Composition for treating cerebral hemorrhage

Abstract

Provided is a composition or the like useful for filling an intracerebral hematoma cavity in a subject. This composition for filling an intracerebral hematoma cavity contains an alginic acid monovalent metal salt, and is used in combination with mesenchymal stem cells.

Description

Composition for the treatment of cerebral hemorrhage

[0001] This invention relates to a composition for the treatment of cerebral hemorrhage. In particular, this invention relates to a composition used to fill a hematoma cavity in the brain.

[0002] Strokes include ischemic strokes, which occur when blood vessels in the brain become blocked, and hemorrhagic strokes, which occur when blood vessels in the brain rupture. Both types of strokes cause significant damage to the brain.

[0003] Among strokes, cerebral hemorrhage is a disease that causes severe neurological sequelae. According to the results of a nationwide stroke survey in Japan (Toyoda K, et al. JAMA Neurol, 2022), the post-treatment outcomes of cerebral hemorrhage patients have not improved over the past 20 years. In fact, the proportion of patients with severe neurological sequelae is on the rise.

[0004] In recent years, cell-based therapies have attracted attention as treatments to minimize or reverse neurological sequelae in patients with cerebral hemorrhage. For example, Non-Patent Documents 1 to 5 disclose various studies applying mesenchymal stem cells suspended in phosphate-buffered saline (PBS) or saline to cerebral hemorrhage model rats or human patients. Patent Document 1 discloses microcapsules that can be used for the treatment of cerebral hemorrhage, which may contain cross-linked alginate and mesenchymal stem cells as components.

[0005] Special table number 2009-536166

[0006] Cell Physiol Biochem 2009;24:307-316Mol Cells. 2017 Feb 28; 40(2): 133-142. PMID: 28190323Stem Cells Int. 2015; 2015: 318269.Exp Ther Med. 2016 Dec; 12(6): 3535-3540.Neural Regen Res 18(9):1999-2004.

[0007] The present invention aims to provide a composition and the like for use in filling a hematoma cavity that has formed in the brain due to a cerebral hemorrhage.

[0008] As a result of intensive studies, the present inventors have completed the present invention. The present invention provides the following compositions for filling intracerebral hematoma cavities (also simply referred to as "compositions").

[0009] [1-1] A composition for filling an intracerebral hematoma cavity, containing a monovalent metal alginate, and used in combination with mesenchymal stem cells. [1-2] The composition according to [1-1], wherein the monovalent metal alginate is a low endotoxin. [1-3] The composition according to [1-1] or [1-2], wherein the monovalent metal alginate is sodium alginate. [1-4] The composition according to any one of [1-1] to [1-3], wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, skeletal muscle, umbilical cord, dental pulp, dermis or peripheral blood. [1-5] The composition according to any one of [1-1] to [1-4], wherein the mesenchymal stem cells are of allogeneic origin. [1-6] The cell density of the mesenchymal stem cells is used so as to be 1.0×10 ４ cells / mL or more after being combined with the composition for filling an intracerebral hematoma cavity. The composition according to any one of [1-1] to [1-5]. [1-7] The composition according to any one of [1-1] to [1-6], which is used in a method for treating cerebral hemorrhage in a subject. [1-8] The composition according to [1-7], wherein the cerebral hemorrhage is acute cerebral hemorrhage. [1-9] The composition according to [1-7] or [1-8], wherein the cerebral hemorrhage includes one or more selected from the group consisting of subcortical hemorrhage, hypothalamic hemorrhage, brainstem hemorrhage, cerebellar hemorrhage and putamen hemorrhage. [1-10] A kit for treating cerebral hemorrhage, comprising mesenchymal stem cells and a monovalent metal alginate. [1-11] The kit according to [1-10], wherein the monovalent metal alginate is in a dry state or a liquid state. [1-12] A composition for filling an intracerebral hematoma cavity, containing mesenchymal stem cells, and used in combination with a biomaterial.

