Preventive and / or therapeutic drug for cerebrovascular disease

Abstract

To provide a drug effective in preventing and / or treating cerebrovascular disease.SOLUTION: The present inventors have focused on the disruption filtrate of mesenchymal stem cells and conducted extensive research, and as a result, have found that the disruption filtrate has a therapeutic effect on cerebrovascular disease, particularly the effect of alleviating symptoms of subacute or chronic cerebrovascular disease, specifically the effect of recovering neurological and motor functions. The preventive and / or therapeutic drug of the present invention has an effect of alleviating symptoms of subacute or chronic cerebrovascular disease, particularly. Furthermore, since the preventive and / or therapeutic drug of the present invention does not contain cells, adverse events due to cell administration can be avoided and handling is easy, providing significant clinical advantages.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a preventive and / or therapeutic drug for cerebrovascular diseases, which contains a filtrate of disrupted mesenchymal stem cell lysate as an active ingredient. [Background technology]

[0002] Cerebrovascular disease is a general term for illnesses caused by abnormalities in cerebral arteries, and is broadly divided into "ischemic cerebrovascular disease," in which blood flow to brain tissue is reduced or lost, and "hemorrhagic cerebrovascular disease," in which bleeding occurs within the skull. Even in today's world of advanced medical technology, cerebrovascular disease remains a major cause of morbidity and mortality, with over 1.1 million patients in Japan, with over 60,000 deaths annually from cerebral infarction, making it a common disease that accounts for a large proportion of deaths in Japan.

[0003] When blood supply to the brain is impaired by cerebrovascular disease, blood cannot reach the brain. This causes brain cells to die or become necrotic, and the function of the damaged part of the brain is reduced. However, brain cells do not regenerate, so lost functions cannot be regained. Therefore, even if the patient survives, they may be left with aftereffects. There are various aftereffects, but typical aftereffects include neurological and motor dysfunction such as cerebral palsy, spasticity, and contracture, higher brain dysfunction that reduces cognitive function, memory function, and language function, and mental disorders such as depression and panic, all of which are said to have aftereffects that interfere with daily life. In addition, cerebral infarction is one of the diseases that is prone to recurrence among cerebrovascular diseases, and there is data that shows that 10 to 30% of cases recur within three years of onset.

[0004] Treatment for cerebrovascular diseases is often divided into four periods. For example, in the case of cerebral infarction, the period within 8 hours after onset is called the "hyperacute phase," the period between 8 hours and 1 week after onset is called the "acute phase," the period between 1 week and 6 months after onset is called the "subacute phase," and the period 6 months after onset is called the "chronic phase."

[0005] In the hyperacute and acute phases, there are thrombolytic therapy using t-PA, which can be administered within 4.5 hours after onset, endovascular treatment, which can be administered within 8 hours after onset, and anticoagulant therapy using heparin and hemodilution therapy, which can be administered within 48 hours after onset. In the subacute phase, brain swelling and high blood pressure will subside to some extent, but in severe cases, serious aftereffects may remain. If aftereffects remain, rehabilitation is given priority, but the current situation is that even with rehabilitation, it is difficult to restore function to the same level as before the onset of the disease.

[0006] Meanwhile, mesenchymal stem cells are cells with pluripotency and self-renewal ability, and can differentiate into various cells including chondrocytes, bone cells, muscle cells, and adipocytes. In the field of regenerative medicine, which has been attracting attention in recent years, mesenchymal stem cells have begun to be used to treat physical disorders that cannot be cured or regenerated. In addition, therapeutic drugs focusing on exosomes secreted by mesenchymal stem cells have also been developed.

[0007] Examples of therapeutic agents for cerebrovascular diseases, such as those containing mesenchymal stem cells as an active ingredient, include Patent Documents 1 to 5, and an example of a composition for treating damaged areas containing stem cell culture supernatant as an active ingredient is Patent Document 6. In addition, examples of therapeutic agents for cerebrovascular diseases, such as those containing stem cell-derived exosomes as an active ingredient, are Patent Documents 7 and 8. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2005 / 007176 [Patent Document 2] International Publication No. 2006 / 085697 [Patent Document 3] International Publication No. 2017 / 188457 [Patent Document 4] Patent Publication No. 2022-126812 [Patent Document 5] Special Publication No. 2022-533781 [Patent Document 6] International Publication No. 2011 / 118795 [Patent Document 7] Special Publication No. 2016-540014 [Patent Document 8] International Publication No. 2016 / 152786 Summary of the Invention [Problem to be solved by the invention]

[0009] There are known treatments for cerebrovascular disease in the acute phase, but these can only be used for patients within a few hours of onset, so further treatments have been required in the medical field. In addition, for the aftereffects in the subacute and chronic phases, rehabilitation and other physical therapy and functional training are the major methods, and there is currently no fundamental treatment.

