Pharmaceutical composition for nasal administration

Abstract

The purpose of the present invention is to provide a pharmaceutical composition having an excellent therapeutic effect on central nervous system diseases. A pharmaceutical composition for nasal administration for the treatment of central nervous system diseases, which contains a purified extracellular vesicle, and which is obtained by the method for producing the purified extracellular vesicle, has an excellent therapeutic effect on the treatment of central nervous system diseases. The production method comprises: (i) a purification step for bringing a liquid to be treated, which contains extracellular vesicles and inclusions, into contact with an exclusion and anion exchange carrier to obtain a treated liquid containing the extracellular vesicles; and (ii) membrane filtration in which the treated liquid is subjected to membrane filtration to obtain a concentrated solution of the extracellular vesicles.

Description

Technical Field

[0001] The present disclosure relates to a pharmaceutical composition for nasal administration that can be used to treat central nervous system diseases. Background Art

[0002] When brain is seriously damaged in the cases of cardiac arrest, myocardial infarction, asphyxia, various shocks, there will be obstacles, causing hypoxic encephalopathy. In the treatment of hypoxic encephalopathy, it is not just to maintain blood pressure, it is also very important to maintain the blood flow to the internal organs and peripheral tissues of the whole body, and with regard to survival rate, it is as low as 14% in Japan and as low as 20-30% in the U.S., and as for reintegration rate, it is only 7% in Japan and only 9% in the U.S. (non-patent literature 1, 2). At present, for hypoxic encephalopathy, actual situation is to have only the coping therapy that avoids damage development such as low temperature therapy.

[0003] Exosomes are a type of extracellular vesicle, granular substances with a diameter of 50-200 nm secreted by cells. Their surface contains lipids and proteins from the cell membrane, while their interior contains intracellular substances such as nucleic acids (miRNA, mRNA, DNA, etc.) and proteins, and they are believed to play a role in the transmission of information between cells.

[0004] Exosomes have been reported to be associated with various diseases, including cancer and neurodegenerative disorders. The types and amounts of functional molecules encapsulated in exosomes or present on the exosome membrane vary depending on the disease, leading to their potential applications as disease detection, prognosis prediction, and therapeutic targets.

[0005] Exosomes are also highly anticipated as natural DDS, unlike artificial substances such as liposomes. Attempts are underway to deliver siRNA, miRNA, and low-molecular-weight compounds to target cells. It has been reported that various cell adhesion molecules and sugar chains are expressed on the surface of exosomes, and their expression patterns determine the specific cell types for which exosomes bind. Furthermore, new DDS are being developed by modifying and utilizing the properties of exosomes.

[0006] While extensive research and development has been conducted on exosomes and extracellular vesicles (EVs), standardized and efficient technologies for their production and isolation are still under development. Because biological samples containing EVs contain numerous proteins and cells that share physical and chemical properties with EVs, the isolation of EVs is inherently complex.

[0007] The main separation methods currently used are methods that utilize differences in density, size, and specific surface markers of extracellular vesicles. Specific examples include ultracentrifugation, precipitation, filtration, size exclusion chromatography, and immunoaffinity.

[0008] Ultracentrifugation is a separation method that exploits differences in density and size between cells, EVs, and proteins and is used as a typical method for isolating extracellular vesicles (Non-Patent Document 3). However, ultracentrifugation has the disadvantages of requiring a long time to recover extracellular vesicles, poor productivity, and the coprecipitation of protein aggregates.

[0009] The precipitation method was developed as a method that avoids ultracentrifugation, which has problems with recovery time and productivity, and has already achieved sales of extracellular vesicle / exosome isolation kits.

[0010] Filtration methods use commercially available membrane filters (e.g., PVDF or polycarbonate filters with a pore diameter of approximately 50 to 450 nm) to separate larger vesicle components such as cells and extracellular vesicles from biological samples. This method is often combined with ultracentrifugation to separate extracellular vesicles and exosomes from proteins. (Non-Patent Documents 4, 5, 6)

[0011] Size exclusion chromatography has been used to separate extracellular vesicles and exosomes from protein aggregates. Typically, after centrifugation and filtration to remove larger vesicle components such as cells and extracellular vesicles, extracellular vesicles such as exosomes are separated using commercially available size exclusion chromatography columns. (Non-Patent Documents 4, 7, 8, 9, 10)

[0012] Immunoaffinity separation is a method for isolating extracellular vesicles such as exosomes by utilizing differences in specific surface markers. Generally speaking, it is not suitable for isolating extracellular vesicles and exosomes from a large number of biological samples. (Non-patent Document 11)

[0013] The reality is that these existing isolation methods require dedicated experimental equipment or experimental reagents, and multi-step operation procedures, and are accompanied by many difficulties in analyzing extracellular vesicles such as exosomes as routine diagnostic tools in clinical sites (Non-Patent Document 12).

[0014] Prior art literature

[0015] Non-patent literature

[0016] Non-patent literature 1: Neurotherapeutics. 2020 Apr; 17(2): 539-562.

[0017] Non-patent document 2: The Lancet Journal volume 369, issue 9577, P1925, 2007

[0018] Non-patent document 3: Biol. Chem. 394, no. 10 (2013): 1253-1262. DOI: 10.1515 / hsz-2013-0141.

[0019] Non-Patent Document 4: Kidney Int. 82, no. 9 (2012): 1024-1032. DOI: 10.1038 / ki.2012.256.

[0020] Non-patent literature 5: Nat. Biotechnol. 32, no. 5, 490-495 (2014). DOI: 10.1038 / nbt.2886

[0021] Non-Patent Literature 6: Cancer 119 (2013): 1159-1167. DOI: 10.1002 / cncr.27895.

[0022] Non-Patent Literature 7: Nanomedicine 11, no. 4 (2015): 879-883. doi.org / 10.1016 / j.nano.2015.01.003.

[0023] Non-Patent Literature 8: J. Immunol. Methods 411 (2014): 55-65. DOI: 10.1016 / j.jim.2014.06.007.

[0024] Non-patent literature 9: PLoS One 10 (2015): e0145686

[0025] Non-Patent Literature 10: J. Extracell. Vesicles 3 (2014): 23430. DOI: 10.3402 / jev.v3.23430.

[0026] Non-Patent Literature 11: Biol. Chem. 394, no. 10 (2013): 1253-1262. DOI: 10.1515 / hsz-2013-0141.