[0010] [2-1] A cerebral hematoma cavity filling agent containing a monovalent metal alginate, which is used in combination with mesenchymal stem cells. [2-2] The cerebral hematoma cavity filling agent according to [2-1], wherein the monovalent metal alginate is low in endotoxin. [2-3] The cerebral hematoma cavity filling agent according to [2-1] or [2-2], wherein the monovalent metal alginate is sodium alginate. [2-4] The cerebral hematoma cavity filling agent according to any one of [2-1] to [2-3], wherein the mesenchymal stem cells are derived from bone marrow, fat, skeletal muscle, umbilical cord, dental pulp, dermis, or peripheral blood. [2-5] The cerebral hematoma cavity filling agent according to any one of [2-1] to [2-4], wherein the mesenchymal stem cells are allogeneic. [2-6] The cell density of the mesenchymal stem cells is 1.0 × 10⁻⁶ after combination with the cerebral hematoma cavity filling agent. ４ [2-1] to [2-5] or higher is used to fill the intracerebral hematoma cavity. [2-7] An intracerebral hematoma cavity filling agent according to any one of [2-1] to [2-6] used in a method for treating the target cerebral hemorrhage. [2-8] An intracerebral hematoma cavity filling agent according to [2-7], wherein the cerebral hemorrhage is acute cerebral hemorrhage. [2-9] An intracerebral hematoma cavity filling agent according to [2-7] or [2-8], wherein the cerebral hemorrhage comprises one or more selected from the group consisting of subcortical hemorrhage, subthalamic hemorrhage, brainstem hemorrhage, cerebellar hemorrhage and putaminal hemorrhage. [2-10] An intracerebral hematoma cavity filling agent containing mesenchymal stem cells, which is used in combination with a biomaterial.

[0011] [3-1] A combination of monovalent metal alginate and mesenchymal stem cells for filling the target intracerebral hematoma cavity. [3-2] The combination according to [3-1], wherein the monovalent metal alginate is low in endotoxin. [3-3] The combination according to [3-1] or [3-2], wherein the monovalent metal alginate is sodium alginate. [3-4] The combination according to any one of [3-1] to [3-3], wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, skeletal muscle, umbilical cord, dental pulp, dermis, or peripheral blood. [3-5] The combination according to any one of [3-1] to [3-4], wherein the mesenchymal stem cells are allogeneic. [3-6] The cell density of the mesenchymal stem cells after combination with monovalent metal alginate is 1.0 × 10⁻⁶.４ A combination according to any one of [3-1] to [3-5], which is cells / mL or more. [3-7] A combination according to any one of [3-1] to [3-6], which is used in a method for treating a target cerebral hemorrhage. [3-8] The combination according to [3-7], wherein the cerebral hemorrhage is acute cerebral hemorrhage. [3-9] The combination according to [3-7] or [3-8], wherein the cerebral hemorrhage includes one or more selected from the group consisting of subcortical hemorrhage, hypothalamic hemorrhage, brain stem hemorrhage, cerebellar hemorrhage, and putamen hemorrhage.

[0012] [4-1] Use of a monovalent metal alginate for filling an intracerebral hematoma cavity, in combination with mesenchymal stem cells. [4-2] The use according to [4-1], wherein the monovalent metal alginate is low endotoxin. [4-3] The use according to [4-1] or [4-2], wherein the monovalent metal alginate is sodium alginate. [4-4] The use according to any one of [4-1] to [4-3], wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, skeletal muscle, umbilical cord, dental pulp, dermis, or peripheral blood. [4-5] The use according to any one of [4-1] to [4-4], wherein the mesenchymal stem cells are of allogeneic origin. [4-6] After combination with the monovalent metal alginate, the cell density of the mesenchymal stem cells is 1.0×10 ４ cells / mL or more, and the use according to any one of [4-1] to [4-5]. [4-7] The use according to any one of [4-1] to [4-6], which is used in a method for treating a target cerebral hemorrhage. [4-8] The use according to [4-7], wherein the cerebral hemorrhage is acute cerebral hemorrhage. [4-9] The use according to [4-7] or [4-8], wherein the cerebral hemorrhage includes one or more selected from the group consisting of subcortical hemorrhage, hypothalamic hemorrhage, brain stem hemorrhage, cerebellar hemorrhage, and putamen hemorrhage.