[0010] The therapeutic drugs for cerebrovascular disease containing mesenchymal stem cells as an active ingredient described in Patent Documents 1 to 5 are described as being effective in the acute, subacute, and chronic phases, but since the cells themselves are used for treatment, it is necessary to start cell culture at the appropriate time for use, and preparation and operation are lacking in convenience. In addition, since live cells are used, there is concern about the occurrence of adverse events such as the risk of tumorigenesis due to undifferentiated cells. Furthermore, the therapeutic drugs described in Patent Documents 1 to 5 are administered into veins or arteries, but in that case, the majority of the mesenchymal stem cells, which are the active ingredient, are captured by the embryo, spleen, liver, etc. Therefore, the dosage (i.e., the number of administered cells) of the therapeutic drugs described in Patent Documents 1 to 5 that are said to be effective may be larger than the amount that would be effective in the first place. There is also concern about side effects because increasing the number of administered cells carries the risk of causing embryo embolism.

[0011] Patent Document 6 confirms the therapeutic effect of stem cell culture supernatant on rats with cerebral infarction, but its effectiveness in humans is unknown. Furthermore, culture supernatants may contain additives that are added to efficiently culture cells, and their safety must be confirmed before administration. Patent Document 6 states that "the effect is achieved without complex and advanced purification," but does not evaluate the side effects of not purifying the cells.

[0012] Patent Documents 7 and 8 relate to exosomes derived from stem cells. Neither document describes stem cell lysate, and Patent Document 7 specifically confirms the inhibitory effect on intraventricular hemorrhage in premature infants, but the extent of the effect on so-called general cerebrovascular diseases, particularly ischemic cerebrovascular diseases, is unclear. Patent Document 8 does not describe or suggest cerebrovascular diseases.

[0013] Therefore, an objective of the present invention is to provide an effective agent for the prevention and / or treatment of cerebrovascular diseases. [Means for solving the problem]

[0014] In order to solve the above problems, the present inventors have focused on the lysate of mesenchymal stem cells and conducted extensive research, and as a result, have found that the lysate has a therapeutic effect on cerebrovascular disease, particularly the effect of alleviating symptoms of subacute or chronic cerebrovascular disease, specifically the effect of recovering neurological and motor functions, and have thus completed the present invention. That is, the present invention has the following configuration.

[0015] [1] A preventive and / or therapeutic agent for cerebrovascular disease, comprising a filtrate of crushed mesenchymal stem cells as an active ingredient. [2] The preventive and / or therapeutic agent for cerebrovascular disease according to [1], wherein the cerebrovascular disease is an ischemic cerebrovascular disease. [3] The filtrate contains 1×10 mesenchymal stem cells. 4 ~1×10 8 The preventive and / or therapeutic agent for cerebrovascular disease according to [1] or [2], which is a filtrate of a lysate of 100 cells / mL. [4] A preventive and / or therapeutic agent for cerebrovascular disease according to [1] or [2], which is administered 14 days or more after the onset of cerebrovascular disease. [5] The preventive and / or therapeutic agent for cerebrovascular disease according to [1] or [2], which is an oral or parenteral formulation. Effect of the Invention

[0016] The preventive and / or therapeutic drug for cerebrovascular disease of the present invention is effective for treating cerebrovascular disease, particularly for alleviating symptoms occurring in patients with cerebrovascular disease in the subacute or chronic phase after the acute phase, specifically for improving neurological dysfunction and motor dysfunction. In addition, since live cells are generally used in cell administration, even if pathogens are present, they cannot be inactivated or removed, which poses the risk of infection. On the other hand, the preventive and / or therapeutic drug for cerebrovascular disease of the present invention uses not cells but their disrupted filtrate, which makes it possible to avoid adverse events caused by cell administration.

