Pharmaceutical composition for intravascular administration

Abstract

The purpose of the present disclosure is to provide a pharmaceutical composition having excellent therapeutic effects in the treatment of central nervous system diseases.　This pharmaceutical composition for intravascular administration is used for treating central nervous system diseases and contains a purified extracellular vesicle product. The purified extracellular vesicle product is obtained by a method for producing a purified extracellular vesicle product, the method comprising: (i) a purification step for bringing a solution to be treated containing extracellular vesicles and foreign substances into contact with size-exclusion and anion-exchange carriers to obtain a treated solution containing the extracellular vesicles; and (ii) a membrane filtration step for subjecting the treated solution to membrane filtration to obtain a concentrated solution of the extracellular vesicles. The pharmaceutical composition has excellent therapeutic effects in the treatment of central nervous system diseases.

Description

Pharmaceutical composition for intravascular administration

[0001] The present disclosure relates to a pharmaceutical composition for intravascular administration used to treat central nervous system disorders.

[0002] Cerebral infarction, which occurs when blood vessels in the brain become blocked, can cause severe after-effects such as paralysis of the limbs and speech impairment, and is the leading cause of patients certified as requiring nursing care levels 3 to 5 (i.e., severely ill patients). Catheter treatment (thrombectomy) to reopen blocked blood vessels has seen explosive growth since its approval in Japan in 2017. Thrombectomy has resulted in a certain number of patients experiencing dramatic recovery, achieving a level of recovery not previously achieved with existing treatments. However, even among patients who undergo thrombectomy, approximately half end up wheelchair-bound or bedridden, making it important to find ways to reduce the number of patients with such severe after-effects.

[0003] Against this background, great expectations are being placed on stem cells. In recent years, it has been reported that part of the mechanism by which stem cells restore neural function lies in exosomes secreted by stem cells. Exosomes are a type of extracellular vesicle, a granular substance with a diameter of 50-200 nm. Their surface contains lipids and proteins derived from the cell membrane, while their interior contains intracellular substances such as nucleic acids (miRNA, mRNA, DNA, etc.) and proteins, and are thought to play a role in intercellular communication. Research on exosomes is progressing as a potential drug that provides neuroprotection by delivering trophic factors or neuroprotective factors.

[0004] While much research and development has been conducted on exosomes and extracellular vesicles, standard and efficient technologies for their production and isolation are still under development. Biological samples containing extracellular vesicles contain many proteins and cells that have similar physical and chemical properties to extracellular vesicles, making the isolation of extracellular vesicles inherently complex.

[0005] The main separation methods currently in use utilize differences in the density, size, or specific surface markers of extracellular vesicles, and specific examples include ultracentrifugation, sedimentation, filtration, size exclusion chromatography, and immunoaffinity.

[0006] Ultracentrifugation is a separation method that exploits the differences in density and size between cells, EVs, and proteins, and is typically used as a separation method for extracellular vesicles (Non-Patent Document 1). However, ultracentrifugation has the drawbacks of requiring a long time to collect extracellular vesicles, low throughput, and co-precipitation of protein aggregates.

[0007] The precipitation method was developed as a method that does not use ultracentrifugation, which has problems with recovery time and throughput, and extracellular vesicles / exosome isolation kits are now commercially available.

[0008] Filtration is a method for separating relatively large vesicular components such as cells and extracellular vesicles from biological samples using commercially available membrane filters (e.g., filters made of PVDF or polycarbonate with pore sizes of approximately 50 to 450 nm), and typically requires further ultracentrifugation to separate extracellular vesicles and exosomes from proteins (Non-Patent Documents 2, 3, 4).

[0009] Size exclusion chromatography is used to separate extracellular vesicles and exosomes from protein aggregates. Typically, centrifugation or filtration is first performed to remove relatively large vesicle components such as cells and extracellular vesicles, and then extracellular vesicles such as exosomes are separated using a commercially available size exclusion column (Non-Patent Documents 2, 4, 5, 6, 7).

[0010] Immunoaffinity isolation is a method for isolating extracellular vesicles such as exosomes by utilizing differences in specific surface markers. Generally, it is not suitable for isolating extracellular vesicles or exosomes from large amounts of biological samples (Non-Patent Document 8).

[0011] These conventional separation methods require specialized laboratory equipment or reagents and multi-step procedures, making it difficult to analyze extracellular vesicles such as exosomes as routine diagnostic tools in clinical settings (Non-Patent Document 9).

[0012] Kidney Int. 82, no. 9 (2012): 1024-1032. DOI: 10.1038 / ki.2012.256.Nat. Biotechnol. 32, no. 5, 490-495 (2014). DOI: 10.1038 / nbt.2886Cancer 119 (2013): 1159-1167. DOI: 10.1002 / cncr.27895. Nanomedicine 11, no. 4 (2015): 879-883. doi.org / 10.1016 / j.nano.2015.01.003. J. Immunol. Methods 411 (2014): 55-65. DOI: 10.1016 / j.jim.2014.06.007.PLoS One 10 (2015): e0145686J. Extracell. Vesicles 3 (2014): 23430. DOI: 10.3402 / jev.v3.23430.Biol. Chem. 394, no. 10 (2013): 1253-1262. DOI: 10.1515 / hsz-2013-0141.CRDS National Research and Development Agency, Japan Science and Technology Agency, Center for Research and Development Strategy, Overview Report, Life Sciences and Clinical Medicine (2021) 499-519

[0013] There is still room for improvement in the therapeutic effects of central nervous system diseases such as cerebral infarction, and the creation of new pharmaceutical compositions that improve the efficacy of such central nervous system diseases is desirable. Furthermore, although research into the relationship between exosomes and diseases has been conducted, the focus of isolation methods for extracellular vesicles such as exosomes has been on improving their efficiency, and no studies have been conducted on production methods for obtaining purified products that are highly effective for specific therapeutic applications.

[0014] The present disclosure aims to provide a pharmaceutical composition that has excellent therapeutic effects in treating central nervous system disorders.

[0015] As a result of extensive research, the present inventors have found that by preparing a purified extracellular vesicle obtained by a unique production method as a composition for intraarterial administration, excellent therapeutic effects can be obtained in the treatment of central nervous system diseases. The present disclosure was completed through further research based on this finding.