[0013] [5-1] A method for treating a target cerebral hemorrhage, comprising injecting a cerebral hematoma cavity filling composition containing a monovalent metal alginate and mesenchymal stem cells into the target cerebral hematoma cavity. [5-2] The method of treatment according to [5-1], wherein the monovalent metal alginate is low endotoxin. [5-3] The method of treatment according to [5-1] or [5-2], wherein the monovalent metal alginate is sodium alginate. [5-4] The method of treatment according to any one of [5-1] to [5-3], wherein the mesenchymal stem cells are derived from bone marrow, fat, skeletal muscle, umbilical cord, dental pulp, dermis, or peripheral blood. [5-5] The method of treatment according to any one of [5-1] to [5-4], wherein the mesenchymal stem cells are allogeneic. [5-6] The cell density of the mesenchymal stem cells after combination with the cerebral hematoma cavity filling composition is 1.0 × 10⁻⁶ ４ A treatment method according to any one of items [5-1] to [5-5], wherein the cells / mL is ≥ [5-7]. A treatment method according to any one of items [5-1] to [5-6], wherein the cerebral hemorrhage is acute cerebral hemorrhage. A treatment method according to any one of items [5-8], wherein the cerebral hemorrhage includes one or more selected from the group consisting of subcortical hemorrhage, subthalamic hemorrhage, brainstem hemorrhage, cerebellar hemorrhage, and putaminal hemorrhage. A treatment method according to any one of items [5-1] to [5-7], wherein the cerebral hemorrhage comprises aspirating and removing a hematoma that has occurred in the brain, washing the hematoma cavity with physiological saline, and injecting a cerebral hematoma cavity filling composition containing monovalent metal alginate and mesenchymal stem cells into the cerebral hematoma cavity of the target.

[0014] [6-1] A composition for filling a cerebral hematoma cavity, comprising mesenchymal stem cells, which is used in combination with a biomaterial. [6-2] The composition according to [6-1], wherein the biomaterial is low in endotoxin. [6-3] The composition according to [6-1] or [6-2], wherein the biomaterial is sodium alginate. [6-4] The composition according to any one of [6-1] to [6-3], wherein the mesenchymal stem cells are derived from bone marrow, fat, skeletal muscle, umbilical cord, dental pulp, dermis, or peripheral blood. [6-5] The composition according to any one of [6-1] to [6-4], wherein the mesenchymal stem cells are allogeneic. [6-6] The cell density of the mesenchymal stem cells after combination with the biomaterial is 1.0 × 10⁻⁶ ４ A composition according to any one of the claims [6-1] to [6-5], used to have a concentration of cells / mL or higher. [6-7] A composition according to any one of the claims [6-1] to [6-6], used in a method for treating the target cerebral hemorrhage. [6-8] The composition according to [6-7], wherein the cerebral hemorrhage is acute cerebral hemorrhage. [6-9] The composition according to [6-7] or [6-8], wherein the cerebral hemorrhage comprises one or more selected from the group consisting of subcortical hemorrhage, subthalamic hemorrhage, brainstem hemorrhage, cerebellar hemorrhage, and putaminal hemorrhage.

[0015] The present invention provides a composition for filling intracerebral hematoma cavities, an intracerebral hematoma cavities filling agent, a combination of monovalent metal alginate and mesenchymal stem cells for filling intracerebral hematoma cavities, the use of monovalent metal alginate, and a method for treating cerebral hemorrhage.

[0016] Figure 1 is a graph showing the evaluation results of the Modified Neurological Severity Score (mNSS). Figure 2 is a graph showing the evaluation results of the rotarod test. Figure 3 is a graph showing the evaluation results of the body swing test. Figure 4 is a graph showing the evaluation results of (a) the total distance moved by the rats and (b) the time spent in the central area in the open field test. Figure 5 is a graph showing the evaluation results of (a) hematoma cavity volume and (b) brain atrophy rate by H&E staining. Figure 6 is a graph showing (a) the number of Iba1-positive cells 3 days after brain hemorrhage and (b) the number of Iba1-positive cells 28 days after brain hemorrhage by immunohistochemistry. Figure 7 is a graph showing (a) the CD68-positive cell area 3 days after brain hemorrhage and (b) the CD68-positive cell area 28 days after brain hemorrhage by immunohistochemistry. Figure 8 is a graph showing the number of MPO-positive cells 3 days after brain hemorrhage by immunohistochemistry. Figure 9 is a graph showing the number of TUNEL-positive cells 3 days after brain hemorrhage by immunohistochemistry. Figure 10 is a graph showing BDNF production on days 1, 3, and 7 after cerebral hemorrhage, as determined by immunohistochemistry. Figure 11 is a graph showing BDNF production and the number of viable cells on day 7 of culture in vitro.

[0017] The numerical ranges described herein can be any combination of upper and lower limits. For example, if the numerical range is described as "3.0 to 10, 3.2 to 8", the ranges of "3.0 to 8" and "3.2 to 10" are also included in the numerical range described herein. Similarly, if the numerical range is described as "3.0 or greater, 3.2 or greater, and 10 or less, 8 or less", the ranges of "3.0 to 8" and "3.2 to 10" are also included in the numerical range described herein. Furthermore, multiple requirements in the various embodiments described herein can be combined. In addition, numerical ranges indicated using "~" in this specification indicate a range that includes the numbers described before and after "~" as the minimum and maximum values, respectively.