[0017] In addition, frozen and thawed cells are rarely used for treatment because they may lose activity or die after being frozen and thawed. Therefore, in order to use live cells, they must be cultured according to the patient's schedule, but the present invention has great clinical advantages, such as no need for preparation when used, easy handling, and long-term storage and freezing and thawing. [Brief description of the drawings]

[0018] [Figure 1] Angiography image of patient A ((B) is an image taken at a different angle than (A)) [Diagram 2] MRI images of patient A. (A) Image used to confirm the location of cerebral infarction, (B) Image immediately after onset (B-1: frontal image, B-2: lateral image), (C) Image one month after administration of nasal drops (C-1: frontal image, C-2: lateral image) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention relates to a drug for preventing and / or treating cerebrovascular disease, which contains a filtrate of a lysate of mesenchymal stem cells as an active ingredient (hereinafter, also referred to as a "medicine for treating cerebrovascular disease"). In this specification, "containing as an active ingredient" means containing a filtrate of a lysate of mesenchymal stem cells in an amount effective for treatment. <Mesenchymal stem cells>

[0020] Mesenchymal stem cells are cells belonging to the mesenchymal system, and are known to have pluripotency and self-renewal capabilities, as well as the ability to differentiate into connective tissue cells such as bone cells, chondrocytes, and adipocytes, as well as into nerve cells and cardiac muscle cells. Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. Of these, adipose tissue-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are preferred, and among these, adipose tissue-derived mesenchymal stem cells are preferred because they can be easily collected.

[0021] Adipose tissue-derived mesenchymal stem cells (also known as ASC, ADSC, AT-MSC, AD-MSC, etc.) are a type of somatic stem cell found in adipose tissue. Adipose tissue-derived mesenchymal stem cells also have the ability to self-renew and differentiate into multiple cell types, and are known to be capable of differentiating into a variety of cells, including not only fat cells, but also bone, cartilage, nerves, muscles, cardiac muscle, blood vessels, hepatocytes, and pancreatic islet cells.

[0022] The mesenchymal stem cells in the present invention may be derived from the same species as the subject (recipient) to which the cerebrovascular disease therapeutic agent of the present invention is administered, or may be derived from a different species. It is preferable to use cells derived from the same species as the recipient. Examples of biological species include humans and non-human mammals (including pet animals, livestock, and experimental animals. Specifically, for example, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, and hamsters). The mesenchymal stem cells may be derived from the subject, i.e., autologous cells, or may be derived from another subject of the same species, i.e., allogeneic cells. Autologous cells are preferable to avoid the problem of immune rejection.

[0023] Adipose tissue-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, or dental pulp-derived mesenchymal stem cells are prepared according to conventional methods. Here, a method for preparing adipose tissue-derived mesenchymal stem cells, which are preferred cells, is specifically described. <Method for preparing adipose tissue-derived mesenchymal stem cells>

[0024] In the present invention, ADSCs also include cells obtained by culturing (including subculture) somatic stem cells, so long as they maintain pluripotency. Usually, ADSCs are prepared in an "isolated state" as cells that constitute a cell population (including cells other than ADSCs derived from adipose tissue) using adipose tissue separated from a living body as the starting material. Here, "isolated state" means a state in which the cells are removed from their original environment (i.e., a state in which they constitute a part of a living body), that is, a state in which the cells exist in a state different from their original state due to artificial manipulation.

[0025] In addition, the preparation of ADSC in the present invention may be performed according to a conventional method. Since ADSC are widely used for various purposes, those skilled in the art can prepare them by referring to literature and books. Cells provided from a public cell bank or commercially available cells may also be used. Below, as an example of a method for preparing cells, a method for preparing adipose tissue-derived mesenchymal stem cells (one example) will be described.

[0026] ADSCs are prepared through steps such as separation, washing, concentration, and culture of stem cells from adipose matrix. The method for preparing ADSCs is not particularly limited. For example, ADSCs can be prepared according to known methods (Fraser JK et al. (2006), Fat tissue: an under appreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr; 24(4): 150-4. Epub 2006 Feb 20. Review.; Zuk PA et al. (2002), Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell; Dec;13(12):4279-95.; Zuk PA et al. (2001), Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Engineering; Apr; 7(2): 211-28., etc. are useful references). In addition, devices for preparing ADSCs from adipose tissue (e.g., Celution (registered trademark) device (Cytori Therapeutics, San Diego, USA)) are commercially available, and ADSCs may be prepared using such devices. Using such devices, a cell population containing ADSCs can be isolated from adipose tissue (K. Lin. Et al. Cytotherapy (2008) Vol. 10, No. 4, 417-426). A specific example of a method for preparing ADSCs is shown below. (1) Preparation of cell populations from adipose tissue

[0027] Adipose tissue is collected from humans and non-human mammals by means of resection, suction, etc. The non-human mammal may be any of the animals mentioned above. The age and sex of the organism are not particularly limited. In humans, ADSCs can also be prepared from tissue pieces aspirated by liposuction during cosmetic surgery, or from excised adipose tissue contained in tissue excised from a living body during surgery. ADSCs exist around large blood vessels, so they can be obtained in larger amounts from excised adipose tissue than from liposuction fluid. On the other hand, preparing stem cells from liposuction fluid leaves smaller surgical scars and places less of a burden on the donor.