[0027] Non-Patent Literature 12: CRDS National Science and Technology Agency Research and Development Strategy Center Overview Report Life Sciences / Clinical Medicine (2021) 499-519 Summary of the Invention

[0028] Problems to be solved by the invention

[0029] There is still room for improvement in the therapeutic effects of central nervous system diseases such as anoxic encephalopathy, and the development of novel pharmaceutical compositions that enhance their effectiveness against such central nervous system diseases is highly desirable. Furthermore, while the relationship between exosomes and disease has been studied, isolation methods for extracellular vesicles such as exosomes have primarily focused on improving their efficiency. Consequently, methods for producing purified products that are highly effective for a given therapeutic application have not been studied.

[0030] The present disclosure aims to provide a pharmaceutical composition having excellent therapeutic effects on central nervous system diseases.

[0031] Solutions to Problems

[0032] The present inventors conducted intensive research and discovered that a composition for nasal administration prepared from purified extracellular vesicles obtained using a unique production method can achieve excellent therapeutic effects in treating central nervous system diseases. The present disclosure is based on this finding and further research.

[0033] That is, the present disclosure provides the following inventions.

[0034] Item 1. A pharmaceutical composition for nasal administration, comprising purified extracellular vesicles and for treating central nervous system diseases.

[0035] The purified extracellular vesicles are obtained by a method for producing a purified extracellular vesicle comprising the following (i) purification step and (ii) membrane filtration step:

[0036] (i) a purification step of contacting a treated solution containing extracellular vesicles and impurities with an exclusion and anion exchange carrier to obtain a treated solution containing the extracellular vesicles; and

[0037] (ii) A membrane filtration step of subjecting the treated solution to membrane filtration to obtain the extracellular vesicle concentrate.

[0038] Item 2. The pharmaceutical composition for nasal administration according to Item 1, wherein the central nervous system disease is anoxic encephalopathy.

[0039] Item 3. The pharmaceutical composition for nasal administration according to Item 2, wherein the hypoxic encephalopathy is global cerebral ischemia.

[0040] Item 4. The pharmaceutical composition for nasal administration according to Item 2 or 3, comprising 1×10 extracellular vesicles 3 More than 100 μL.

[0041] Item 5. The pharmaceutical composition for nasal administration according to any one of Items 1 to 4, wherein the liquid to be treated is a culture supernatant of cells or tissues secreting the extracellular vesicles.

[0042] Item 6. The pharmaceutical composition for nasal administration according to any one of Items 1 to 5, wherein the exclusion and anion exchange carrier is a porous carrier having positive charges inside the pores.

[0043] Item 7. The pharmaceutical composition for nasal administration according to any one of Items 1 to 6, wherein the molecular weight cutoff of the membrane used for the membrane filtration is 100,000 to 1,000,000.

[0044] Item 8. The pharmaceutical composition for nasal administration according to any one of Items 1 to 7, wherein the membrane used for the membrane filtration has a gamma globulin permeability of 10 to 80%.

[0045] Item 9. The pharmaceutical composition for nasal administration according to any one of Items 1 to 8, wherein the membrane used for the membrane filtration has a pure water permeation rate of 500 to 1500 L / m at 0.1 MPa. 2 / Hr.

[0046] Item 10. The pharmaceutical composition for nasal administration according to any one of Items 1 to 9, wherein the membrane used for the membrane filtration is a cellulose-based hydrophilic membrane.

[0047] Item 11. The pharmaceutical composition for nasal administration according to any one of Items 1 to 10, wherein the membrane used for the membrane filtration is a hollow fiber membrane.

[0048] Item 12. The pharmaceutical composition for nasal administration according to any one of Items 1 to 11, wherein the membrane filtration is tangential flow filtration.

[0049] Item 13. The pharmaceutical composition for nasal 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.

[0050] Item 14. The pharmaceutical composition for nasal administration according to any one of Items 1 to 13, 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 treated solution.

[0051] Item 15. The pharmaceutical composition for nasal administration according to any one of Items 1 to 14, wherein the concentration of total protein contained in the extracellular vesicle concentrate is 0.3 times or less the concentration of total protein contained in the treated solution.

[0052] Item 16. A method for producing a pharmaceutical composition for nasal administration for treating global cerebral ischemia, the method comprising:

[0053] (i) a purification step of contacting a treated solution containing extracellular vesicles and impurities with an exclusion and anion exchange carrier to obtain a treated solution containing the extracellular vesicles; and

[0054] (ii) A membrane filtration step of subjecting the treated solution to membrane filtration to obtain the extracellular vesicle concentrate.

[0055] Effects of the Invention

[0056] According to the present disclosure, a pharmaceutical composition with excellent therapeutic effects for treating central nervous system diseases can be provided. Furthermore, according to the present disclosure, since a pharmaceutical composition with excellent therapeutic effects for treating central nervous system diseases can be rapidly obtained, the concentration and amount of extracellular vesicles required for treatment can be quickly secured, particularly in clinical settings where treatment must be initiated within a matter of seconds, such as in global cerebral ischemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The outline of the apparatus used for the membrane filtration step in the method for producing the purified extracellular vesicles produced in Test Example 1 is shown.

[0058] Figure 2 The schematic diagram of an apparatus for measuring the gamma globulin permeability of a hollow fiber membrane used in a membrane filtration step is shown.

[0059] Figure 3 The cross section of brain tissue of hypoxic encephalopathy model rats 2 days after nasal administration of the pharmaceutical composition for nasal administration containing the purified extracellular vesicles produced in Test Example 3 is shown.

[0060] Figure 4 The results show the effect of improving the survival rate or proliferation rate of microglia by the purified extracellular vesicles produced in Test Example 4 (exosome purified concentrate A).

[0061] Figure 5 The inhibitory effect of the purified extracellular vesicles produced in Test Example 4 (exosome purified concentrate A) on the secretion of inflammatory cytokines is shown. DETAILED DESCRIPTION

[0062] The pharmaceutical composition disclosed herein comprises purified extracellular vesicles and is a pharmaceutical composition for nasal administration for treating central nervous system diseases. The pharmaceutical composition is characterized in that the purified extracellular vesicles is obtained by a given production method.

[0063] 1. Given manufacturing method

[0064] The given production method includes the following: (i) a purification step and (ii) a membrane filtration step. The purified extracellular vesicles obtained by the given production method have excellent therapeutic effects for treating central nervous system diseases. In addition, since the given production method can quickly prepare the purified extracellular vesicles at the concentration and amount required for treatment, it can also accelerate the initiation of treatment for central nervous system diseases.

[0065] (i) A purification step of contacting a treatment solution containing extracellular vesicles and impurities with an exclusion and anion exchange carrier to obtain a treatment solution containing the extracellular vesicles.