[0018] 1. Composition containing a monovalent metal alginate salt (First Composition) The present invention provides a composition containing a monovalent metal alginate salt, before being combined with mesenchymal stem cells (hereinafter also referred to as "First Composition"). The First Composition of the present invention is used in combination with mesenchymal stem cells. The various components that constitute the First Composition of the present invention, or that are used in combination with the First Composition of the present invention, and the physical properties of the First Composition will be described in detail below.

[0019] 1-1. Monovalent metal alginate salts The "monovalent metal alginate salt" contained in the first composition of the present invention is a salt in which the hydrogen atom of the carboxylic acid at position 6 of alginic acid is replaced with Na ＋ Ya K ＋ It is a water-soluble salt produced by ion exchange with monovalent metal ions such as [specific examples of monovalent metal salts of alginate]. Specifically, examples of monovalent metal salts of alginate include sodium alginate and potassium alginate, with sodium alginate being preferred.

[0020] The first composition of the present invention may be in a solid state or a liquid state. In the solid state, it may be a freeze-dried body or a freeze-dried powder, and in the liquid state, it may be liquid or non-gel. When the first composition of the present invention is in a liquid state, it may gel due to heat, temperature, light or chemical action (e.g., ionic crosslinking, chemical crosslinking, etc.), but it is desirable that it be in a fluid liquid or non-gel state at least when injected into the intracerebral hematoma cavity. When the first composition of the present invention is mixed with mesenchymal stem cells and then injected into the intracerebral hematoma cavity, it is also desirable that it be fluid and in a liquid or non-gel state.

[0021] The concentration of monovalent metal alginate salt contained in the first composition of the present invention cannot be generalized as it is affected by the molecular weight, but is preferably 0.1 to 5.0 w / w%, more preferably 0.5 to 4.0 w / w%, even more preferably 0.8 to 3.0 w / w%, and particularly preferably 1.0 to 2.5 w / w%. In another embodiment, it is preferably 0.1 to 5.0 w / v%, more preferably 0.5 to 4.0 w / v%, even more preferably 0.8 to 3.0 w / v%, and particularly preferably 1.0 to 2.5 w / v%.

[0022] In this invention, "alginic acid" refers to a biodegradable high-molecular-weight polysaccharide, a polymer formed by the linear polymerization of two types of uronic acids, D-mannuronic acid (M) and L-guluronic acid (G). More specifically, it is a block copolymer in which a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a fraction in which D-mannuronic acid and L-guluronic acid are randomly arranged (MG fraction) are arbitrarily bonded together.

[0023] The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid in alginic acid varies mainly depending on the type of organism from which it is derived, such as seaweed, and is also influenced by the habitat and season of the organism, resulting in a wide range from a high-G type with an M / G ratio of about 0.1 to a high-M type with an M / G ratio of about 5. The M / G ratio of the alginic acid used in the present invention is about 0.1 to 5.0, preferably about 0.2 to 4.0, and more preferably about 0.3 to 3.0, from the viewpoint of easily adjusting the apparent viscosity of the composition to a predetermined numerical range.

[0024] Alginic acid may be of natural origin or synthetic, but it is preferable that it be of natural origin. Examples of naturally derived alginic acid include that extracted from brown algae. Examples of brown algae that can be used as raw materials for alginic acid include the genera Lessonia, Macrocystis, Laminaria (kelp), Ascophyllum, Durvillea, Eisenia, and Ecklonia, and more preferably Lessonia, and particularly preferably Lessonia nigressens.

[0025] Monovalent metal alginate salts are high-molecular-weight polysaccharides. The molecular weight of the monovalent metal alginate salt contained in the first composition of the present invention is preferably in the range of 10,000 to 1,000,000, more preferably 80,000 to 800,000, and even more preferably 90,000 to 500,000, in absolute molecular weight.

[0026] It is generally known that the molecular weight of polymeric substances can vary depending on the measurement method. For example, the weight-average molecular weight of the monovalent metal alginate salt used in this invention, measured by gel permeation chromatography (GPC) or gel filtration chromatography (collectively referred to as "size exclusion chromatography (SEC)"), is preferably 100,000 to 5,000,000, more preferably 500,000 to 3,500,000, and even more preferably 1,000,000 to 3,000,000. When using gel permeation chromatography for molecular weight measurement, for example, a GMPW-XL x 2 + G2500PW-XL (7.8 mm I.D. x 300 mm) column can be used, for example, a 200 mM sodium nitrate aqueous solution can be used as the eluent, and for example, pullulan can be used as the molecular weight standard.