[0028] Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Among these, subcutaneous fat is a preferred cell source because it can be collected very easily under local anesthesia and places less of a burden on the donor during collection. Usually, one type of adipose tissue is used, but it is also possible to use two or more types of adipose tissue in combination. In addition, adipose tissues collected in multiple batches (not necessarily the same type of adipose tissue) may be mixed and used in subsequent operations.

[0029] The amount of adipose tissue to be collected can be determined taking into consideration the type of donor, the type of tissue, or the amount of ADSC required, and is, for example, about 0.5 to 500 g. However, taking into consideration the burden on the donor, it is preferable to collect an amount of about 3 to 20 g or less at one time. The collected adipose tissue is subjected to the removal of blood components attached thereto and to fragmentation as necessary, and then subjected to the following enzyme treatment. Blood components can be removed by washing the adipose tissue in an appropriate buffer solution or culture medium.

[0030] The enzyme treatment is carried out by digesting the adipose tissue with an enzyme such as collagenase, trypsin, or dispase. Such enzyme treatment may be carried out by a method and under conditions known to those skilled in the art (see, for example, RI Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). The cell population obtained by the above enzyme treatment includes pluripotent stem cells, endothelial cells, stromal cells, blood cells, and / or their precursor cells. The types and ratios of cells constituting the cell population depend on the origin and type of the adipose tissue used. (2) Acquisition of sedimented cell populations (SVF fractions: stromal vascular fractions)

[0031] The cell population is then subjected to centrifugation. The sediment from the centrifugation is collected as a sedimented cell population (also referred to as "SVF fraction" in this specification). The conditions for centrifugation vary depending on the type and amount of cells, but are, for example, 1 to 10 minutes and 800 to 1,500 rpm. It is preferable to filter the cell population after the enzyme treatment prior to centrifugation to remove any tissues that are not digested by the enzyme. The "SVF fraction" obtained here contains ADSC. The type and ratio of cells that make up the SVF fraction depend on the origin and type of the adipose tissue used, the conditions of the enzyme treatment, and the like. The characteristics of the SVF fraction are also shown in the pamphlet of International Publication No. 2006 / 006692A1. (3) Selective culture of adherent cells (ADSC) and cell recovery

[0032] The SVF fraction contains other cell components (endothelial cells, interstitial cells, blood cells, their precursor cells, etc.) in addition to ADSCs. Therefore, in one embodiment of the present invention, the following selective culture is performed to remove unnecessary cell components from the SVF fraction. The cells obtained as a result are used as ADSCs in the present invention. First, the SVF fraction is suspended in an appropriate medium, then seeded on a culture dish and cultured overnight. Floating cells (non-adherent cells) are removed by changing the medium. Thereafter, the culture is continued while appropriately changing the medium (for example, once every 2 to 4 days). Subculture is performed as necessary. The number of passages is not particularly limited, but excessive repeating of passages is not preferable from the viewpoint of maintaining pluripotency and proliferation ability (it is preferable to limit it to about 6 passages). In addition, a medium for normal animal cell culture can be used as the culture medium. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., Ltd., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Institute), etc. may be used. A medium supplemented with serum (fetal bovine serum, human serum, sheep serum, etc.) or a serum substitute (Knockout serum replacement (KSR), etc.) may also be used. The amount of serum or serum substitute added can be set within the range of, for example, 5 to 30% (v / v).

[0033] The above procedure allows the adherent cells to selectively survive and proliferate. The proliferated cells are then harvested. The harvesting procedure can be performed according to conventional methods. For example, the cells can be easily harvested by detaching them with a cell scraper or pipette after enzyme treatment (trypsin or dispase treatment). In addition, when sheet culture is performed using a commercially available temperature-sensitive culture dish, it is also possible to harvest the cells in a sheet form without enzyme treatment. By using the cells (ADSC) harvested in this way, a cell population containing ADSC at high purity can be prepared. (4) Low-serum culture (selective culture in low-serum medium) and cell recovery

[0034] In one embodiment of the present invention, instead of or after the above operation (3), the following low serum culture is performed. The cells obtained as a result are used in the present invention as ADSCs. In the low serum culture, the SVF fraction (when this step is performed after (3), the cells collected in (3) are used) is cultured under low serum conditions to selectively proliferate the target pluripotent stem cells (i.e., ADSCs). Since the low serum culture method requires only a small amount of serum, when the ADSCs obtained by the method of the present invention are used for therapeutic purposes, it is possible to use the subject's (patient's) own serum. In other words, culture using autologous serum is possible. Here, "under low serum conditions" refers to conditions in which the medium contains 5% (v / v) or less of serum. Preferably, the cells are cultured in a culture medium containing 2% (v / v) or less of serum. More preferably, the cells are cultured in a culture medium containing 2% (v / v) or less of serum and 1 to 100 ng / mL of fibroblast growth factor-2 (bFGF). A serum-free medium may be used.