[0066] (ii) A membrane filtration step of subjecting the treated solution to membrane filtration to obtain the extracellular vesicle concentrate.

[0067] 1-1. Processed liquid

[0068] The liquid to be processed, which is a material for a given production method, contains extracellular vesicles and inclusions.

[0069] Examples of the types of extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. Among them, exosomes are preferred from the viewpoint of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure.

[0070] The source organism of the extracellular vesicles is not particularly limited, and examples thereof include mammals such as mice, cows, and humans.

[0071] The tissues, cells or body fluids from which extracellular vesicles are derived are not particularly limited. For example, examples of tissues or cells include bone marrow, dental pulp, fat, amnion, umbilical cord, periosteum, perichondrium, and cells thereof. 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, peritoneal effusion, joint effusion, aqueous humor, and vitreous humor.

[0072] The type of cells from which the extracellular vesicles are derived is not particularly limited, and may be any cell among germ cells, somatic cells, and stem cells. From the viewpoint of further improving the therapeutic effect of the pharmaceutical composition disclosed herein, stem cells are preferably used. It should be noted that, for the treatment of central nervous system diseases, the cells may be any cell among autologous cells and allogeneic cells. In addition, as stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, etc. may be cited. From the viewpoint of further improving the therapeutic effect of the pharmaceutical composition disclosed herein, mesenchymal stem cells may be preferably cited. In addition, as the source of mesenchymal stem cells, there is no particular limitation, and examples thereof include bone marrow, dental pulp, blood, fat, amniotic membrane, umbilical cord, umbilical cord blood, periosteum, perichondrium, etc.

[0073] The treated liquid is not particularly limited as long as it is a biological sample containing extracellular vesicles and inclusions. A biological sample refers to a sample collected from a cell or tissue culture or an organism. From the perspective of further improving 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.

[0074] Inclusions are components that are mixed in along with extracellular vesicles during the preparation of the biological sample. Examples include culture medium components, components produced by culture, components contained in tissues, cells, or body fluids from which the sample was collected, and include a variety of components, including proteins.

[0075] In the present disclosure, from the viewpoint of further improving the therapeutic effect of the pharmaceutical composition, the treated liquid is particularly preferably the culture supernatant of cells or tissues secreting the extracellular vesicles, and most preferably the culture supernatant of mesenchymal stem cells.

[0076] 1-2. Exclusion and anion exchange carriers

[0077] The treated liquid is brought into contact with the exclusion and anion exchange carriers, thereby obtaining a treated liquid containing extracellular vesicles.

[0078] Exclusion and anion exchange carriers refer to carriers that can perform both size exclusion and anion exchange. In order to achieve size exclusion, a carrier with pores can be used. As the exclusion size, for example, 400 kDa or more, preferably 500 kDa or more, more preferably 600 kDa or more, and further preferably 650 kDa or more can be cited. In addition, in order to achieve anion exchange, a positively charged carrier can be used. The exclusion and anion exchange carrier can be a combination of a carrier that can only perform size exclusion and a carrier that can only perform anion exchange, or a carrier that can perform both size exclusion and anion exchange.

[0079] From the perspective 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 with positively charged pores. Porous carriers with positively charged pores are more preferably particles with positively charged pores and non-positively charged areas outside the pores. Even more preferably, multilayer particles have an inner layer modified with a positively charged ligand and a porous shell layer not modified with the ligand.

[0080] The shape of the exclusion and anion exchange carrier is not particularly limited, but a particulate form is preferred. Examples of the average particle size of the particulate exclusion and anion exchange carrier include 1 to 1000 μm, 10 to 500 μm, 20 to 300 μm, and 20 to 150 μm. It should be noted that the average particle size refers to the particle size (D50, median particle size) at which the cumulative value of the volume-based particle size distribution obtained by laser diffraction / scattering is 50%.

[0081] 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, polyacrylonitrile, polyethylene, polypropylene, and polystyrene; and inorganic polymers such as glass, porous glass, silica gel, and hydroxyapatite. Polysaccharides are preferred, and agarose and silica gel are more preferred.

[0082] Note that purification using exclusion and anion exchange carriers can be performed by chromatography or by a batch method.

[0083] The concentration of extracellular vesicles in the treated solution obtained by the purification step (step (i)) can be, for example, 0.65 times or more (based on weight) the concentration of extracellular vesicles in the treated solution used in this step. From the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, it can be preferably 0.70 times or more, more preferably 0.75 times or more, even more preferably 0.80 times or more, particularly 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 extracellular vesicle concentration in the treated solution can be 1 times or less the concentration of extracellular vesicles in the treated solution.

[0084] The total protein concentration in the treated liquid obtained by the purification step (step (i)) can be, for example, 0.5 times or less (based on weight) the total protein concentration in the treated liquid subjected to this step. From the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, it can be preferably 0.4 times or less, more preferably 0.3 times or less, even more preferably 0.2 times or less, and particularly preferably 0.15 times or less. The lower limit of the total protein concentration in the treated liquid is not particularly limited, and can be, for example, 0.05 times or more, 0.08 times or more, or 0.1 times or more of the total protein concentration in the treated liquid.

[0085] 1-3. Membrane Filtration

[0086] The molecular weight cutoff of the membrane used for membrane filtration is not particularly limited, and examples thereof include 100,000 to 1,000,000. From the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, it is preferably 150,000 to 800,000, more preferably 200,000 to 600,000, further preferably 230,000 to 400,000, and particularly preferably 250,000 to 350,000.

[0087] The gamma globulin permeability of the membrane used for membrane filtration is not particularly limited, but can be, for example, 5% to 95%. From the perspective of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, it is preferably 10% to 80%, more preferably 20% to 70%, even more preferably 30% to 60%, and particularly preferably 40% to 50%. The gamma globulin permeability, when filtering a gamma globulin aqueous solution through a hollow fiber membrane at a filtration pressure of 0.1 MPa, is the value (%) derived from the following formula: (gamma globulin concentration in the permeate / gamma globulin concentration in the aqueous solution × 100).

[0088] The pure water permeation rate of the membrane used for membrane filtration at 0.1 MPa can be, for example, 500 to 1500 L / m 2 / Hr, from the viewpoint of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, it is preferably 600 to 1300 L / m 2 / Hr, more preferably 800~1100L / m 2 / Hr.