[0027] Furthermore, for example, the absolute molecular weight of the monovalent metal alginate salt used in the present invention, as measured by the GPC-MALS method (also called the SEC-MALS method), which combines gel permeation chromatography (GPC / SEC) and multi-angle light scattering (MALS), is preferably 10,000 to 1,000,000, more preferably 80,000 to 800,000, and even more preferably 90,000 to 500,000. When using GPC-MALS for molecular weight measurement, for example, an RI detector and a light scattering detector (MALS) can be used as detectors.

[0028] The apparent viscosity (also simply called "viscosity") of the monovalent metal alginate used in the present invention is preferably 10 mPa·s to 800 mPa·s, more preferably 30 mPa·s to 700 mPa·s, and even more preferably 50 mPa·s to 600 mPa·s, of the monovalent metal alginate used in the present invention is obtained by dissolving it in Milli-Q water to make a 1 w / w% or 1 w / v% solution, and measuring it at 20°C using a cone-plate viscometer.

[0029] When the first composition of the present invention is in a liquid state, its viscosity is preferably 20 mPa·s to 10,000 mPa·s, more preferably 100 mPa·s to 8,000 mPa·s, and even more preferably 300 mPa·s to 6,000 mPa·s, measured at 20°C using a cone-plate viscometer. It is more desirable to measure the apparent viscosity of monovalent metal alginate and the first composition of the present invention using a cone-plate viscometer. For example, the following procedure can be used as the measurement conditions. The sample solution is prepared using MilliQ water. The measurement temperature is 20°C. The rotation speed of the cone-plate viscometer is set to 1 rpm when measuring a 1 w / w% or 1 w / v% solution of monovalent metal alginate, and to 0.5 rpm when measuring a 2 w / w% or 2 w / v% solution, and this is used as a guideline to determine the measurement speed. For 1 w / w% or 1 w / v% solutions of monovalent metal alginate, the measurement time is 2 minutes, and the average value from the first 1 minute to the 2 minute mark is used. For 2 w / w% or 2 w / v% solutions, the measurement time is 2.5 minutes, and the average value from the first 0.5 minutes to the 2.5 minute mark is used. The test value is the average of three measurements.

[0030] The apparent viscosity of the first composition of the present invention can be adjusted, for example, by changing the concentration, molecular weight, or M / G ratio of the monovalent metal alginate salt. The apparent viscosity of the first composition of the present invention is high when the concentration of the monovalent metal alginate salt in the solution is high, and low when the concentration is low. Furthermore, the apparent viscosity of the first composition of the present invention is high when the molecular weight of the monovalent metal alginate salt is large, and low when the molecular weight is small. The apparent viscosity of the first composition of the present invention can be adjusted by selecting alginate with an M / G ratio of about 0.1 to about 5.0, preferably about 0.2 to about 4.0, and more preferably about 0.3 to about 3.0.

[0031] The monovalent metal alginate used in the present invention is preferably a monovalent metal alginate with low endotoxin. "Low endotoxin" means that the endotoxin level is low to such an extent that it does not substantially cause inflammation or fever. For the method of low endotoxin treatment and the method of measuring the endotoxin level, reference can be made to, for example, the description in PCT / JP2017 / 002925 (International Publication No. 2017 / 163603).

[0032] When measuring the endotoxin content of the monovalent metal alginate by, for example, using Limulus reagent (LAL), it is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, and even more preferably 50 EU / g or less. The sodium alginate treated with low endotoxin can be obtained, for example, from commercially available products such as Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA ＴＭ UP LVG (FMC BioPolymer), etc.

[0033] 1-2. Mesenchymal stem cells The mesenchymal stem cells used in combination with the first composition of the present invention are one type of somatic stem cells and are stem cells having pluripotency and self-renewal ability existing in the stromal cells of mesenchymal tissues. Also, mesenchymal stem cells are known to have the ability to differentiate into various cells such as osteocytes, chondrocytes, adipocytes, cardiomyocytes, and neurons.

[0034] The origin of mesenchymal stem cells includes, for example, bone marrow, dental pulp, adipose tissue, skeletal muscle, umbilical cord, dermis, or peripheral blood, etc. Also, the mesenchymal stem cells can be of autologous origin or allogeneic origin, but for example, when the first composition of the present invention is used for the treatment of acute cerebral hemorrhage, it is preferably of allogeneic origin. Also, the mesenchymal stem cells are preferably in an undifferentiated state.