[0035] The serum is not limited to fetal bovine serum, and human serum, sheep serum, etc. can be used. When the activated sperm obtained by the method of the present invention is used for human treatment, preferably human serum, more preferably serum of the subject to be treated (i.e., autologous serum) is used. The medium can be a medium for normal animal cell culture, provided that the amount of serum contained at the time of use is low. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., Ltd., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Institute), etc. can be used.

[0036] By culturing in the above manner, ADSCs can be selectively proliferated. In addition, since ADSCs proliferated under the above culture conditions have high proliferation activity, the number of cells required for the present invention can be easily prepared by subculture. In addition, WO 2006 / 006692A1 shows the characteristics of cells selectively proliferated by low serum culture of SVF fraction. Next, the cells selectively proliferated by the above low serum culture are collected. The collection operation may be performed in the same manner as in (3) above. By using the collected ADSCs, a cell population containing ADSCs at high purity can be obtained.

[0037] In the above method, the cells proliferated by low serum culture of the SVF fraction are used, but the cells proliferated by directly culturing a cell population obtained from adipose tissue in low serum (without going through centrifugation to obtain the SVF fraction) may be used as ADSCs. That is, in one aspect of the present invention, the cells proliferated when a cell population obtained from adipose tissue is cultured in low serum are used as ADSCs. Also, instead of the pluripotent stem cells obtained by selective culture (above (3) and (4)), the SVF fraction (containing adipose tissue-derived mesenchymal stem cells) may be used as it is. Here, "using as it is" means using in the present invention without going through selective culture. <Method for preparing filtrate of mesenchymal stem cell lysate>

[0038] The therapeutic agent for cerebrovascular disease of the present invention contains as an active ingredient a filtrate (sometimes referred to as "stem cell filtrate") obtained by filtering the lysate obtained by lysing mesenchymal stem cells.

[0039] A general cell disruption method can be used to disrupt mesenchymal stem cells. For example, a treatment method using freeze-thaw (a process of freezing and then thawing), ultrasound, a French press, a mortar, a homogenizer, glass beads, or the like can be used. In addition, the cells to be subjected to the disruption process are not limited to living cells, and dead cells or damaged cells may be used. Among the above-mentioned disruption processes, freeze-thawing and ultrasound treatment are preferred. In particular, freeze-thawing is particularly preferred because it is simple and can avoid contamination due to contact between the machine and the cells, and is hygienic. When disrupting by freeze-thawing, the preferred conditions described below can be used. When disrupting by ultrasound treatment, it is preferable to use unfrozen cells for the disruption process, but since the heat generated by the equipment often causes protein denaturation and aggregation, it is preferable to repeatedly perform the short-term treatment while cooling the cell suspension in ice. Specifically, it is preferable to repeat multiple times the disruption for 5 to 15 seconds at an output of 200 to 300 W and a pause for 10 to 30 seconds.

[0040] In addition, during the freezing process, cells expand and ice crystals are formed, and the ice crystals destroy the cells, which dissolve when thawed. Therefore, it is preferable to repeat the freezing and thawing process in order to dissolve the cells sufficiently. Specifically, it is preferable to repeat the freezing and thawing process 1 to 5 times. The freezing conditions in the freezing and thawing process are not particularly limited, but freezing at, for example, -20 to -196°C is preferable. The thawing conditions are also not particularly limited. For example, thawing by leaving the cells in a refrigerator at 5°C or less overnight, thawing in a hot water bath (for example, 35 to 40°C), thawing at room temperature, etc. can be used. The concentration of the cell suspension used for disruption is 1 x 10 4 ~1×10 8 Preferably, the concentration of mesenchymal stem cells is 10×10 4 ~500×10 4 cells / mL is more preferable, as it is a more workable concentration and allows for a sufficient amount of filtrate to be obtained in one run.