[0016] That is, the present disclosure provides the following aspects of the invention. Item 1. A pharmaceutical composition for intravascular administration used to treat a central nervous system disease, comprising a purified extracellular vesicle product, wherein the purified extracellular vesicle product is obtained by a method for producing a purified extracellular vesicle product, the method comprising the following (i) purification step and (ii) membrane filtration step: (i) a purification step in which a treatment liquid containing extracellular vesicles and impurities is contacted with an exclusion and anion exchange carrier to obtain a treatment liquid containing the extracellular vesicles, and (ii) a membrane filtration step in which the treatment liquid is subjected to membrane filtration to obtain a concentrated solution of the extracellular vesicles. Item 2. The pharmaceutical composition for intravascular administration according to Item 1, wherein the central nervous system disease is an ischemic cerebrovascular disorder. Item 3. The pharmaceutical composition for intravascular administration according to Item 2, wherein the ischemic cerebrovascular disorder is cerebral infarction. Item 4. The pharmaceutical composition for intravascular administration according to Item 1, wherein the extracellular vesicles are concentrated in a concentration of 1 x 10 3 Item 5. The pharmaceutical composition for intravascular administration according to any one of Items 1 to 4, wherein the liquid to be treated is a culture supernatant of cells secreting extracellular vesicles or tissue thereof. Item 6. The pharmaceutical composition for intravascular administration according to any one of Items 1 to 5, wherein the exclusion and anion exchange carrier is a porous carrier having positively charged pores inside. Item 7. The pharmaceutical composition for intravascular administration according to any one of Items 1 to 6, wherein the membrane used for membrane filtration has a molecular weight cutoff of 100,000 to 1,000,000. Item 8. The pharmaceutical composition for intravascular administration according to any one of Items 1 to 7, wherein the membrane used for membrane filtration has a gamma globulin permeability of 10 to 80%. Item 9. The membrane used for membrane filtration has a pure water permeation flow rate of 500 to 1,500 L / m at 0.1 MPa. 2 / Hr. Item 10. The pharmaceutical composition for intravascular administration according to any one of Items 1 to 9, wherein the membrane used for the membrane filtration is a cellulose-based hydrophilic membrane. Item 11. The pharmaceutical composition for intravascular administration according to any one of Items 1 to 10, wherein the membrane used for the membrane filtration is a hollow fiber membrane. Item 12. The pharmaceutical composition for intravascular administration according to any one of Items 1 to 11, wherein the membrane filtration is tangential flow filtration. Item 13. The pharmaceutical composition for intravascular administration according to any one of Items 1 to 12, wherein the membrane surface velocity in the membrane filtration is 0.3 m / sec to 2 m / sec. Item 14. The pharmaceutical composition for intravascular administration according to any one of Items 1 to 13, wherein the concentration of extracellular vesicles contained in the extracellular vesicle concentrate is 10 times or more the concentration of extracellular vesicles contained in the treatment solution. Item 15. Item 16. The pharmaceutical composition for intravascular administration according to any one of Items 1 to 15, wherein the concentration of total protein contained in the concentrate of extracellular vesicles is 0.3 times or less the concentration of total protein contained in the liquid to be treated. Item 17. A method for producing a pharmaceutical composition for intravascular administration for treating ischemic cerebrovascular disease, comprising: (i) a purification step of contacting a liquid to be treated containing extracellular vesicles and impurities with an exclusion and anion exchange carrier to obtain a treated liquid containing the extracellular vesicles; and (ii) a membrane filtration step of subjecting the treated liquid to membrane filtration to obtain a concentrated liquid of extracellular vesicles. Item 18. A method for treating a central nervous system disease, comprising administering an effective amount of a purified extracellular vesicle product to a subject requiring intravascular administration for the treatment of the disease, wherein the purified extracellular vesicle product is obtained by a method for producing a purified extracellular vesicle product, the method comprising the following steps: (i) a purification step, in which a treatment liquid containing extracellular vesicles and impurities is contacted with an exclusion and anion exchange carrier to obtain a treatment liquid containing the extracellular vesicles; and (ii) a membrane filtration step, in which the treatment liquid is subjected to membrane filtration to obtain a concentrated solution of the extracellular vesicles.Item 19. Use of a purified extracellular vesicle product for the production of a pharmaceutical composition for intravascular administration used in the treatment of central nervous system diseases, wherein the purified extracellular vesicle product is obtained by a method for producing a purified extracellular vesicle product, the method comprising the following (i) purification step and (ii) membrane filtration step: (i) a purification step of contacting a treatment liquid containing extracellular vesicles and impurities with an exclusion and anion exchange carrier to obtain a treatment liquid containing the extracellular vesicles, and (ii) a membrane filtration step of subjecting the treatment liquid to membrane filtration to obtain a concentrated solution of the extracellular vesicles. A purified extracellular vesicle product for use in intravascular administration for the treatment of central nervous system diseases, which is obtained by a method for producing a purified extracellular vesicle product, comprising the following (i) purification step and (ii) membrane filtration step: (i) a purification step in which a treatment liquid containing extracellular vesicles and impurities is contacted with an exclusion and anion exchange carrier to obtain a treatment liquid containing the extracellular vesicles; and (ii) a membrane filtration step in which the treatment liquid is subjected to membrane filtration to obtain a concentrated solution of the extracellular vesicles.

[0017] According to the present disclosure, a pharmaceutical composition having excellent therapeutic effects for treating central nervous system disorders is provided.

[0018] 1 shows an outline of an apparatus for performing the membrane filtration step in the method for producing the purified extracellular vesicles produced in Test Example 1. 1 shows an outline of an apparatus for measuring the gamma globulin permeability of the hollow fiber membrane used in the membrane filtration step. 1 shows the effect of the purified extracellular vesicles (exosome purified concentrate A) produced in Test Example 3 on improving the survival rate or proliferation rate of microglial cells. 1 shows the effect of the purified extracellular vesicles (exosome purified concentrate A) produced in Test Example 3 on suppressing the secretion of inflammatory cytokines. 1 shows an intravascularly administered pharmaceutical composition containing the purified extracellular vesicles (exosome purified concentrate A) produced in Test Example 3 administered intraarterially to a rat model of cerebral infarction (2.0 x 10 exosome amount). 9 The results of evaluating motor function after intravascular administration of a pharmaceutical composition containing the purified extracellular vesicles (exosome purified concentrate A) produced in Test Example 3 to cerebral infarction model rats were shown. 9The figure shows the change in body weight after intravascular administration of a pharmaceutical composition containing the purified extracellular vesicles (exosome purified concentrate A) produced in Test Example 3 to a cerebral infarction model rat (2.0 × 10 exosome amount). 9 The figure shows a cross section of brain tissue after intravascular administration of a pharmaceutical composition containing the purified extracellular vesicles (exosome purified concentrate A) produced in Test Example 3 to a cerebral infarction model rat (5.0 × 10 exosome amount). 9 The results of evaluating motor function after intravascular administration of a pharmaceutical composition containing the purified extracellular vesicles (exosome purified concentrate A) produced in Test Example 3 to cerebral infarction model rats (5.0 × 10 exosome amounts) were shown. 9 The figure shows the change in body weight after intravascular administration of a pharmaceutical composition containing the purified extracellular vesicles (exosome purified concentrate A) produced in Test Example 3 to a cerebral infarction model rat (5.0 × 10 exosome amount). 9 The figure shows the rate of CD68-positive cells in the peri-cerebral infarction lesion after intra-arterial administration of a pharmaceutical composition for intravascular administration containing the purified extracellular vesicles (exosome purified concentrate A) produced in Test Example 3 to a cerebral infarction model rat (5.0 × 10 exosome amount). 9 The figures show the number of apoptosis in the peri-cerebral infarction lesion after intra-arterial administration of a pharmaceutical composition for intravascular administration containing the purified extracellular vesicles (exosome purified concentrate A) produced in Test Example 3 to a cerebral infarction model rat (5.0 × 10 exosome amount). 9 This shows the ability of the blood-brain barrier to be maintained in the infarcted hemisphere after MRI (individuals).

[0019] The pharmaceutical composition of the present disclosure is a pharmaceutical composition for intravascular administration used to treat central nervous system diseases, containing a purified extracellular vesicle product, and is characterized in that the purified extracellular vesicle product is obtained by a predetermined manufacturing method.

[0020] 1. Prescribed Manufacturing Method The prescribed manufacturing method includes the following (i) purification step and (ii) membrane filtration step. The purified extracellular vesicles obtained by the prescribed manufacturing method have excellent therapeutic effects in the treatment of central nervous system disorders. Furthermore, the prescribed manufacturing method makes it possible to quickly prepare purified extracellular vesicles in the concentration and amount required for treatment, thereby speeding up the start of treatment of central nervous system disorders. (i) A purification step in which a treatment liquid containing extracellular vesicles and impurities is contacted with an exclusion and anion exchange carrier to obtain a treatment liquid containing the extracellular vesicles. (ii) A membrane filtration step in which the treatment liquid is subjected to membrane filtration to obtain a concentrated solution of the extracellular vesicles.

[0021] 1-1. Liquid to be processed The liquid to be processed, which is the material for the predetermined manufacturing method, contains extracellular vesicles and impurities.

[0022] Types of extracellular vesicles include exosomes, microvesicles, apoptotic bodies, etc., and among these, exosomes are preferred from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure.

[0023] The organism from which the extracellular vesicles are derived is not particularly limited, and examples include mammals such as mice, cows, and humans.

[0024] The tissues, cells, or body fluids from which the extracellular vesicles are derived are not particularly limited, and examples of tissues or cells include bone marrow, dental pulp, fat, amniotic membrane, umbilical cord, periosteum, perichondrium, and cells thereof, preferably amniotic cells. Examples of body fluids include blood, umbilical cord blood, plasma, serum, saliva, urine, tears, sweat, breast milk, amniotic fluid, cerebrospinal fluid, bone marrow fluid, pleural effusion, ascites, synovial fluid, aqueous humor, and vitreous body.