[0089] The material of the membrane used for membrane filtration is not particularly limited, and examples thereof include hydrophobic membranes such as polyethersulfone membranes and polysulfone membranes, and hydrophilic membranes such as cellulose membranes. From the perspective of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, hydrophilic membranes such as cellulose membranes are preferred. Specific examples of the cellulose membranes include regenerated cellulose membranes and cellulose ester membranes (specifically, cellulose acetate membranes, cellulose propionate membranes, cellulose butyrate membranes, and cellulose benzoate membranes). From the perspective 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.

[0090] The shape of the membrane used for membrane filtration is not particularly limited, but from the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, a hollow fiber membrane is preferably used. The inner diameter of the hollow fiber membrane can be, for example, 0.2 mm to 1.4 mm. From the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, it can be 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.

[0091] As the form of the membrane used for membrane filtration, particularly when a hollow fiber membrane is used, a bundle of multiple (e.g., 10 to 500, 10 to 200, 20 to 100, 50 to 100, 60 to 90, or 70 to 80) hollow fiber membranes is preferably used.

[0092] The membrane filtration method may be either dead-end filtration or tangential flow (cross flow) filtration. From the viewpoint of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, tangential flow filtration is preferred.

[0093] Examples of the membrane surface velocity (membrane surface flow rate) in membrane filtration include 0.3 m / s to 2 m / s. From the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, it is preferably 0.4 m / s to 1.5 m / s, more preferably 0.5 m / s to 1.3 m / s, further preferably 0.6 m / s to 1.2 m / s, and particularly preferably 0.8 to 1.2 m / s.

[0094] Examples of the inlet pressure during membrane filtration include 0.01 MPa to 0.2 MPa. From the perspective of further enhancing the therapeutic effect of the pharmaceutical composition of the present disclosure, the inlet pressure is preferably 0.015 MPa to 0.12 MPa, and more preferably 0.02 MPa to 0.07 MPa.

[0095] 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 (based on weight) the concentration of extracellular vesicles in the treatment solution used in this step. From the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, it can be preferably 20 times or more, more preferably 30 times or more, even more preferably 40 times or more, particularly preferably 45 times or more, 50 times or more, 80 times or more, or 100 times or more. The upper limit of the extracellular vesicle concentration in the concentrate is not particularly limited, but can be, for example, 60 times or less or 55 times or less of the concentration of extracellular vesicles in the treatment solution.

[0096] The total protein concentration in the extracellular vesicle concentrate obtained by the membrane filtration step (step (ii)) can be, for example, 0.3 times or less (based on weight) the total protein concentration in the treated liquid subjected to the purification step (step (i)). From the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, it can be preferably 0.25 times or less, and more preferably 0.2 times or less. The lower limit of the total protein concentration in the extracellular vesicle concentrate is not particularly limited, but can be, for example, 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 of the total protein concentration in the treated liquid.

[0097] 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 treated solution used in the same step (step (ii)). From the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, it can be preferably 1.8 times or less, and more preferably 1.6 times or less. The lower limit of the total protein concentration in the extracellular vesicle concentrate is not particularly limited, but can be, for example, 0.4 times or more, 0.7 times or more, or 1 times or more of the total protein in the treated solution.

[0098] 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)). From the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, it can be preferably 5% or less, more preferably 3% or less, even more preferably 2% or less, or 1% or less. Examples of the volume of the extracellular vesicle concentrate obtained by the membrane filtration step (step (ii)) include 0.005% or more, 0.001% or more, 0.01% or more, 0.1% or more, 0.5% or more, or 1% or more.

[0099] 2. Extracellular Vesicle Purification

[0100] The purified extracellular vesicles contained in the pharmaceutical composition of the present disclosure are obtained by the above-described production method. Specific forms of the purified extracellular vesicles include a concentrated extracellular vesicle solution obtained by the above-described production method, a superconcentrated solution obtained by further removing water from the concentrated solution, or a dried product. Methods for obtaining the dried product include freeze-drying, spray drying, and air drying.

[0101] Examples of the types of extracellular vesicles contained in the purified extracellular vesicles include exosomes, microvesicles, and apoptotic bodies, which may be included alone or in combination. Among these extracellular vesicles, exosomes are preferred from the perspective of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure.

[0102] Regarding the size of the extracellular vesicles contained in the extracellular vesicle purification, the average particle size of the extracellular vesicles can be, for example, 15 to 250 nm. From the viewpoint of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, preferably 40 to 200 nm, more preferably 50 to 190 nm, 55 to 170 nm, further preferably 65 to 150 nm, particularly preferably 75 to 130 nm, and particularly more preferably 80 to 120 nm, 110 to 120 nm, or 100 to 120 nm. It should be noted that in the present disclosure, the average particle size refers to the 50% cumulative value (D) calculated from the volume-based particle size distribution measured by the laser diffraction / scattering method. 50 The mode diameter of the extracellular vesicles contained in the extracellular vesicle purification product may be, for example, 10 to 200 nm. From the viewpoint of further improving the therapeutic effect of the pharmaceutical composition of the present invention, preferably, it may be 30 to 190 nm, 30 to 180 nm, more preferably 50 to 160 nm, further preferably 60 to 140 nm, particularly preferably 70 to 120 nm, further preferably 76 to 115 nm, 85 to 116 nm, or 93 to 115 nm. In addition, as the particle size distribution width ((D 90 -D 10 ) / D 50 ), for example, 0.1 to 1.6, and from the viewpoint of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, preferably 0.2 to 1.4, more preferably 0.3 to 1.2, further preferably 0.4 to 0.95, particularly preferably 0.5 to 0.85, and particularly further preferably 0.6 to 0.9, 0.65 to 0.85, or 0.7 to 0.79.

[0103] The content of the purified extracellular vesicles in the pharmaceutical composition of the present disclosure is not particularly limited as long as it is an amount that is permissible in a composition for nasal administration and that is effective for treating central nervous system diseases. For example, the amount of the purified extracellular vesicles is 1×10 3 cells / μL or more, preferably 1×10 4 cells / μL or more, more preferably 1×10 5 The upper limit of the content of the purified extracellular vesicles in the pharmaceutical composition of the present disclosure is not particularly limited, and the amount of extracellular vesicles can be, for example, 1×10 13 less than 1×10 12 less than 1×10 11 less than 1×10 10 less than 1 μg / μL.

[0104] In addition, the content of the purified extracellular vesicles in the pharmaceutical composition of the present disclosure can be, for example, 1 to 100,000 μg / mL, 4 to 10,000 μg / mL, or 8 to 5,600 μg / mL in terms of the amount of extracellular vesicles.

[0105] 3. Other ingredients

[0106] The pharmaceutical composition of the present disclosure may contain the purified extracellular vesicles as an active ingredient and may or may not contain other ingredients such as bases and / or additives that are permitted and pharmacologically acceptable in compositions for nasal administration.