[0041] The disruption solution may be centrifuged and the supernatant may be used in the next step (filter treatment). By centrifuging the disruption solution, nuclei and other particles are removed and clogging of the filter can be prevented, allowing efficient filter treatment. When centrifuging in advance, it is preferable to centrifuge at 100 to 1,500×g for 3 to 10 minutes after disrupting the mesenchymal stem cells. The temperature during centrifugation is not particularly limited.

[0042] The therapeutic agent for cerebrovascular disease of the present invention uses the filtrate obtained by filtering the cell lysate or the supernatant obtained by centrifugation. Unnecessary components can be removed by filtering. Furthermore, by using a filter with an appropriate pore size, removal of unnecessary components and sterilization filtration can be performed simultaneously. The material of the filter used for filtering is not particularly limited, but cellulose acetate and metal filters that are less likely to adsorb proteins are preferred. Cellulose acetate is particularly preferred. The filter pore size is preferably 0.1 to 0.45 μm, more preferably 0.15 to 0.3 μm. When sterilization filtration is also performed at the same time, a pore size of 0.2 μm is preferred.

[0043] If the filtrate is not to be used immediately for treatment, it can be frozen and stored until use. It is preferably stored at -100 to -60°C. In general, repeated freezing and thawing of cells tends to reduce cell activity and increase the number of dead cells, but since the therapeutic agent for cerebrovascular disease of the present invention is a filtrate and does not contain stem cells, its quality does not change even if it is frozen and thawed many times. <Method of manufacturing therapeutic drug for cerebrovascular disease>

[0044] The therapeutic agent for cerebrovascular disease of the present invention can be produced by the following steps: The method for culturing mesenchymal stem cells, the method for homogenization, the method for centrifugation and the method for filtration have been described above. (1) A step of disrupting mesenchymal stem cells. (2) A step of filtering the disruption solution obtained in step (1) or a supernatant obtained by centrifuging the disruption solution to obtain a filtrate. (3) A step of formulating the filtrate obtained in step (2).

[0045] The therapeutic agent for cerebrovascular disease of the present invention is formulated by mixing with non-toxic and inactive medicamentously acceptable excipients, such as solid, semi-solid or liquid diluents, dispersants, fillers and carriers.Furthermore, within the range that does not impair the effect of the present invention, stabilizers, preservatives, pH adjusters, binders, disintegrants, surfactants, lubricants, flow enhancers, flavorings, colorants, flavorings, preservatives, antibacterial agents, media, physiological saline, and other medicinal agents may be included as additives. <Administration method and dosage>

[0046] The dosage form of the therapeutic agent for cerebrovascular disease of the present invention is not particularly limited, and oral or parenteral preparations are preferred. Oral preparations include dosage forms that can be administered orally, such as tablets, pills, powders, granules, capsules, syrups, emulsions, and suspensions. Parenteral preparations include dosage forms such as ointments, injections, intravenous injections (infusions), intramuscular injections, subcutaneous injections, and nasal drops (nasal sprays). Among these, nasal drops are preferred. When administered intravenously as a parenteral preparation, the active ingredient may be captured in the lungs, spleen, liver, etc., making it difficult for the active ingredient to reach the lesion and exert a sufficient effect. In this respect, nasal drops are preferred because they are less invasive and can efficiently deliver the active ingredient to the brain, which is the target tissue. When used as a nasal drop, it is preferable to prepare the filtrate using PBS and / or physiological saline.

[0047] The dosage of the therapeutic agent for cerebrovascular disease of the present invention can be appropriately adjusted taking into consideration the method of administration, the sex, age, weight, symptoms, etc. of the subject to be administered.

[0048] For administration to humans, the concentration of mesenchymal stem cells is 1×10 4 ~1×10 8It is preferable that the cell suspension is a filtrate of a cell suspension of 1000000000000000000 cells / mL. It may be administered once to three times a day, or intermittently every one to two days. Since the dosage varies depending on various conditions, a dosage or number of administrations less than the above range may be sufficient, or a dosage or number of administrations exceeding the above range may be necessary. When administering to humans as a nasal drop, it is preferable that the amount sprayed per shot is 0.05 mL to 0.20 mL. It is also preferable to spray one shot into each of the left and right nostrils twice a day, in the morning and evening.

[0049] The therapeutic agent for cerebrovascular disease of the present invention is particularly effective in the treatment of subacute or chronic phases, and is therefore preferably administered 14 days or later after the onset of cerebrovascular disease. By administering at such a time, it is possible to improve the neurological and motor functions impaired by cerebrovascular disease. Furthermore, it is also effective in promoting angiogenesis and improvement of vascular occlusion in the lesion, and is therefore particularly effective in the treatment of ischemic cerebrovascular disease.