[0025] The type of cells from which the extracellular vesicles are derived is not particularly limited, and may be any of germ cells, somatic cells, and stem cells. However, from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, stem cells are preferred. The cells may be either autologous or allogeneic cells for the treatment of central nervous system diseases. Furthermore, stem cells include hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, and skin stem cells. However, from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, mesenchymal stem cells are preferred. Furthermore, the origin of mesenchymal stem cells is not particularly limited, and examples include bone marrow, dental pulp, blood, fat, amniotic membrane, umbilical cord, umbilical cord blood, periosteum, perichondrium, and the like, and preferably amniotic membrane.

[0026] The liquid to be treated is not particularly limited as long as it is a biological sample containing extracellular vesicles and impurities. The biological sample refers to a cell or tissue culture or a specimen collected from an organism, and from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, a cell or tissue culture is preferred, and a cell culture is more preferred.

[0027] Contaminants are components that are mixed in with extracellular vesicles during the preparation of the biological sample, and include, for example, medium components, components produced by culture, and components contained in the tissues, cells, or body fluids from which the sample was collected, and include a wide variety of components including proteins.

[0028] In the present disclosure, from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition, particularly preferred treatment liquids include culture supernatants of cells or tissues thereof that secrete the extracellular vesicles, and most preferably culture supernatants of mesenchymal stem cells.

[0029] 1-2. Exclusion and anion exchange carrier The liquid to be treated is brought into contact with the exclusion and anion exchange carrier, thereby obtaining a treated liquid containing extracellular vesicles.

[0030] The exclusion and anion exchange carrier refers to a carrier capable of both size exclusion and anion exchange. To enable size exclusion, a carrier having pores may be used. Examples of the exclusion size include 400 kDa or more, preferably 500 kDa or more, more preferably 600 kDa or more, and even more preferably 650 kDa or more. To enable anion exchange, a positively charged carrier may be used. The exclusion and anion exchange carrier may be a combination of a carrier capable of only size exclusion and a carrier capable of only anion exchange, or may be a carrier capable of both size exclusion and anion exchange.

[0031] From the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, the exclusion and anion exchange carrier is preferably a carrier capable of both size exclusion and anion exchange, and more preferably a porous carrier in which the inside of the pores is positively charged. More preferably, the porous carrier in which the inside of the pores is positively charged includes particles in which the inside of the pores is positively charged and the portion other than the pores is not positively charged, and even more preferably, includes multilayer particles having an inner layer modified with a positively charged ligand and a porous shell layer not modified with the ligand.

[0032] The shape of the exclusion and anion exchange carrier is not particularly limited, but is preferably particulate. The average particle diameter of the particulate exclusion and anion exchange carrier is, for example, 1 to 1000 μm, 10 to 500 μm, 20 to 300 μm, or 20 to 150 μm. The average particle diameter refers to the particle diameter at 50% of the integrated value in the volume-based particle size distribution determined by a laser diffraction / scattering method (D50, median diameter).

[0033] The material of the exclusion and anion exchange carrier is not particularly limited, and examples thereof include polysaccharides such as cellulose, agarose, starch, amylose, dextran, pullulan, and glucomannan; synthetic organic polymers such as polyacrylic acid and its derivatives, polyvinyl alcohol, nylon, polysulfone, polyacrylnitrile, polyethylene, polypropylene, and polystyrene; and inorganic polymers such as glass, porous glass, silica gel, and hydroxyapatite, with polysaccharides being preferred, and agarose and silica gel being more preferred.

[0034] The exclusion and purification using an anion exchange carrier may be carried out by a chromatography method or a batch method.

[0035] The concentration of extracellular vesicles in the treatment solution obtained by the purification step (step (i)) can be, for example, 0.65 times or more (by weight) the concentration of extracellular vesicles in the treatment solution subjected to the step. From the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, the concentration is preferably 0.70 times or more, more preferably 0.75 times or more, even more preferably 0.80 times or more, even more preferably 0.9 times or more, and even more preferably 0.92 times or more or 0.94 times or more. The upper limit of the concentration of extracellular vesicles in the treatment solution can be 1 times or less the concentration of extracellular vesicles in the treatment solution. Specific examples of the concentration range of extracellular vesicles in the treatment solution include 0.65 to 1 times, 0.70 to 1 times, more preferably 0.75 to 1 times, even more preferably 0.80 to 1 times, even more preferably 0.9 to 1 times, and even more preferably 0.92 to 1 times or 0.94 to 1 times the concentration of extracellular vesicles in the treatment solution.

[0036] The total protein concentration in the treatment solution obtained by the purification step (step (i)) is, for example, 0.5 times or less (by weight) the total protein concentration in the treatment solution subjected to the step. From the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, the concentration is preferably 0.4 times or less, more preferably 0.3 times or less, even more preferably 0.2 times or less, and even more preferably 0.15 times or less. The lower limit of the total protein concentration in the treatment solution is not particularly limited, and examples include 0.05 times or more, 0.08 times or more, or 0.1 times or more of the total protein concentration in the treatment solution. Specific examples of the concentration range of the total protein in the treatment solution include 0.05 to 0.5 times, preferably 0.05 to 0.4 times, more preferably 0.05 to 0.3 times, even more preferably 0.05 to 0.2 times, and even more preferably 0.05 to 0.15 times, 0.08 to 0.15 times, or 0.1 to 0.15 times the total protein concentration in the treatment solution.

[0037] The molecular weight cutoff of the membrane used for membrane filtration is not particularly limited, but may be, for example, 100,000 to 1,000,000, and from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, preferably 150,000 to 800,000, more preferably 200,000 to 600,000, even more preferably 230,000 to 400,000, and even more preferably 250,000 to 350,000.

[0038] The gamma globulin permeability of the membrane used for membrane filtration is not particularly limited, and may be, for example, 5% to 95%. From the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, the permeability is preferably 10 to 80%, more preferably 20 to 70%, even more preferably 30 to 60%, and even more preferably 40 to 50%. When an aqueous gamma globulin solution is subjected to membrane filtration through a hollow fiber membrane at a filtration pressure of 0.1 MPa, the gamma globulin permeability is a value (%) derived from the following formula: (gamma globulin concentration in the permeate / gamma globulin concentration in the aqueous solution×100.

[0039] The pure water permeation flow rate of the membrane used for membrane filtration at 0.1 MPa is, for example, 500 to 1500 L / m 2 / Hr, and from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, it is preferably 600 to 1300 L / m 2 / Hr, more preferably 800 to 1100 L / m 2 / Hr.

[0040] The material of the membrane used for membrane filtration is not particularly limited, but examples thereof include hydrophobic membranes such as polyethersulfone membranes and polysulfone membranes, and hydrophilic membranes such as cellulose-based membranes. From the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, hydrophilic membranes such as cellulose-based membranes are preferred. Specific examples of the cellulose-based membranes include regenerated cellulose membranes and cellulose ester membranes (specifically, cellulose acetate membranes, cellulose propionate membranes, cellulose butyrate membranes, cellulose benzoate membranes, etc.). From the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, cellulose ester membranes are more preferred, and cellulose acetate membranes are particularly preferred.

[0041] The shape of the membrane used for membrane filtration is not particularly limited, but from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, a hollow fiber membrane is preferred. The inner diameter of the hollow fiber membrane is, for example, 0.2 mm to 1.4 mm, and from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, preferably 0.4 mm to 1.2 mm, more preferably 0.6 mm to 1.0 mm, and even more preferably 0.7 mm to 0.9 mm.

[0042] As the form of the membrane used for membrane filtration, particularly when a hollow fiber membrane is used, a bundle of a plurality of hollow fiber membranes (for example, 10 to 500, 10 to 200, 20 to 100, 50 to 100, 60 to 90, or 70 to 80) is preferably used.

[0043] The membrane filtration method may be either dead-end filtration or tangential flow (cross-flow) filtration, but from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, tangential flow filtration is preferred.

[0044] The membrane surface velocity (membrane surface flow velocity) in membrane filtration is, for example, 0.3 m / sec to 2 m / sec, and from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, is preferably 0.4 m / sec to 1.5 m / sec, more preferably 0.5 m / sec to 1.3 m / sec, even more preferably 0.6 m / sec to 1.2 m / sec, and even more preferably 0.8 to 1.2 m / sec.