[0035] The culture medium used for culturing mesenchymal stem cells can be appropriately selected from known standard media depending on the cell type, etc. Specifically, examples include Dulbecco's modified Eagle medium (DMEM), NPBM, and α-MEM. Serum such as FBS, nutritional factors such as amino acids, antibiotics, growth factors, etc. can be added to the medium. Mesenchymal stem cells may be used as a suspension recovered from the culture medium, or as a suspension that has been thawed from frozen. Commercial products may be used, or high-purity human mesenchymal stem cells (REC) obtained using cell separation technology may be used. The suspension of mesenchymal stem cells may contain a solution suitable for cell preservation. It is desirable that the mesenchymal stem cells be in a fluid liquid state, at least when injected into the intracerebral hematoma cavity.

[0036] After combining the mesenchymal stem cells with the first composition of the present invention, the cell density becomes 1.0 × 10⁻⁶. ３ cells / mL or more, 2.5 x 10 ３ cells / mL or more, 5.0×10 ３ cells / mL or more, 1.0×10 ４ cells / mL or more, 2.5 x 10 ４ cells / mL or more, 5.0×10 ４ cells / mL or more, 1.0×10 ５ cells / mL or more, 2.5 x 10 ５ cells / mL or more, 5.0×10 ５ cells / mL or higher, or 1.0 × 10 ６ cells / mL or higher, and 1.0 × 10 ９ cells / mL or less, 5.0×10 ８ cells / mL or less, 2.5×10 ８ cells / mL or less, 1.0×10 ８ cells / mL or less, 5.0×10 ７ cells / mL or less, 2.5×10 ７ cells / mL or less, 1.0×10 ７ cells / mL or less, 5.0×10 ６ cells / mL or less, 2.5×10 ６ cells / mL or less, or 1.0 × 10 ６cells / mL or less, preferably 1.0 × 10 ６ cells / mL or more, 1.0×10 ９ cells / mL or less or 1.0 × 10 ３ cells / mL or more, 1.0×10 ６ cells / mL or less, more preferably 5.0 × 10 ５ cells / mL or more, 5.0×10 ６ It is preferable to use it so that the cells / mL is ≤.

[0037] 1-3. Other Components The first composition of the present invention may also contain factors that promote cell growth (hereinafter also referred to as "growth factors"). Examples of growth factors include NGF, BDNF, BMP, FGF, VEGF, HGF, TGF-β, IGF-1, PDGF, CDMP (cartilage-derived-morphogenic protein), CSF, EPO, IL, PRP (Platelet Rich Plasma), SOX, and IF. Growth factors may be produced by recombinant methods or purified from protein compositions. The first composition of the present invention may also not contain growth factors. Even without growth factors, a good brain tissue regeneration effect can be obtained through various factors secreted by mesenchymal stem cells, and it is safer compared to cases where cell growth is actively promoted.

[0038] The first composition of the present invention may also contain a factor that suppresses cell death. Examples of factors that induce cell death include casspace and TNFα, while examples of factors that suppress cell death include antibodies and siRNA. Factors that suppress cell death may be produced by recombinant DNA or purified from a protein composition. The first composition of the present invention may also not contain a factor that suppresses cell death. Even without a factor that suppresses cell death, a good regenerative effect on brain tissue can be obtained through various factors secreted by mesenchymal stem cells, and it is safer than when cell growth is actively promoted.

[0039] The first composition of the present invention may optionally contain other pharmaceutically active ingredients, as well as ingredients commonly used in pharmaceuticals, such as stabilizers, emulsifiers, osmotic pressure regulators, buffers, isotonic agents, preservatives, analgesics, and colorants. In one embodiment of the present invention, the first composition may not contain collagen. In this embodiment, the collagen content is less than about 0.1% by mass on a total basis of the first composition, less than about 0.05% by mass in another embodiment, less than about 0.01% by mass in yet another embodiment, and 0% by mass in yet another embodiment.

[0040] 2. Composition containing mesenchymal stem cells (second composition) The present invention provides a composition containing mesenchymal stem cells before being combined with a biomaterial (hereinafter also referred to as the "second composition"). The second composition of the present invention is used in combination with a biomaterial. The various components that constitute the second composition of the present invention, or that are used in combination with the second composition of the present invention, and the physical properties of the second composition will be described in detail below.

[0041] 2-1. Mesenchymal stem cells The "mesenchymal stem cells" contained in the second composition of the present invention are the same as the mesenchymal stem cells used in combination with the first composition of the present invention as described in 1-2.

