the body muscle strength. The mouse was hung head down with their hind paws on a 50 ml centrifuge tube, and three factors were used as assessment parameters: 1. the hanging time; 2. the number of pulls up (out of the tube); and 3. the hindlimb score (HLS score). HLS score: 4 points: normal hindlimb separation, and tail raised; 3 points: evident weakness, hindlimbs stay close but rarely touching; 2 points: hindlimbs stay close, often touching; 1 point: evident weakness, hindlimbs always in a holding position, and tail raised; 0 point: hindlimbs held continuously, tail drooping; The HLS score is 0 point if a mouse cub can not hold the test tube. TTS=(hang time+number of pull-ups×10)×(HLS+1)/4 [14].
The results show that the TTS score of the mice in the vehicle group is significantly lower than that of the wild-type mice in the blank control group, and after the SMA mice in the plasminogen group is administered with plasminogen, the score is increased, and the degradation trend of the motor function of the mice is improved (
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/089631 | May 2020 | WO | international |
This application is a national stage application under 35 USC § 371 of International Application No. PCT/CN2021/093039, filed May 11, 2021, which claims priority to International Application No. PCT/CN2020/089631, filed May 11, 2020, the entire contents of each priority application are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/093039 | 5/11/2021 | WO |