[0045] The inlet pressure in membrane filtration may be, for example, 0.01 MPa to 0.2 MPa, and from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, preferably 0.015 MPa to 0.12 MPa, more preferably 0.02 MPa to 0.07 MPa.

[0046] The concentration of extracellular vesicles in the extracellular vesicle concentrate obtained by the membrane filtration step (step (ii)) can be, for example, 10 times or more (by weight) the concentration of extracellular vesicles in the treatment solution used in the step. From the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, the concentration can be preferably 20 times or more, more preferably 30 times or more, even more preferably 40 times or more, and even more preferably 45 times or more, 50 times or more, 80 times or more, or 100 times or more. The upper limit of the concentration of extracellular vesicles in the concentrate is not particularly limited, and can be, for example, 300 times or less, 200 times or less, 100 times or less, 60 times or less, or 55 times or less the concentration of extracellular vesicles in the treatment solution. Specific examples of the concentration range of extracellular vesicles in the concentrated solution include 10 to 300 times, 20 to 300 times, more preferably 30 to 300 times, even more preferably 40 to 300 times, and even more preferably 45 to 300 times, 45 to 200 times, 45 to 100 times, 45 to 60 times, 45 to 55 times, 50 to 300 times, 80 to 300 times, 80 to 200 times, 80 to 100 times, or 100 to 300 times the concentration of extracellular vesicles in the treatment solution.

[0047] The total protein concentration in the extracellular vesicle concentrate obtained by the membrane filtration step (step (ii)) is, for example, 0.3 times or less (by weight) the total protein concentration in the treated liquid subjected to the purification step (step (i)), and from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, is preferably 0.25 times or less, more preferably 0.2 times or less. The lower limit of the total protein concentration in the extracellular vesicle concentrate is not particularly limited, and examples thereof include 0.0001 times or more, 0.0005 times or more, 0.001 times or more, 0.05 times or more, 0.1 times or more, or 0.15 times or more the total protein concentration in the treated liquid. Specific examples of the concentration range of total protein in the extracellular vesicle concentrate include 0.0001 to 0.3 times, preferably 0.0001 to 0.25 times, more preferably 0.0001 to 0.2 times, 0.0005 to 0.2 times, 0.001 to 0.2 times, 0.05 to 0.2 times, 0.1 to 0.2 times, or 0.15 to 0.2 times the concentration of total protein in the liquid to be treated.

[0048] The amount of total protein in the extracellular vesicle concentrate obtained by the membrane filtration step (step (ii)) can be, for example, 2 times or less (by weight) the amount of total protein in the treatment solution used in the step (step (ii)). From the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, the amount is preferably 1.8 times or less, and more preferably 1.6 times or less. The lower limit of the amount of total protein in the extracellular vesicle concentrate is not particularly limited, and can be, for example, 0.4 times or more, 0.7 times or more, or 1 times or more the amount of total protein in the treatment solution. Specific examples of the concentration range of total protein in the extracellular vesicle concentrate include 0.4 to 2 times, preferably 0.4 to 1.8 times, more preferably 0.4 to 1.6 times, 0.7 to 1.6 times, or 1 to 1.6 times the amount of total protein in the treatment solution.

[0049] The volume of the extracellular vesicle concentrate obtained by the membrane filtration step (step (ii)) can be, for example, 10% (by volume) or less of the treated liquid subjected to the purification step (step (i)), and from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, is preferably 5% or less, more preferably 3% or less, and even more preferably 2% or less or 1% or less. The lower limit of the volume of the extracellular vesicle concentrate obtained by the membrane filtration step (step (ii)) is not particularly limited, and examples thereof include 0.005% or more, 0.001%, 0.01% or more, 0.1% or more, 0.5% or more, or 1% or more of the treated liquid subjected to the purification step (step (i)). Specific examples of the volume range of the concentrated solution of extracellular vesicles obtained by the membrane filtration step (step (ii)) include 0.005 to 10%, preferably 0.005 to 5%, more preferably 0.005 to 3%, and even more preferably 0.005 to 2%, 0.005 to 1%, 0.001 to 1%, 0.01 to 1%, 0.1 to 1%, 0.5 to 1%, or 1 to 2% of the treated solution subjected to the purification step (step (i)).

[0050] 2. Purified Extracellular Vesicle Product The purified extracellular vesicles contained in the pharmaceutical composition of the present disclosure are obtained by the above-mentioned predetermined production method. Specific forms of the purified extracellular vesicles include a concentrated solution of extracellular vesicles obtained by the above-mentioned predetermined production method, and an ultra-concentrated solution or dried product obtained by further removing water from the concentrated solution. Methods for obtaining the dried product include freeze-drying, spray drying, and air drying.

[0051] The types of extracellular vesicles contained in the purified extracellular vesicles include exosomes, microvesicles, apoptotic bodies, etc., and these may be contained alone or in combination. Among these extracellular vesicles, exosomes are preferred from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure.

[0052] Regarding the size of the extracellular vesicles contained in the purified extracellular vesicles, the average particle diameter of the extracellular vesicles is, for example, 15 to 250 nm. From the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, the average particle diameter is preferably 40 to 200 nm, more preferably 50 to 190 nm, 55 to 170 nm, even more preferably 65 to 150 nm, still more preferably 75 to 130 nm, and even more preferably 80 to 120 nm, 110 to 120 nm, or 100 to 120 nm. In the present disclosure, the average particle diameter is the 50% cumulative value (D 50 ) The mode diameter of the extracellular vesicles contained in the purified extracellular vesicles is, for example, 10 to 200 nm, and from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, is preferably 30 to 190 nm, 30 to 180 nm, more preferably 50 to 160 nm, even more preferably 60 to 140 nm, even more preferably 70 to 120 nm, and even more preferably 76 to 115 nm, 85 to 116 nm, or 93 to 115 nm. In addition, the particle size distribution width ((D 90 -D 10 ) / D 50 ) is, for example, 0.1 to 1.6, and from the viewpoint of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, is preferably 0.2 to 1.4, more preferably 0.3 to 1.2, even more preferably 0.4 to 0.95, even more preferably 0.5 to 0.85, and still more preferably 0.6 to 0.9, 0.65 to 0.85, or 0.7 to 0.79.

[0053] The content of the purified extracellular vesicles in the pharmaceutical composition of the present disclosure is not particularly limited as long as it is acceptable for a composition for intravascular administration and can be administered at a dose effective for treating central nervous system disorders. For example, the amount of extracellular vesicles may be 1 x 10 5 particles / μL or more, preferably 1×10 6 particles / μL or more, more preferably 1×10 7 particles / μL or more, more preferably 1×10 8 particles / μL or more, more preferably 1×10 9The upper limit of the content of the purified extracellular vesicles in the pharmaceutical composition of the present disclosure is not particularly limited, but the amount of extracellular vesicles can be, for example, 1 × 10 13 pcs / μL or less, 1×10 12 pcs / μL or less, 1×10 11 pcs / μL or less, or 1×10 10 Specific examples of the range of the content of the purified extracellular vesicles include 1 x 10 extracellular vesicles / μL or less. 5 ~1 x 10 13 / μL, preferably 1 × 10 6 ~1 x 10 13 particles / μL, more preferably 1×10 7 ~1 x 10 13 cells / μL, more preferably 1×10 8 ~1 x 10 13 cells / μL, more preferably 1×10 9 ~1 x 10 13 pieces / μL, 1×10 9 ~1 x 10 12 pieces / μL, 1×10 9 ~1 x 10 11 cells / μL, or 1 x 10 9 ~1 x 10 10 Examples include:

[0054] Furthermore, the content of the purified extracellular vesicle product in the pharmaceutical composition of the present disclosure may be, in terms of the amount of extracellular vesicles, for example, 1 to 100,000 μg / mL, 4 to 10,000 μg / mL, or 8 to 5,600 μg / mL.

[0055] 3. Other Components The pharmaceutical composition of the present disclosure contains the above-described purified extracellular vesicles as an active ingredient, and may or may not contain, as other components, pharmacologically acceptable bases and / or additives that are acceptable in compositions for intravascular administration.