[0107] Pharmacologically acceptable bases and / or additives include, for example, excipients, thickeners, lubricants, binders, disintegrants, solvents, dissolution aids, suspending agents, emulsifiers, isotonic agents, buffers, analgesics, stabilizers, preservatives (preservatives), pH adjusters, cooling agents, antioxidants, wetting agents, adhesives, flavoring agents, etc. These bases and additives may be used alone or in combination.

[0108] 4. Formulation

[0109] The pharmaceutical composition of the present invention can be in any form of a liquid or powder that can be used in a composition for nasal administration, and a liquid can be preferably mentioned. In the case of preparing a liquid, it can be prepared by mixing the purified extracellular vesicles and a solvent, a dissolution aid, a suspending agent, an isotonic agent, a buffer, and / or an analgesic used as needed and dissolving, suspending or emulsifying it. A thickening agent can also be added to increase viscosity and impart retention. In the case of preparing a powder, it can be prepared by mixing the purified extracellular vesicles and an excipient, a binder, a disintegrant and / or other appropriate additives used as needed and further drying it as needed.

[0110] The pharmaceutical composition of the present disclosure can be filled in a container for nasal administration. As a container for nasal administration, an appropriate commercially available container can be used.

[0111] 5. Purpose

[0112] The pharmaceutical composition of the present disclosure can be used for the treatment of central nervous system diseases. As central nervous system diseases, for example, ischemic cerebrovascular disorder and neurodegenerative diseases can be mentioned. As ischemic cerebrovascular disorder, for example, cerebral embolism, transient ischemic stroke, subclavian steal syndrome, Wallenberg syndrome (lateral medullary syndrome), cerebral thrombosis, lacunar infarction (lacunar infarction), reversible ischemic neurological disorder, cerebral infarction, moyamoya disease (occlusive disease in circle of Willis), anoxic encephalopathy, venous sinus thrombosis, postoperative spinal cord ischemia, etc. can be mentioned. As neurodegenerative diseases, Alzheimer's type cognitive impairment, Lewy body type cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis, etc. can be mentioned. Among these central nervous system diseases, from the viewpoint of further improving the therapeutic effect of the pharmaceutical composition of the present disclosure, ischemic cerebrovascular disorder can be preferably mentioned, and anoxic encephalopathy can be more preferably mentioned. In addition, the method for producing the pharmaceutical composition of the present disclosure can rapidly prepare purified extracellular vesicles at the concentration and amount required for treatment. Therefore, it is particularly suitable for central nervous system diseases, such as global cerebral ischemia, which is a highly urgent symptom requiring urgent treatment.

[0113] The pharmaceutical composition of the present disclosure can be used for nasal administration. The dosage of the pharmaceutical composition of the present disclosure for human administration can be appropriately determined according to the level of the applicable disease and symptoms, and can be, for example, 1×10 4 pcs / kg or more, preferably 1×10 5 More than 1×10 6 More than 1×107 More than 5 × 10 7 The upper limit of the dosage of the pharmaceutical composition of the present disclosure, in terms of the amount of extracellular vesicles, can be, for example, 1×10 11 less than 1×10 10 less than 1×10 9 less than 1×10 8 Less than pcs / kg.

[0114] Each aspect disclosed in this specification may also be combined with any other features disclosed in this specification.

[0115] Example

[0116] The present invention will be described in more detail below with reference to the following embodiments. However, the various configurations and combinations thereof in the various embodiments are merely examples, and additions, omissions, substitutions, and other modifications to the configurations may be made as appropriate without departing from the spirit of the present invention. The present disclosure is not limited to the embodiments but is defined solely by the claims.

[0117] Test Example 1

[0118] (I) Preparation of culture supernatant of mesenchymal stem cells (processed solution containing extracellular vesicles and inclusions)

[0119] Human mesenchymal stem cells (obtained from 56.4 mL of bone marrow fluid via mononuclear cell isolation and plating) were cultured in a cell culture medium (MEMα + 5% allogeneic platelet lysates (PL) + 1% antibiotics (penicillin-streptomycin; P / S)). The culture period was 21 days, and 3.5 L of culture supernatant was collected midway.

[0120] (II) Purification step (step (i))

[0121] To 250 mL of the culture supernatant 3.5 L obtained in (I) above (470.5 mg of total protein based on the Bradford method) was added a carrier (a porous granular material with positively charged pores, which is an adsorbent capable of adsorbing and retaining negatively charged unnecessary substances other than extracellular vesicles) 50 mL was equilibrated with a 50 mM Tris-HCl (pH 7.2) solution, and after being dispensed into 50 mL tubes, the mixture was poured and mixed at room temperature at a speed of 10 rpm using a rotator for 10 minutes. Then, the supernatant was centrifuged at 10,000 x g for 2 minutes at room temperature, and the supernatant was further filtered using a STERICUP 0.22 μm filter unit (S2GPU11RE, Merck Millipore) to remove the carrier and obtain a treatment solution containing extracellular vesicles. The amount of total protein based on the Bradford method in the obtained treatment solution was 195.3 mg, which was reduced to 42% before the purification step.

[0122] (III) Membrane filtration step (step (ii))

[0123] use Figure 1 The membrane filtration apparatus 1 shown above was used to perform membrane filtration on 250 mL of the treatment liquid obtained in the above-mentioned step (II).

[0124] Figure 1 The membrane filtration device 1 shown comprises:

[0125] Hollow fiber membrane module 30,

[0126] The first tank 10 and the second tank 20 are in liquid communication with both ends of the hollow fiber membrane module 30,

[0127] A buffer tank 40 that is in liquid communication with the first tank 10 in an openable and closable manner via a buffer liquid delivery line 45 having an opening and closing valve 46,

[0128] A first tank gas supply line 53 in gas communication with the first tank 10,

[0129] A second tank gas supply line 54 in gas communication with the second tank 20,

[0130] A three-way valve 61 that connects the gas supply line 52 with the pressure gauge 51 to the first tank gas supply line 53 or the second tank gas supply line 54 in an openable and closable manner;

[0131] A first exhaust line 55 having an on-off valve 62 that is in gas communication with the first tank 10 in an openable and closable manner, and

[0132] A second exhaust line 56 having an on-off valve 63 that is in gas communication with the second tank 20 in an openable and closable manner.

[0133] The details of the components of the membrane filtration device 1 are as follows.