[0042] The second composition of the present invention is a suspension containing mesenchymal stem cells, which may be in a liquid or frozen state. The second composition of the present invention may also contain a solution suitable for cell preservation. When the second composition of the present invention is injected into the intracerebral hematoma cavity, it is desirable that it be in a fluid liquid state. When the second composition of the present invention is mixed with a biomaterial and then injected into the intracerebral hematoma cavity, it is also desirable that it be fluid and in a liquid or non-gel state.

[0043] The second composition of the present invention, when combined with a biomaterial, results in a mesenchymal stem cell density of 1.0 × 10⁻⁶. ３ cells / mL or more, 2.5 x 10 ３ cells / mL or more, 5.0×10 ３ cells / mL or more, 1.0×10 ４ cells / mL or more, 2.5 x 10 ４cells / mL or more, 5.0×10 ４ cells / mL or more, 1.0×10 ５ cells / mL or more, 2.5 x 10 ５ cells / mL or more, 5.0×10 ５ cells / mL or higher, or 1.0 × 10 ６ cells / mL or higher, and 1.0 × 10 ９ cells / mL or less, 5.0×10 ８ cells / mL or less, 2.5×10 ８ cells / mL or less, 1.0×10 ８ cells / mL or less, 5.0×10 ７ cells / mL or less, 2.5×10 ７ cells / mL or less, 1.0×10 ７ cells / mL or less, 5.0×10 ６ cells / mL or less, 2.5×10 ６ cells / mL or less, or 1.0 × 10 ６ cells / mL or less, preferably 1.0 × 10 ６ cells / mL or more, 1.0×10 ９ cells / mL or less or 1.0 × 10 ３ cells / mL or more, 1.0×10 ６ cells / mL or less, more preferably 5.0 × 10 ５ cells / mL or more, 5.0×10 ６ It is preferable that the concentration be such that it is 1 / 2 or less cells / mL, and that the composition is such that this is possible.

[0044] 2-2. Biomaterials The second composition of the present invention is used in combination with biomaterials. "Biomaterial" refers to a polymer material intended for introduction into living organisms and possesses biocompatibility. Examples of biomaterials include proteins, animal-derived polysaccharides, and plant-derived polysaccharides. Specifically, proteins include collagen, gelatin, fibronectin, elastin, tenacin, laminin, and vitronectin. Specifically, animal-derived polysaccharides include hyaluronic acid (hyaluronan), heparan sulfate, heparin, chitin, chitosan, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, and glycogen. Specifically, plant-derived polysaccharides include alginic acid, carrageenan, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, gellan gum, xanthan gum, galactomannan, guar gum, locust bean gum, and tara gum. In addition, polyglutamic acid, polylactic acid, polyglycolic acid, and lactic acid-glycolic acid copolymers can also be used as biomaterials. These materials may be used individually or in combination of two or more. Derivatives of these materials, such as metal salts, may also be used. When alginic acid is used among these materials, monovalent metal alginate is preferred, and the monovalent metal alginate is the same as that described in 1-1. Furthermore, in one embodiment of the present invention, the biomaterial may not contain collagen. In this embodiment, the collagen content is less than about 0.1% by mass on a total basis of the second composition, less than about 0.05% by mass in another embodiment, less than about 0.01% by mass in yet another embodiment, and 0% by mass in yet another embodiment.

[0045] Biomaterials may be in a solid or liquid state. In the solid state, they may be freeze-dried or freeze-dried powder, and in the liquid state, they may be liquid or non-gel. When biomaterials are in a liquid state, they may gel due to heat, temperature, light, or chemical action (e.g., ionic crosslinking, chemical crosslinking, etc.), but it is desirable that they have fluidity and be liquid or non-gel at least when injected into a hematoma cavity in the brain.

[0046] The concentration of the biomaterial varies depending on the type of material, but is preferably 0.1 to 5.0 w / w%, more preferably 0.5 to 4.0 w / w%, even more preferably 0.8 to 3.0 w / w%, and particularly preferably 1.0 to 2.5 w / w%. In another embodiment, it is preferably 0.1 to 5.0 w / v%, more preferably 0.5 to 4.0 w / v%, even more preferably 0.8 to 3.0 w / v%, and particularly preferably 1.0 to 2.5 w / v%.

[0047] The apparent viscosity of the biomaterial used in the present invention is preferably 10 mPa·s to 30,000 mPa·s, more preferably 100 mPa·s to 20,000 mPa·s, even more preferably 300 mPa·s to 10,000 mPa·s, even more preferably 500 mPa·s to 8,000 mPa·s, and especially preferably 1,000 mPa·s to 6,000 mPa·s, when dissolved in MilliQ water to a 1 w / w% or 1 w / v% solution and measured at 20°C using a cone-plate viscometer.