[0056] Examples of pharmacologically acceptable bases and / or additives include excipients, thickeners, solvents, solubilizers, suspending agents, emulsifiers, isotonicity agents, buffers, soothing agents, stabilizers, preservatives (antiseptics), pH adjusters, cooling agents, antioxidants, humectants, adhesives, etc. These bases and additives may be used alone or in combination of two or more.

[0057] 4. Formulation The pharmaceutical composition of the present disclosure may be either a liquid or a powder acceptable for intravascular administration, preferably a liquid. When preparing a liquid, the purified extracellular vesicles may be mixed with a solvent, a solubilizer, a suspending agent, an isotonic agent, a buffer, and / or a soothing agent, etc., as needed, followed by dissolution, suspension, or emulsification. When preparing a powder, the purified extracellular vesicles may be mixed with an excipient, a binder, a disintegrant, and / or other suitable additives as needed, and then dried as needed.

[0058] 5. Uses The pharmaceutical composition of the present disclosure is used for the treatment of central nervous system disorders. Examples of central nervous system disorders include ischemic cerebrovascular disorders and neurodegenerative disorders. Examples of ischemic cerebrovascular disorders include cerebral embolism, transient cerebral ischemia, subclavian steal syndrome, Wallenberg syndrome (lateral medullary syndrome), cerebral thrombosis, lacunar infarction, reversible ischemic neuropathy, cerebral infarction, Moyamoya disease (circle of Willis occlusion), hypoxic encephalopathy, sinus thrombosis, and postoperative spinal cord ischemia. Examples of neurodegenerative diseases include Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease, and amyotrophic lateral sclerosis. Since the production method for obtaining the pharmaceutical composition of the present disclosure enables rapid preparation of purified extracellular vesicles at the concentration and amount required for treatment, the central nervous system disorder is preferably ischemic cerebrovascular disorders, which are highly urgent conditions requiring immediate treatment, and more preferably cerebral infarction.

[0059] In addition, the pharmaceutical composition of the present disclosure has an excellent anti-inflammatory effect, and therefore is preferably used for suppressing post-ischemic inflammation in ischemic cerebrovascular disorders such as cerebral infarction. Furthermore, the pharmaceutical composition of the present disclosure also has an excellent effect of improving cerebral edema that occurs after ischemic cerebrovascular disorders such as cerebral infarction, and therefore is preferably used for improving cerebral edema that occurs after ischemic cerebrovascular disorders such as cerebral infarction.

[0060] The pharmaceutical composition of the present disclosure is used for intravascular administration. That is, the pharmaceutical composition of the present disclosure is used for intraarterial administration or intravenous administration. Preferably, the pharmaceutical composition of the present disclosure is used for intraarterial administration. In particular, when the pharmaceutical composition of the present disclosure is used for the treatment of cerebral infarction, the pharmaceutical composition of the present disclosure can be administered intraarterially via a catheter placed in the carotid artery leading to the brain immediately after catheter treatment for cerebral infarction.

[0061] The dosage of the pharmaceutical composition of the present disclosure to humans can be determined appropriately depending on the disease to be treated, the level of symptoms, etc., but the amount of extracellular vesicles is, for example, 1 × 10 7 pieces / kg or more, preferably 1 x 10 8 pieces / kg or more, more preferably 1 x 10 9 pieces / kg or more, more preferably 1 x 10 10 pieces / kg or more, more preferably 1 x 10 11 pieces / kg or 1 x 10 12 The upper limit of the dose of the pharmaceutical composition of the present disclosure is not particularly limited, but may be, for example, 1 x 10 extracellular vesicles / kg or more. 14 pieces / kg or less, 1×10 13 pieces / kg or less, 5×10 12 pieces / kg or less, 5×10 11 pieces / kg or less, or 5 x 10 10 Specific examples of dosage ranges for humans of the pharmaceutical compositions of the present disclosure include 1 x 10 extracellular vesicles / kg or less. 7 ~1 x 10 14 pieces / kg, preferably 1 x 10 8 ~1 x 10 14 pieces / kg, more preferably 1 x 10 9 ~1 x 10 14 pieces / kg, more preferably 1 x 1010 ~1 x 10 14 pieces / kg, 1×10 11 ~1 x 10 14 pieces / kg, 1×10 12 ~1 x 10 14 pieces / kg, 1×10 12 ~1 x 10 13 pieces / kg, 1×10 10 ~5 x 10 12 pieces / kg, 1×10 10 ~5 x 10 11 pieces / kg, or 1 x 10 9 ~5 x 10 10 Examples include pieces / kg.

[0062] Each feature disclosed herein may be combined with any other feature disclosed herein.

[0063] The present invention will be described in more detail below with reference to examples, but the configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations may be made as appropriate within the scope of the present invention. The present disclosure is not limited by the embodiments, but is limited only by the scope of the claims.

[0064] Test Example 1 (I) Preparation of mesenchymal stem cell culture supernatant (liquid to be treated containing extracellular vesicles and impurities) Human-derived mesenchymal stem cells (obtained from 56.4 mL of bone marrow fluid via mononuclear cell isolation and seeding) were cultured using a cell culture medium (MEMα + 5% allogeneic platelet lysates (PL) + 1% antibiotics (penicillin streptomycin; P / S)). The culture period was 21 days, during which 3.5 L of culture supernatant was obtained.

[0065] (II) Purification step (step (i)) 250 mL of the 3.5 L of culture supernatant obtained in (I) above (470.5 mg of total protein by the Bradford method) was added to 50 mL of a carrier (a porous granular material with positively charged pores inside, capable of adsorbing and retaining negatively charged unnecessary substances excluding extracellular vesicles) equilibrated with 50 mM Tris-HCl (pH 7.2) solution, and the mixture was then dispensed into a 50 mL tube. The mixture was then mixed by inversion at a rotation speed of 10 rpm at room temperature for 10 minutes using a rotator. The mixture was then centrifuged at 10,000 x g for 2 minutes at room temperature, and the supernatant was filtered using a Stericup 0.22 μm filtration unit (S2GPU11RE, Merck Millipore) to remove the carrier, yielding a treatment solution containing extracellular vesicles. The total protein amount in the resulting treated solution, as determined by the Bradford method, was 195.3 mg, which was reduced to 42% of the amount before the purification step.

[0066] (III) Membrane Filtration Step (Step (ii)) 250 mL of the treated liquid obtained in (II) above was subjected to membrane filtration using the membrane filtration device 1 shown in FIG.

[0067] The membrane filtration device 1 shown in FIG. 1 includes: a hollow fiber membrane module 30; a first tank 10 and a second tank 20 in liquid communication with both ends of the hollow fiber membrane module 30; a buffer tank 40 in liquid communication with the first tank 10 via a buffer solution delivery line 45 with an on-off valve 46 that can be opened and closed; a first tank gas supply line 53 in gas communication with the first tank 10; a second tank gas supply line 54 in gas communication with the second tank 20; a three-way valve 61 that openably and closedly connects a gas supply line 52 with a pressure gauge 51 to the first tank gas supply line 53 or the second tank gas supply line 54; a first gas vent line 55 with an on-off valve 62 that can be opened and closed and connected by gas to the first tank 10; and a second gas vent line 56 with an on-off valve 63 that can be opened and closed and connected by gas to the second tank 20.