[0134] ·10 for the first can and 20 for the second can

[0135] Material: Acrylic resin

[0136] Size: Length 25cm, Inner diameter 0.25cm, Capacity 120cm 3

[0137] Buffer tank 40

[0138] Capacity: 1.6L

[0139] Hollow fiber membrane module 30

[0140] Hollow fiber size: inner diameter 0.8mm, outer diameter 1.3mm, length 17cm

[0141] Material: Cellulose acetate (CA)

[0142] Molecular weight cut-off: 300,000

[0143] The gamma globulin permeability was determined by the following method: 54%

[0144] Pure water permeation flow rate at 0.1MPa: 980L / m 2 / Hr

[0145] Form: A hollow fiber membrane bundle consisting of 15 cellulose acetate (CA) hollow fiber membranes (manufactured by DAICEN MEMBRANE-SYSTEMS) was placed in a polycarbonate cylindrical container. The membrane area was 0.0062 m 2

[0146] γ-Globulin permeability measurement

[0147] use Figure 2 The γ-globulin permeability measuring device 300 was used to measure the γ-globulin permeability according to the following operation.

[0148] 1) Add a γ-globulin aqueous solution (γ-globulin concentration: 100 ppm) to the tank 302.

[0149] 2) Connect the hollow fiber membrane 303 to the flow path 305 .

[0150] 3) Nitrogen gas was injected from valve V-1, and a pressure of 0.1 MPa was applied to tank 302. The aqueous solution was filtered from the inner surface to the outer surface of hollow fiber membrane 303. The filtrate was discarded into container 307, and the permeate was recovered into container 306. The initial permeate (approximately 100 mL) was discarded, and the subsequent permeate was used as a sample for permeability measurement.

[0151] After the permeate sample was prepared by the apparatus 300 , the γ-globulin permeability was measured by the following method.

[0152] 4) The absorbance at 280 nm (A1) of the γ-globulin aqueous solution (100 ppm) and the absorbance at 280 nm (A2) of the permeate were measured using a spectrophotometer (manufactured by Shimadzu Corporation, product name "UV2450"). The γ-globulin transmittance (%) was calculated using the formula (A2 / A1×100).

[0153] Membrane filtration was performed at approximately 20°C according to the following procedure.

[0154] (1) The treatment liquid obtained in the above (II) is added into the first tank 10.

[0155] (2) Nitrogen gas is supplied to the upper space of the first tank 10 at a pressure of 0.04 MPa, so that the diluted liquid of the treatment liquid in the first tank 10 passes through the hollow fiber membrane module 30, thereby performing tangential flow filtration. At this time, the on-off valve 63 is opened to open the second tank 20 to the atmosphere, and the pressure is zero. In addition, the membrane surface linear velocity flowing through the hollow fiber membrane module 30 is 1.0 m / s. It should be noted that the membrane surface linear velocity is calculated based on the rate of increase of the concentrated liquid amount in the second tank 20. The permeate of the hollow fiber membrane module 30 is stored in the permeate tank 35, and the concentrated liquid is transferred to the second tank 20. Thus, the diluted liquid of the treatment liquid in the first tank 10 passes through the hollow fiber membrane module 30 and is subjected to membrane filtration.

[0156] (3) When most of the treated liquid in the first tank 10 had been 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. Simultaneously, 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 passed through the hollow fiber membrane module 30, thereby performing tangential flow filtration in the opposite direction to that of (2) above. The permeate was stored in the permeate tank 35, and the concentrated liquid was transferred to the first tank 10. Thus, the concentrated liquid in the second tank 20 passed through the hollow fiber membrane module 30 and underwent membrane filtration.

[0157] (4) The concentrate transferred to the first tank 10 was further subjected to alternating tangential flow filtration from the first tank 10 side toward the second tank 20 side and tangential flow filtration from the second tank 20 side toward the first tank 10 side, as described in (2) and (3) above, until the volume of the concentrate reached 10 ml. For backwashing, 90 mL of phosphate buffer solution (PBS) was supplied to the hollow fiber membrane module 30 from a tank (not shown). After further repeating alternating tangential flow filtration and supplying 90 mL of phosphate buffer solution twice, 11.2 mL of a purified concentrate (purified extracellular vesicles) was obtained by alternating tangential flow filtration.

[0158] The total protein concentration of the final purified exosome concentrate (extracellular vesicle purification) was 22.5 mg, and the protein content was reduced to 4.8% through the purification and membrane filtration steps.

[0159] (IV) OGD experiment

[0160] Cryopreserved neuroblastoma cell line (SH-SY5Y, ATCC) was cultured in SY5Y-specific medium (eMEM: 45%, F12: 45%, FBS: 10%, P / S: 1%) and 1.2 × 10 5 Cells were seeded into 24-well plates (Falcon) under 1:1 / well conditions. The culture medium was then replaced with glucose-free DMEM in a hypoxic chamber (Invivo2 300) under an atmosphere of 1% oxygen, 5% carbon dioxide, and 94% nitrogen for 24 hours of hypoxia / low glucose stress (OGD). Following OGD, the cells were returned to a conventional incubator (21% oxygen) and the culture medium replaced with DMEM high glucose (FBS-free).

[0161] When the culture medium was replaced, the purified exosome concentrate (extracellular vesicle purification product) obtained in step (III) was added to the culture medium so that the exosome count reached 1.7 × 10 10 / 10μL / well. As a control, wells without exosome addition were also set up. After 72 hours of culture, the culture medium was aspirated, and 500μL of stripping solution (TrypLE Select, GIBCO) was added to each well, incubated at 37°C for about 5 minutes, and neutralized by adding 500μL of the above culture medium. The pellet obtained by centrifugation was diluted with 1ml of culture medium, and the cell number and survival rate were measured using Luna (Logos Biosystems). The results are shown in Table 1 below.

[0162]

[0163] As shown in Table 1, the purified extracellular vesicles obtained through steps (i) and (ii) significantly increased the proliferation rate of neuroblasts under hypoxia / low glucose stress (OGD). Furthermore, the purified extracellular vesicles increased not only the total cell count but also the ratio of viable cells (survival rate) in OGD, demonstrating that the number of viable cells was significantly increased.

[0164] Test Example 2

[0165] The purified exosome concentrate (extracellular vesicle purification) obtained in Test Example 1 was subjected to particle size analysis using the nanoparticle multianalyzer qNano (IZON). The average particle size (50% cumulative value (D) calculated from the volume-based particle size distribution measured by the laser diffraction / scattering method) was 1.0447 nm. 50 )) is 84nm, the mode particle size is 79nm, and the particle size distribution width ((D 90 -D 10 ) / D 50 ) is 0.68, and the number of particles is 1.7×10 12 pieces / mL.