[0050] When the therapeutic agent for cerebrovascular disease of the present invention is administered, it may be administered in combination with other therapeutic agents such as antiplatelet agents and antihypertensive agents. The term "co-administration" refers to administration simultaneously with the administration of the therapeutic agent for cerebrovascular disease of the present invention, or administration before or after administration of the therapeutic agent for cerebrovascular disease of the present invention. Alternatively, the therapeutic agent for cerebrovascular disease of the present invention and the other therapeutic agents may be mixed to prepare a single preparation. <Applicable to>

[0051] Cerebrovascular disease is a general term for diseases caused by abnormalities in cerebral arteries, and is broadly divided into "ischemic cerebrovascular disease," in which blood flow to brain tissue is reduced or lost, and "hemorrhagic cerebrovascular disease," in which bleeding occurs within the skull. Ischemic cerebrovascular disease includes cerebral infarction, cerebral arteriosclerosis, cerebral thrombosis, and cerebral embolism, and causes loss of brain function due to occlusion or stenosis of the artery, which causes a stagnation of blood supply. Hemorrhagic cerebrovascular disease includes cerebral hemorrhage and subarachnoid hemorrhage, and causes loss of brain function due to compression of brain cells caused by bleeding from hematomas or rupture of cerebral aneurysms.

[0052] The therapeutic agent for cerebrovascular disease of the present invention is effective for ischemic cerebrovascular disease and / or hemorrhagic cerebrovascular disease, and is particularly effective for ischemic cerebrovascular disease. In particular, it is effective for treating cerebral infarction. Cerebral infarction includes lacunar infarction caused by intracerebral small artery lesion, atherothrombotic cerebral infarction caused by atherosclerosis of relatively large intracranial arteries, and cardiogenic cerebral embolism caused by heart disease, and the therapeutic agent for cerebral infarction of the present invention is preferably used in any of the above cases.

[0053] There are four periods in cerebrovascular disease. In the present invention, the period within 8 hours after onset is the "hyperacute period", the period from 8 hours to 1 week after onset is the "acute period", the period from 1 week to 6 months after onset is the "subacute period", and the period after 6 months after onset is the "chronic period". The therapeutic drug for cerebrovascular disease of the present invention can be used in any of the hyperacute, acute, subacute, and chronic periods, but is particularly suitable for use in the subacute and chronic periods. In the subacute and chronic periods, symptoms are almost fixed and rehabilitation and prevention of recurrence are the main focus. By administering the therapeutic drug for cerebrovascular disease of the present invention, improvement of neurological and motor functions, and angiogenesis in cerebrovascular lesions and improvement and promotion of vascular occlusion can be expected. EXAMPLES

[0054] The present invention will be described in detail below by way of examples, but the present invention is not limited to these examples in any way. <Preparation of nasal drops>

[0055] (1) Preparation of filtrate from ADSC lysate A filtrate was prepared using human adipose stem cells by the method described herein. Specifically, the human adipose stem cells were cultured in Messenger Pro RS medium to proliferate ADSCs. The cells were cultured at a concentration of 5 × 10 6 The cells were subcultured for 6 passages until 1 × 10 6A cell suspension of 1000 cells / mL was prepared. It was left overnight at -30°C and frozen. The frozen cell solution was left overnight in a 4°C refrigerator and thawed. After the cells were disrupted in this way, they were filtered through a cellulose acetate membrane filter (pore size 0.2 μm) to obtain the filtrate of ADSC disruption. Serum-free medium (KBM ADSC-5, Kohjin Bio Co., Ltd.) was used as a control. (2) Preparation of nasal drops

[0056] The ADSC lysate obtained in (1) (1 × 10 6 cells / mL), 1 mL of saline and an antibiotic (manufacturer: Gibco TM Antibiotic-Antimycotic (100X)) 25ug / mL was added and filled into a nasal drop bottle of 20mL. One shot can spray 0.13mL (±0.05mL). <Testing method>

[0057] (1) Modified Water Swallowing Test The procedure was carried out as follows. 1. Place 3 mL of cold water in the subject's mouth. 2. Ask the subject to swallow. 3. Evaluate whether the swallowing reflex can be induced, choking, or changes in breathing. 4. Based on the following criteria, if the score is 4 or more, repeat the test up to two times (total of three times), and the worst case will be the evaluation result. The criteria for the revised water drinking test are shown in Table 1. [Table 1] (2)National Institutes of Health Stroke Scale (NIHSS)