[0068] The details of each component of the membrane filtration device 1 are as follows: First tank 10 and second tank 20: Material: Acrylic ester resin; Size: Length 25 cm, inner diameter 0.25 cm, capacity 120 cm 3Buffer solution tank 40 Capacity: 1.6 L Hollow fiber membrane module 30 Hollow fiber size: inner diameter 0.8 mm, outer diameter 1.3 mm, length 17 cm Material: cellulose acetate (CA) Molecular weight cutoff: 300,000 Gamma globulin permeability measured by the following method: 54% Pure water permeation flow rate at 0.1 MPa: 980 L / m 2 / Hr Form: A bundle of 15 cellulose acetate (CA) hollow fiber membranes (manufactured by Daisen Membrane Systems Co., Ltd.) was placed in a polycarbonate cylindrical container, and the membrane area was 0.0062 m 2 <Measurement of γ-globulin permeability> Using the γ-globulin permeability measuring device 300 of FIG. 2, γ-globulin permeability was measured by the following procedure. 1) A γ-globulin aqueous solution (γ-globulin concentration 100 ppm) was charged into the tank 302. 2) The hollow fiber membrane 303 was connected to the flow path 305. 3) Nitrogen gas was injected through the valve V-1 to apply a pressure of 0.1 MPa to the tank 302. The aqueous solution was filtered from the inner surface to the outer surface of the hollow fiber membrane 303, the filtrate was discarded in the container 307, and the permeate was recovered in the container 306. The initial permeate (approximately 100 mL) was discarded, and the permeate discharged thereafter was used as a sample for permeability measurement. After preparing a sample of the permeate using the device 300, the γ-globulin permeability was measured by the following method. 4) Using a spectrophotometer (Shimadzu Corporation, product name "UV2450"), the absorbance (A1) at 280 nm of the γ-globulin aqueous solution (100 ppm) and the absorbance (A2) at 280 nm of the permeated solution were measured, and the γ-globulin transmittance (%) was calculated using the formula (A2 / A1×100).

[0069] Membrane filtration was performed at approximately 20°C using the following procedure. (1) The treated liquid obtained in (II) above was placed in the first tank 10. (2) Nitrogen gas was supplied to the upper space of the first tank 10 at a pressure of 0.04 MPa, and the diluted treated liquid in the first tank 10 was passed through the hollow fiber membrane module 30 to perform tangential flow filtration. During this process, the second tank 20 was open to the atmosphere with the on-off valve 56 open, and the pressure was zero. The membrane surface linear velocity flowing through the hollow fiber membrane module 30 was 1.0 m / s. The membrane surface linear velocity was calculated from the rate of increase in the volume of concentrated liquid in the second tank 20. The permeated liquid from the hollow fiber membrane module 30 was stored in the permeated liquid tank 35, and the concentrated liquid was transferred to the second tank 20. As a result, the diluted treated liquid in the first tank 10 passed through the hollow fiber membrane module 30 and was membrane filtered. (3) When most of the treated liquid in the first tank 10 was membrane filtered, nitrogen gas was supplied to the second tank 20 at a pressure of 0.04 MPa by switching the three-way valve 61, and at the same time, the pressure in the first tank 10 was released by opening the on-off valve 62. As a result, the concentrated liquid in the second tank 20 was passed through the hollow fiber membrane module 30, thereby performing tangential flow filtration in the opposite direction to that described in (2) above. The permeated liquid was stored in the permeated liquid tank 35, and the concentrated liquid was transferred to the first tank 10. As a result, the concentrated liquid in the second tank 20 was passed through the hollow fiber membrane module 30 and membrane filtered. (4) As described in (2) and (3) above, the concentrated liquid that had transferred to the first tank 10 was subjected to tangential flow filtration from the first tank 10 to the second tank 20 and from the second tank 20 to the first tank 10, alternately and repeatedly, until the volume of the concentrated liquid reached 10 ml. To perform backwashing, 90 mL of phosphate buffer solution (PBS) was supplied to the hollow fiber membrane module 30 from a tank not shown, and then alternating tangential flow filtration and the supply of 90 mL of phosphate buffer solution were repeated two more times, after which 11.2 mL of purified concentrate (extracellular vesicle purified product) was obtained by alternating tangential flow filtration.

[0070] The total protein concentration of the finally obtained purified exosome concentrate (purified extracellular vesicles) was 22.5 mg, and the protein content had been reduced to 4.8% through the purification and membrane filtration processes.

[0071] (IV) OGD Experiments Cryopreserved neuroblastoma cell lines (SH-SY5Y, ATCC) were cultured in SY5Y-specific medium (eMEM: 45%, F12: 45%, FBS: 10%, P / S: 1%) and plated at 1.2 × 10 cells per well in a 24-well dish (Falcon). 5 The cells were seeded under 1 / well conditions. Then, in a hypoxic chamber (Invivo2 300) under an atmosphere of 1% oxygen, 5% CO2, and 94% nitrogen, the medium was replaced with glucose-free DMEM medium, and hypoxia and low glucose depletion (OGD) was performed for 24 hours. After OGD, the cells were returned to a normal incubator (21% oxygen), and the medium was replaced with DMEM high glucose (without FBS) medium.

[0072] At the time of medium change, the purified concentrate of exosomes (purified extracellular vesicles) obtained in (III) above was added to the medium containing 1.7 × 10 exosomes. 10 The cells were added to the culture medium at a concentration of 10 μL per well. A control well was also set up without exosomes. After 72 hours of culture, the medium was aspirated, and 500 μL of detachment solution (Triple Select, GIBCO) was added to each well. The cells were incubated at 37°C for approximately 5 minutes, and then 500 μL of the above medium was added to neutralize the cells. The pellet obtained after centrifugation was diluted with 1 ml of medium, and the cell count, viability, etc. were measured using Luna (Logos Biosystems). The results are shown in Table 1 below.

[0073]

[0074] As shown in Table 1, the purified extracellular vesicles obtained through steps (i) and (ii) significantly improved the proliferation rate of neuroblastoma cells under hypoxia and low glucose loading (OGD). Furthermore, the purified extracellular vesicles improved not only the total cell number but also the ratio of viable cells (survival rate) under OGD, resulting in a significantly increased number of viable cells.

[0075] Test Example 2 The purified exosome concentrate (extracellular vesicle purified product) obtained in Test Example 1 was subjected to particle size analysis using a nanoparticle multi-analyzer qNano (IZON). The mean particle diameter (50% cumulative value (D) calculated from the volume-based particle size distribution measured by laser diffraction / scattering method) was used. 50 )) is 84 nm, the mode diameter is 79 nm, and the particle size distribution width ((D 90 -D 10 ) / D 50 ) is 0.68, and the number of particles is 1.7 x 10 12 The number was 1 / mL.

[0076] Test Example 3 (1) Preparation of Purified Exosome Concentrate A (Purified Extracellular Vesicle Product) A treatment solution was prepared in the same manner as in "(II) Purification Step (Step (i))" of Test Example 1, using a culture supernatant of human-derived amniotic mesenchymal stem cells (MSCs).

[0077] Approximately 3,185 mL of the resulting treated solution (5,159.7 mg of total protein measured by the BCA method, 512.8 ng of CD9 / CD63-positive exosomes measured by the ELISA method (equivalent to the CD9 / CD63 fusion standard protein used in the calibration curve)) was filtered using a Stericup 0.22 μm filtration unit (S2GPU11RE, Merck Millipore) and subjected to membrane filtration in the same manner as in Test Example 1. However, the hollow fiber membrane module used had the following specifications.

[0078] Hollow fiber size: inner diameter 0.8 mm, outer diameter 1.3 mm, length 17 cm Material: cellulose acetate (CA) Molecular weight cutoff: 300,000 Gamma globulin permeability measured by the following method: 54% Pure water permeation flow rate at 0.1 MPa: 980 L / m 2 / Hr Form: A bundle of 125 cellulose acetate (CA) hollow fiber membranes (manufactured by Daisen Membrane Systems Co., Ltd.) was placed in a polycarbonate cylindrical container, and the membrane area was 0.05 m 2

[0079] The total protein amount of the finally obtained 26 mL of purified exosome concentrate A (extracellular vesicle purified product) was 35.1 mg, and the protein amount had decreased to 0.7%. The exosome amount was 372.2 ng, and the recovery rate was 73%. Furthermore, when the obtained purified exosome concentrate was analyzed using a nanoparticle imaging analyzer VIDEO DROP, the average particle diameter (50% cumulative value (D) calculated from the volume-based particle size distribution measured by laser diffraction / scattering method) was 1.0 μg. 50 )) is 187 nm, the mode diameter is 185 nm, and the particle size distribution width ((D 90 -D 10 ) / D 50 ) is 1.14, and the number of particles is 5.2 x 10 10 The number was 1 / mL.