[0166] Test Example 3

[0167] (I) Preparation of culture supernatant of bone marrow-derived mesenchymal stem cells (processed solution containing extracellular vesicles and inclusions)

[0168] Rat bone marrow-derived mesenchymal stem cells (BMSCs) were cultured in DMEM + 10% FBS + 1% P / S. When they reached 80% confluence, the medium was switched to FBS-free medium and cultured for 24 hours. The supernatant was collected and passed through a 0.45 μm filter.

[0169] (II) Purification step (step (i))

[0170] To 150 mL of the culture supernatant obtained in (I) above (350.6 mg of total protein based on the Bradford method), 30 mL of a carrier (a porous granular material with positively charged pores, which is an adsorbent capable of adsorbing and retaining negatively charged unnecessary substances other than extracellular vesicles) equilibrated with a 50 mM Tris-HCl (pH 7.2) solution was added, and after being dispensed into 50 mL tubes, the mixture was poured and mixed at room temperature at a speed of 10 rpm using a rotator for 10 minutes. Then, the supernatant was centrifuged at room temperature for 2 minutes at 10,000 x g, and the supernatant was further filtered using a STERICUP 0.22 μm filter unit (S2GPU11RE, Merck Millipore) to remove the carrier and obtain a treatment solution containing extracellular vesicles. The amount of total protein based on the Bradford method in the obtained treatment solution was 159.3 mg, which was reduced to 45% before the purification step.

[0171] (III) Membrane filtration step (step (ii))

[0172] The treated liquid obtained in (II) was subjected to the same membrane filtration as in Test Example 1 (III). The amount of total protein in the purified exosome concentrate (extracellular vesicle purified product) finally obtained was 4.8 mg. The amount of protein was reduced to 1.4% by the purification process and membrane filtration process. In addition, the obtained purified exosome concentrate (extracellular vesicle purified product) was subjected to particle size analysis using a nanoparticle multi-analyzer qNano (IZON). The average particle size (50% cumulative value (D) calculated based on the volume-based particle size distribution measured by the laser diffraction / scattering method) was 2.374 nm. 50 )) is 113nm, the mode particle size is 105nm, and the particle size distribution width ((D 90 -D 10 ) / D 50 ) is 0.70, and the number of particles is 1.6×10 10 pieces / mL.

[0173] (IV) Animal Experimentation

[0174] The vertebral arteries of rats (SD rats, 379 g, 392 g (about 10 weeks old), male) were completely occluded by electrocoagulation and cutting. The next day, the internal carotid arteries of the rats were temporarily occluded for 30 minutes with a clamp. The occlusion was then released and the purified exosome concentrate (extracellular vesicles purified) obtained in (III) was immediately administered intranasally to a concentration of 3.2 × 10 exosomes. 6 / 5μL / unilateral nasal cavity (6.2×10 6Alternatively, 5 μL of PBS was administered per nasal cavity (10 μL per nasal cavity) as a control. This dose is equivalent to 8×10 7 The conversion to the human dose can be reasonably derived based on the weight of the brain (set as 2 g for rats and 1300 g for humans) and the body weight of humans (set as 50 kg).

[0175] Two days after administration, paraffin sections of rat brain were prepared and apoptotic cells were fluorescently stained and observed using Apoptag (Merck) (Keyence BZ-X). Figure 3 .exist Figure 3 In the figure, an enlarged image of the tissue near the hippocampus is also shown. Figure 3 As shown, compared with the control (PBS), the number of dead cells in the hippocampus was significantly reduced in rats administered with exosomes (Exosome).

[0176] Test Example 4

[0177] (1) Preparation of exosome purification concentrate A (extracellular vesicle purification)

[0178] A treatment solution was prepared in the same manner as in Test Example 1 using the culture supernatant of human amnion-derived mesenchymal stem cells (AMSC).

[0179] Approximately 3185 mL of the resulting treated liquid (5159.7 mg of total protein as determined by the BCA method, 512.8 ng of CD9 / CD63-positive exosomes as determined by ELISA (converted to the CD9 / CD63 fusion standard protein used in the calibration curve)) was filtered using a STERICUP 0.22 μm filter unit (S2GPU11RE, Merck Millipore) and subjected to the same membrane filtration as in Experimental Example 1. A hollow fiber membrane module with the following specifications was used.

[0180] Hollow fiber size: inner diameter 0.8mm, outer diameter 1.3mm, length 17cm

[0181] Material: Cellulose acetate (CA)

[0182] Molecular weight cut-off: 300,000

[0183] The gamma globulin permeability was determined by the following method: 54%

[0184] Pure water permeation flow rate at 0.1MPa: 980L / m 2 / Hr

[0185] Form: A hollow fiber membrane bundle consisting of 125 cellulose acetate (CA) hollow fiber membranes (manufactured by Daicen Membrane-Systems) was placed in a polycarbonate cylindrical container. The membrane area was 0.05 m 2

[0186] The final exosome purified concentrate A (extracellular vesicle purification) 26 mL contained 35.1 mg of total protein, and the protein content was reduced to 0.7%. In addition, the exosome amount was 372.2 ng, and the recovery rate was 73%. Furthermore, the exosome purified concentrate obtained was analyzed using the nanoparticle imaging analyzer VIDEO DROP. The results showed that the average particle size (50% cumulative value (D) calculated based on the volume-based particle size distribution measured by the laser diffraction / scattering method) was 2.3477 μg / mL. 50 )) is 187nm, the mode particle size is 185nm, and the particle size distribution width ((D 90 -D 10 ) / D 50 ) is 1.14, and the number of particles is 5.2×10 10 pieces / mL.

[0187] (2) Preparation of exosome purification concentrate B for comparison based on ultracentrifugation

[0188] The culture supernatant of human amniotic membrane-derived mesenchymal stem cells (AMSC) used in (1) was centrifuged and the supernatant was recovered twice (conditions for the first centrifugation: 2000g, 10 minutes, 4°C; conditions for the second centrifugation: 10000g, 30 minutes, 4°C). The obtained supernatant was subjected to ultracentrifugation under the following conditions (conditions for ultracentrifugation: 100000g, 70 minutes, 4°C). The supernatant was discarded, and the water in the tube was wiped off as much as possible to obtain an exosome precipitate attached to the inner wall of the tube. It was suspended in about 200μl of PBS to obtain a comparative exosome purification concentrate B.