[0058] The NIHSS is a quantitative evaluation of neurological symptoms specific to stroke, scored. The NIHSS is broadly divided into three categories: consciousness and eyes, motor, and sensory and language, but here we evaluated dysarthria. Dysarthria was evaluated by showing the subjects "word cards" and having them read the words written on them. Normal was rated "0," mild to moderate (there was dysarthria, but they could understand what was being said) was rated "1," severe (they could not understand what was being said) was rated "2," and if there was intubation or a physical barrier, it was rated "N." <Administration to patients with cerebrovascular disease>

[0059] For three patients with subacute or chronic cerebrovascular disease as shown in Table 2, the above-mentioned nasal spray bottle was sprayed into each of the left and right nostrils every morning and evening for a certain period of time. [Table 2] <Patient A>

[0060] Patient A is a patient with atherothrombotic cerebral infarction. Figure 1 shows an angiography image of Patient A. The length of the vertebral artery stenosis was 240 mm, so catheter therapy was not applicable. Figure 1(B) is an image taken at a different angle than Figure 1(A). This patient was administered nasal drops from January 6, 2023 to February 3, 2023. The test results before and after administration of the nasal drops are shown in Figure 2 and Table 3. Patient A has been taking medication continuously since the onset of the disease.

[0061] Figure 2 (A) is an MRI image of Patient A's brain at the time of onset. Cerebral infarction can be seen where the arrow indicates. (B) is an MRI image of the blood flow in the vertebral artery taken immediately after the onset of symptoms, and (C) is an image taken one month after administration of the nasal drops. As is clear from the image in (C), the blood flow signals in the vertebral artery and basilar artery can be clearly confirmed, and a dramatic improvement was observed. (C-1) is a frontal image of the patient's head, and (C-2) is a lateral image, and in both images, improvement in the blood flow signals was observed.

[0062] Table 3 shows the test results before and after administration of the nasal spray. The administration of the nasal spray reduced blood pressure and triglyceride levels in particular, and also significantly improved subjective symptoms such as dizziness. These improvements continued even several months after administration. [Table 3] <Patient B>

[0063] Patient B is a patient with cerebral hemorrhage. After the onset of the disease, he underwent rehabilitation and occupational therapy (walking training, upper limb training) almost every day, and although the pain had improved, he suffered from aftereffects such as numbness and speech disorder. Therefore, from February 7, 2023 to July 3, 2023, he was administered nasal drops. The results of interviews with the patient after administration of the nasal drops are shown in Table 4. As a result of administering the nasal drops, there was a significant improvement in subjective symptoms such as numbness, speech disorder, and walking on flat ground. [Table 4] <Patient C>

[0064] Patient C is a cerebral infarction patient. Although he has a high degree of independence in daily life, he was particularly troubled by numbness and speech disorder. Therefore, from February 1, 2023 to March 3, 2023, nasal drops were administered. The results of interviews with the patient after administration of the nasal drops and the speech disorder score are shown in Table 5. As a result of administration of the nasal drops, there was a significant improvement in subjective symptoms such as numbness, speech disorder, and walking on flat ground. [Table 5]

[0065] From the above results, it was confirmed that the therapeutic agent of the present invention has an effect of increasing blood flow, decreasing blood pressure and neutral fat levels, and improving subjective symptoms such as neurological dysfunction such as numbness and motor dysfunction such as walking, when administered to patients with cerebrovascular disease. It is particularly effective for patients with cerebrovascular disease who cannot undergo surgery at the occluded part. It is also effective in improving the occluded part, which is difficult to improve with diet therapy or exercise therapy. [Industrial Applicability]

[0066] The therapeutic agent for cerebrovascular disease of the present invention is effective in treating cerebrovascular disease.

Claims

1. A preventive and / or therapeutic agent for cerebrovascular disease, comprising a filtrate of pulverized mesenchymal stem cells as an active ingredient.

2. The preventive and / or therapeutic drug for cerebrovascular disease according to claim 1, wherein the cerebrovascular disease is an ischemic cerebrovascular disease.

3. The filtrate is diluted with 1×10 4 ~1×10 8 3. The preventive and / or therapeutic drug for cerebrovascular disease according to claim 1 or 2, which is a filtrate of a homogenate solution of 100 cells / mL.

4. 3. The preventive and / or therapeutic drug for cerebrovascular disease according to claim 1 or 2, which is administered 14 days or later after the onset of cerebrovascular disease.

5. 3. The preventive and / or therapeutic agent for cerebrovascular disease according to claim 1 or 2, which is an oral or parenteral formulation.

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