[0080] (2) Preparation of Comparative Exosome Purification Concentrate B by Ultracentrifugation The culture supernatant of human-derived mesenchymal stem cells (MSCs) used in (1) above was centrifuged and the supernatant was collected twice (first centrifugation conditions: 2000 g, 10 minutes, 4°C; first centrifugation conditions: 10,000 g, 30 minutes, 4°C). The resulting supernatant was ultracentrifuged under the following conditions (ultracentrifugation conditions: 100,000 g, 70 minutes, 4°C). The supernatant was discarded, and as much moisture as possible was wiped off from the tube to obtain exosome precipitates adhering to the inner wall of the tube. This was suspended in approximately 200 μl of PBS to obtain Comparative Exosome Purification Concentrate B.

[0081] The total protein amount of the finally obtained comparative exosome purified concentrate B was 140.4 μg. Analysis using the nanoparticle imaging analyzer VIDEO DROP revealed that the mean particle diameter (50% cumulative value (D) calculated from the volume-based particle size distribution measured by laser diffraction / scattering method) was 140.4 μg. 50 )) is 137 nm, the mode diameter is 135 nm, and the particle size distribution width ((D 90 -D 10 ) / D 50 ) is 1.08, and the number of particles is 2.3 x 10 11 The number was 1 / mL.

[0082] (3) Effect of Improving Survival Rate or Proliferation Rate of Microglial Cells Microglial cell line BV2 (Elabscience) was cultured in a 96-well dish (Falcon) at 2.0 × 10 4 One day later, E. coli 026:B6-derived lipopolysaccharide (L8274, Sigma-Aldrich) was added to a concentration of 1,000 ng / well, and 2.8 x 10 9 Exosome purified concentrate A obtained in (1) above or comparative exosome purified concentrate B obtained in (2) above was added to the cells so that the cells were counted at 1000 / well. After further culturing for 1 day, the number of viable cells was counted. The number of viable cells was counted using CK04 Cell Counting Kit-8 (Dojindo Laboratories), and the absorbance at 450 nm was measured as the number of viable cells. The same procedure was performed except that PBS solution was added without lipopolysaccharide or exosomes (Control-1). The same procedure was also performed except that no exosomes were added (lipopolysaccharide was added) (Control-2). The results are shown in Figure 3.

[0083] As shown in Figure 3, it was revealed that the addition of the purified exosome concentrate A obtained in (1) above could improve the survival rate or proliferation rate of microglial cells, and that this effect was greater than that achieved when the comparative purified exosome concentrate B obtained in (2) above was added.

[0084] Microglial cells become activated during pathological conditions, dramatically changing their cellular properties, accumulating and proliferating at the lesion site, and phagocytosing and eliminating damaged neurons. Based on the effect of improving the survival rate or proliferation rate of microglial cells confirmed by the addition of purified exosome concentrate A, it can be reasonably inferred that purified extracellular vesicles obtained by (i) the purification process and (ii) the membrane filtration process, such as purified exosome concentrate A, can better promote the phagocytosis and elimination of damaged neurons than purified extracellular vesicles obtained by ultracentrifugation, such as comparative purified exosome concentrate B.

[0085] (4) Inhibitory effect on secretion of inflammatory cytokines. Microglial cell line BV2 (Elabscience) was cultured in a 96-well dish (Falcon) at 2.0 × 10 4 One day later, E. coli 026:B6-derived lipopolysaccharide (L8274, Sigma-Aldrich) was added to a concentration of 10 ng / well, and 2.8 x 10 9 The purified exosome concentrate A obtained in (1) above or the comparative purified exosome concentrate B obtained in (2) above was added to the cells so that the cells were counted at 100 / well. After culturing for another day, the culture supernatant was collected, and the amounts of the inflammatory cytokines TNFα and IL-6 contained in the supernatant were quantified by ELISA (using a Mouse TNF-alpha Quantikine ELISA Kit and a Mouse IL-6 Quantikine ELISA Kit (both manufactured by R&D Systems)). The same procedure was performed, except that PBS solution was added without lipopolysaccharide or exosomes (Control-1). The same procedure was also performed, except that no exosomes were added (lipopolysaccharide was added) (Control-2). The results are shown in Figure 4.

[0086] As shown in Figure 4, it was revealed that the addition of the purified exosome concentrate A obtained in (1) above was able to suppress the secretion of inflammatory cytokines, and that the effect was greater than that when the comparative purified exosome concentrate B obtained in (2) above was added.

[0087] When the brain is damaged, ischemic necrotic brain cells release DAMPs such as HMGB1 and PRX. HMGB1 disrupts the blood-brain barrier, allowing macrophages in the blood to infiltrate brain tissue. DAMPs activate microglia or macrophages via pattern recognition receptors such as TLRs, inducing the production of inflammatory cytokines, leading to, for example, cerebral edema or further neurological damage after cerebral infarction. The inhibitory effect of microglial TNF-α and IL-6 secretion confirmed by the addition of purified exosome concentrate A suggests that extracellular vesicle purified products obtained by (i) the purification process and (ii) the membrane filtration process, such as purified exosome concentrate A, are superior active ingredients for ischemic cerebrovascular disorders such as cerebral infarction due to their greater suppression of inflammatory cytokine secretion compared to extracellular vesicle purified products obtained by ultracentrifugation, such as comparative exosome purified concentrate B.

[0088] Test Example 4 Rats (SD rats, 260-290 g (8 weeks old)) underwent middle cerebral artery occlusion (MCAO) and then recanalization 120 minutes later. Rats showing a 70% or greater decrease in CBF and neurological symptoms of Bederson score 3 or greater were used as cerebral infarction models.

[0089] Immediately after the closure, transarterial administration was performed. For transarterial administration, the purified exosome concentrate A (purified extracellular vesicles) prepared in Test Example 3 was administered at a concentration of 2.0 × 10 exosomes. 9 2.0 × 10 cells / 500 μL of PBS or 500 μL of PBS as a control were administered. 9 The dose of 4 × 10 cells / 500 μL of PBS for humans is 11 ~14 x 10 11 The dose equivalent to the human dose is reasonably calculated based on the body weight ratio (approximately 300 g for mice vs. 60 kg for humans = approximately 200 times) or brain weight ratio (2 g for mice vs. 1350 g for humans = 700 times).

[0090] For the group administered purified exosome concentrate A and the control group, motor function was evaluated based on the modified neurological severity score (mNSS) neuropathy level before MCAO (day 0) and on days 1, 4, and 7 after MCAO. The results are shown in Figure 5.

[0091] As shown in Figure 5, an improvement in the motor function recovery effect was observed in the group administered with purified exosome concentrate A.

[0092] Body weights were measured for the group administered purified exosome concentrate A and the control group before MCAO (day 0) and on days 1, 4, and 7 after MCAO. The results are shown in Figure 6.

[0093] It is known that there is a negative correlation between cerebral edema and body weight after MCAO, and weight loss after MCAO is considered to be a sign of high levels of cerebral edema and a poor prognosis. As shown in Figure 6, the group administered purified exosome concentrate A showed good recovery in body weight, indicating a high improvement in cerebral edema after cerebral infarction and a good prognosis.

[0094] Paraffin sections of rat brains were prepared 7 days after MCAO for the group administered purified exosome concentrate A and the control group, and Nissl stained. The percentage of non-infarcted area was calculated using the following formula. The results are shown in Figure 7. Percentage of non-infarcted area (%) = infarcted area on the infarcted side / hemispherical area on the non-infarcted side × 100

[0095] As shown in Figure 7, it was revealed that cerebral infarction can be reduced by intraarterial administration of purified exosome concentrate A.

[0096] Test Example 5 (1) Preparation of Cerebral Infarction Model Rats (SD rats, 265-280 g, male, 8 weeks old) were anesthetized with isoflurane and nitrous oxide gas, and subjected to middle cerebral artery occlusion (MCAO) according to the following procedure.

[0097] With the rat in the lateral position, the skin on the right side of the head was incised and the temporalis muscle exposed. A high-speed drill was used to drill out the area just above the bony sutures on the right skull, and cerebral blood flow (CBF) in the middle cerebral artery (MCA) branch was measured using a laser Doppler. Then, with the rat in the supine position, the right neck was approached, the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were identified, and the superior thyroid artery and ascending pharyngeal artery were coagulated and separated. The external carotid artery was also ligated and separated. The pterygopalatine artery (PPA) and CCA were clipped, and a 0.37 mm-tipped Doccol was guided from the ECA to the ICA and placed when resistance was felt.