[0189] The total protein content of the final comparative exosome purified concentrate B was 140.4 μg. The results of analysis using the nanoparticle imaging analyzer VIDEO DROP showed that the average particle size (50% cumulative value (D) calculated from the volume-based particle size distribution measured by the laser diffraction / scattering method) was 2.37 μg. 50 )) is 137nm, the mode particle size is 135nm, and the particle size distribution width ((D 90 -D 10 ) / D 50 ) is 1.08, and the number of particles is 2.3×10 11 pieces / mL.

[0190] (3) Effect of improving the survival rate or proliferation rate of microglia

[0191] Microglial cell line BV2 (Elabscience) was diluted with culture medium (MEMα, P / S: 1%) and seeded in a 96-well plate (Falcon) to a concentration of 2.0 × 10 4 / well. One day later, lipopolysaccharide derived from Escherichia coli 026:B6 (L8274, Sigma-Aldrich) was added to a concentration of 1000 ng / well, and the purified exosome concentrate A obtained in (1) or the comparative exosome purified concentrate B obtained in (2) was added to a concentration of 2.8 × 10 9 / well, and 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 Chemical Research Institute Co., Ltd.), and the absorbance at 450nm was measured as the number of viable cells. On the other hand, the same operation was performed except that PBS solution was added instead of lipopolysaccharide and exosomes (control-1). In addition, the same operation was performed except that exosomes were not added (lipopolysaccharide was added) (control-2). The results are shown in Figure 4 .

[0192] like Figure 4 As shown, by adding the purified exosome concentrate A obtained in (1) above, the survival rate or proliferation rate of microglia can be improved, and it is clear that the effect is higher than when the comparative exosome purified concentrate B obtained in (2) above is added.

[0193] Microglia become active in diseased states, undergoing dramatic changes in their cellular properties. They aggregate and proliferate in lesions, engulfing damaged nerve cells and eliminating them. Based on the observed effect of improving microglial survival and proliferation rates by adding purified exosome concentrate A, it is reasonable to infer that purified exosome concentrates obtained through (i) purification and (ii) membrane filtration, such as purified exosome concentrate A, can further promote the engulfment and elimination of damaged nerve cells compared to purified exosome concentrates obtained by ultracentrifugation, such as purified exosome concentrate B.

[0194] (4) Inhibitory effect on the secretion of inflammatory cytokines

[0195] Microglial cell line BV2 (Elabscience) was diluted with culture medium (MEMα, P / S: 1%) and seeded in a 96-well plate (Falcon) to a concentration of 2.0 × 10 4 / well. One day later, lipopolysaccharide derived from Escherichia coli 026:B6 (L8274, Sigma-Aldrich) was added to a concentration of 10 ng / well, and the purified exosome concentrate A obtained in (1) or the comparative exosome purified concentrate B obtained in (2) was added to a concentration of 2.8 × 10 9 After culturing for another day, the culture supernatant was recovered, and the amount of inflammatory cytokines TNFα and IL-6 contained in the supernatant was quantified by ELISA (using Mouse TNF-alpha Quantikine ELISA Kit and Mouse IL-6 Quantikine ELISA Kit (both manufactured by R&D systems)). On the other hand, the same operation was performed except that PBS solution was added instead of lipopolysaccharide and exosomes (control-1). In addition, the same operation was performed except that exosomes were not added (lipopolysaccharide was added) (control-2). The results are shown in Figure 5 .

[0196] like Figure 5 As shown, the secretion of inflammatory cytokines can be suppressed by adding the purified and concentrated exosomes obtained in (1) above, and it was found that the effect was higher than that of adding the purified and concentrated exosomes for comparison obtained in (2) above.

[0197] When the brain is damaged, microglia are activated, releasing inflammatory cytokines such as TNF-α and IL-6, triggering an inflammatory response. Inflammatory responses are thought to aggravate neurological diseases such as multiple sclerosis, stroke, and Alzheimer's disease, as well as psychiatric disorders such as depression, schizophrenia, and autism. Based on the inhibitory effect of TNF-α and IL-6 secretion by microglia confirmed by adding purified exosome concentrate A, it is reasonable to infer that purified exosome concentrates obtained through (i) purification steps and (ii) membrane filtration steps, such as purified exosome concentrate A, can further suppress the secretion of inflammatory cytokines, thereby further inhibiting the progression of neurological and psychiatric diseases, compared to purified exosome concentrates obtained by ultracentrifugation, such as purified exosome concentrate B.

Claims

1. A pharmaceutical composition for nasal administration, comprising purified extracellular vesicles and for treating central nervous system diseases. The purified extracellular vesicles are obtained by a method for producing a purified extracellular vesicle comprising the following (i) purification step and (ii) membrane filtration step: (i) a purification step of contacting a treated solution containing extracellular vesicles and impurities with an exclusion and anion exchange carrier to obtain a treated solution containing the extracellular vesicles; and (ii) a membrane filtration step of subjecting the treated solution to membrane filtration to obtain a concentrated solution of the extracellular vesicles.

2. The pharmaceutical composition for nasal administration according to claim 1, wherein The central nervous system disease is anoxic encephalopathy.

3. The pharmaceutical composition for nasal administration according to claim 2, wherein The hypoxic encephalopathy is global cerebral ischemia.

4. The pharmaceutical composition for nasal administration according to claim 2, comprising 1×10 extracellular vesicles 3 More than 100 μL.

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

6. The pharmaceutical composition for nasal administration according to claim 1, wherein The exclusion and anion exchange carrier is a porous carrier with positive charges inside the pores.

7. The pharmaceutical composition for nasal administration according to claim 1, wherein The molecular weight cut-off of the membrane used for the membrane filtration is 100,000 to 1,000,000.

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

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

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

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

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

13. The pharmaceutical composition for nasal administration according to claim 1, wherein The membrane surface velocity in the membrane filtration is 0.3 m / s to 2 m / s.

14. The pharmaceutical composition for nasal administration according to claim 1, wherein The concentration of the extracellular vesicles contained in the concentrated extracellular vesicle solution is 10 times or more the concentration of the extracellular vesicles contained in the treated solution.

15. The pharmaceutical composition for nasal administration according to claim 1, wherein The concentration of total proteins contained in the extracellular vesicle concentrate is 0.3 times or less of the concentration of total proteins contained in the treated liquid.

16. A method for producing a pharmaceutical composition for nasal administration for treating global cerebral ischemia, the method comprising: (i) a purification step of contacting a treated solution containing extracellular vesicles and impurities with an exclusion and anion exchange carrier to obtain a treated solution containing the extracellular vesicles; and (ii) a membrane filtration step of subjecting the treated solution to membrane filtration to obtain a concentrated solution of the extracellular vesicles.

Images