[0098] The rats were again placed in a lateral position, and CBF after MCAO was monitored from the right side of the skull. Those with CBF less than 30% of pre-occlusion levels were selected. The rats were then awakened and anesthetized again 2 hours later to remove the thrombus. A 27G blunt ophthalmic needle was placed from the ECA to the ICA. Rats with a Bederson score of grade 3 were used as cerebral infarction models.

[0099] (2) Administration of purified exosome concentrate A Immediately after opening, the purified exosome concentrate A (purified extracellular vesicles) prepared in Test Example 3 was administered using an autoinjector in an amount of 5.0 × 10 exosomes. 9 5.0 × 10 cells / 500 μL of PBS, or 500 μL of PBS as a control, were administered intravenously over 10 minutes. 9 The dose of 10 x 10 cells / 500 μL of PBS is 10 x 10 for humans. 11 ~35 x 10 11 The study was blinded as to whether purified concentrate A or the control was administered.

[0100] (3) Mortality The mortality rate 7 days after MCAO was 12.5% ​​(1 / 8) in the group administered purified exosome concentrate A and 40% (4 / 11) in the control group.

[0101] (4) Motor function Motor function was evaluated based on the modified neurological severity score (mNSS) for rats (N = 7 in the exosome purified concentrate A administration group and N = 7 in the control group) 7 days after MCAO. The results are shown in Figure 8.

[0102] As shown in Figure 8, a significant improvement in neural function was observed in the group administered with purified exosome concentrate A (5.43 in the group administered with purified exosome concentrate A vs. 8.43 in the control group, p = 0.0107 by t-test).

[0103] (5) Body Weight The results of measuring the body weight of rats (N = 7 in the group administered with purified exosome concentrate A, N = 7 in the control group) 7 days after MCAO are shown in Figure 9. As shown in Figure 9, a favorable trend toward improvement in body weight was observed in the group administered with purified exosome concentrate A.

[0104] (6) CD68 (blood-derived macrophage count) Infarct volume was evaluated using tissue sections taken 7 days after MCAO. 5 μm slices were prepared directly below the Bregma, reacted with anti-CD68 antibodies, and stained with DAB. The peri-infarct lesion was defined as the area 2000 μm from the midline, and 10 ROIs (100 μm x 100 μm) were placed in the basal ganglia. The positive cell rate in each ROI was examined. The results are shown in Figure 10. As shown in Figure 10, the positive rate was significantly lower in the exosome purified concentrate A administration group (n = 6 per group, exosome purified concentrate A administration group 8.7% vs. control group 12.1%, p = 0.0055 by t-test).

[0105] (7) Number of Apoptosis Infarct volume was evaluated using tissue sections taken 7 days after MCAO. 5 μm slices were prepared directly below the Bregma and stained with the ApopTag Fluorescein In Situ Apoptosis Detection Kit (S7110, Chemicon International, Temecula, CA, USA). The area 2000 μm from the midline was defined as the peri-infarct focus, and 10 ROIs (100 μm × 100 μm) were placed in the basal ganglia. The number of positive cells in each ROI was verified. The results are shown in Figure 11. As shown in Figure 11, the positive rate was significantly lower in the group administered purified exosome concentrate A. (n = 3 per group, 12.1 cells in the exosome concentrate A group vs. 48 cells in the control group, p = 0.0007 by t-test)

[0106] (8) Exosome distribution in the body (PET measurement) Instead of the above (2), exosomes 64 Similar to Purified Concentrate A except labeled with Cu 64 Purified concentrate A' of Cu-labeled exosomes was used.

[0107] 64 0.3 ml of purified concentrated solution A' of Cu-labeled exosomes was administered intra-arterially over 8 minutes, and the animals were euthanized 1 hour after administration and autoradiographed for brain imaging. 64 Accumulation of Cu was confirmed.

[0108] ​(9) Blood-brain barrier maintenance ability: A cerebral infarction model was prepared in the same manner as in (1) and (2) above, and purified exosome concentrate A was administered intravenously. Three days after MCAO, 2% Evans blue was administered intravenously via the femoral vein at a volume of 5 ml / kg. After 24 hours, the animals were euthanized, and the brain was removed after normal saline perfusion. The infarcted hemisphere was homogenized with 1.5 ml of 50% trichloroacetic acid and centrifuged at 15,000 × g for 30 minutes. 1 ml of absolute ethanol was added to 0.5 ml of the supernatant, and the mixture was left to stand at 4 ° C for 24 hours. It was then centrifuged again at 15,000 × g for 30 minutes, and the absorbance of the supernatant was measured (620 nm). The results are shown in Figure 12. As shown in Figure 12, the group administered with purified exosome concentrate A had significantly lower absorbance, indicating improved blood-brain barrier maintenance ability (group administered with purified exosome concentrate A: 0.0047 vs. control group: 0.0126).

Claims

1. A pharmaceutical composition for intravascular administration containing a purified extracellular vesicle product and used to treat central nervous system diseases, wherein the purified extracellular vesicle product is obtained by a method for producing a purified extracellular vesicle product, the method comprising the following steps: (i) a purification step, in which a treatment liquid containing extracellular vesicles and impurities is contacted with an exclusion and anion exchange carrier to obtain a treatment liquid containing the extracellular vesicles; and (ii) a membrane filtration step, in which the treatment liquid is subjected to membrane filtration to obtain a concentrated solution of the extracellular vesicles.

2. The pharmaceutical composition for intravascular administration according to claim 1, wherein the central nervous system disease is ischemic cerebrovascular disease.

3. The pharmaceutical composition for intravascular administration according to claim 2, wherein the ischemic cerebrovascular disorder is cerebral infarction.

4. 1 x 10 extracellular vesicles 3 The pharmaceutical composition for intravascular administration according to claim 2, comprising more than 1000 pieces / μL.

5. The pharmaceutical composition for intravascular administration according to claim 1, wherein the liquid to be treated is a culture supernatant of cells or tissues thereof that secrete the extracellular vesicles.

6. A pharmaceutical composition for intravascular administration according to claim 1, wherein the exclusion and anion carrier is a porous carrier in which the interior of the pores is positively charged.

7. The pharmaceutical composition for intravascular administration according to claim 1, wherein the membrane used for said membrane filtration has a molecular weight cutoff of 100,000 to 1,000,000.

8. The pharmaceutical composition for intravascular administration according to claim 1, wherein the membrane used for said membrane filtration has a gamma globulin permeability of 10 to 80%.

9. The pure water permeation flow rate of the membrane used for the membrane filtration at 0.1 MPa is 500 to 1500 L / m 2 2. The pharmaceutical composition for intravascular administration according to claim 1, wherein the total blood flow rate is 1000 kcal / Hr.

10. A pharmaceutical composition for intravascular administration according to claim 1, wherein the membrane used for membrane filtration is a cellulose-based hydrophilic membrane.

11. The pharmaceutical composition for intravascular administration according to claim 1, wherein the membrane used for membrane filtration is a hollow fiber membrane.

12. The pharmaceutical composition for intravascular administration according to claim 1, wherein the membrane filtration is tangential flow filtration.

13. The pharmaceutical composition for intravascular administration according to claim 1, wherein the membrane surface velocity during membrane filtration is 0.3 m / sec to 2 m / sec.

14. A pharmaceutical composition for intravascular administration according to claim 1, wherein the concentration of the extracellular vesicles contained in the extracellular vesicle concentrate is 10 times or more the concentration of the extracellular vesicles contained in the treatment solution.

15. A pharmaceutical composition for intravascular administration as described in claim 1, wherein the concentration of total protein contained in the concentrated extracellular vesicle solution is 0.3 times or less the concentration of total protein contained in the treated solution.

16. A pharmaceutical composition for intravascular administration according to claim 1, which is for intraarterial administration.

17. A method for producing a pharmaceutical composition for intravascular administration for treating ischemic cerebrovascular disease, comprising: (i) a purification step of contacting a treated liquid containing extracellular vesicles and impurities with an exclusion and anion exchange carrier to obtain a treated liquid containing the extracellular vesicles; and (ii) a membrane filtration step of subjecting the treated liquid to membrane filtration to obtain a concentrated liquid of the extracellular vesicles.

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