[0048] The apparent viscosity of the biomaterial used in this invention is preferably measured using a cone-plate viscometer. For example, the following procedure can be used for measurement conditions: The sample solution is prepared using Milli-Q water. The measurement temperature is 20°C. The rotation speed of the cone-plate viscometer is set to 1 rpm when measuring a 1 w / w% or 1 w / v% solution of the biomaterial, and to 0.5 rpm when measuring a 2 w / w% or 2 w / v% solution, and this is used as a guideline for determining the optimal speed. For 1 w / w% or 1 w / v% solutions of the biomaterial, the measurement is performed for 2 minutes, and the average value from the first 1 to 2 minutes is used. For 2 w / w% or 2 w / v% solutions, the measurement is performed for 2.5 minutes, and the average value from the first 0.5 minutes to 2.5 minutes is used. The test value is the average of three measurements.

[0049] The apparent viscosity of the biomaterial used in this invention can be adjusted, for example, by changing the concentration or molecular weight of the biomaterial. The apparent viscosity of the biomaterial used in this invention is high when the concentration of the biomaterial in the solution is high, and low when the concentration is low. Furthermore, the apparent viscosity of the biomaterial used in this invention is high when the molecular weight of the biomaterial is large, and low when the molecular weight is small.

[0050] The biomaterials described above may be synthesized using known methods or be commercially available products. Furthermore, similar to monovalent metal alginate salts, it is preferable that the biomaterials be treated to low endotoxin levels. The endotoxin content should fall within the numerical range described for monovalent metal alginate salts.

[0051] 2-3. Other Components The second composition of the present invention may also contain growth factors. The growth factors are the same as those described in 1-3. The second composition of the present invention may also not contain growth factors. Even without growth factors, a good brain tissue regeneration effect can be obtained through various factors secreted by mesenchymal stem cells, and it is safer compared to cases where cell growth is actively promoted.

[0052] The second composition of the present invention may also contain factors that suppress cell death. The factors that cause cell death are the same as the factors that suppress cell death described in 1-3. The second composition of the present invention may also not contain factors that suppress cell death. Even without factors that suppress cell death, a good regenerative effect on brain tissue can be obtained through various factors secreted by mesenchymal stem cells, and it is safer compared to cases where cell growth is actively promoted.

[0053] The second composition of the present invention may, if necessary, also contain other pharmaceutically active ingredients, stabilizers, emulsifiers, osmotic pressure regulators, buffers, isotonic agents, preservatives, analgesics, colorants, and other ingredients commonly used in pharmaceuticals.

[0054] 3. Application of the Compositions The first composition of the present invention is used in combination with mesenchymal stem cells, and the second composition of the present invention is used in combination with a biomaterial, for injection and filling into a target intracerebral hemorrhage cavity, and for treating a target intracerebral hemorrhage. Hereinafter, the first and second compositions of the present invention may be collectively referred to as "the composition of the present invention."

[0055] "Subjects" include humans, non-human mammals (e.g., dogs, cats, rats, mice, rabbits, monkeys, chimpanzees, cattle, horses, pigs, sheep, and goats), and birds (e.g., chickens).

[0056] The term "hematoma cavity" refers to the space remaining in the brain after a hematoma has been removed for the treatment of cerebral hemorrhage. The hematoma cavity is also called the hematoma excision cavity. While not bound by theory, for example, by combining the first composition of the present invention with mesenchymal stem cells, or the second composition of the present invention with a biomaterial, and injecting and filling the hematoma cavity, it is expected that the monovalent metal alginate salt or biomaterial will form a mesh-like network that acts as a scaffold, allowing mesenchymal stem cells to survive in the hematoma cavity for a longer period. As a result, it is expected that the regeneration of nerve cells at the bleeding site will be promoted through the effects of factors such as cytokines secreted by mesenchymal stem cells, including anti-inflammatory effects, antioxidant effects, angiogenesis, anti-gliotic effects, and nutrient supply, thereby achieving a higher level of functional recovery. Furthermore, while not bound by theory, it is expected that mesenchymal stem cells injected into the hematoma cavity by combining the first composition of the present invention with mesenchymal stem cells, or the second composition of the present invention with a biomaterial, will suppress the excessive accumulation of macrophages such as microglia around the hematoma cavity. Phagocytosis of necrotic tissue by microglia is expected to lead to scarring of the hematoma cavity. By using the present invention, the presence of mesenchymal stem cells may suppress scar formation at the bleeding site, promote nerve cell regeneration, and potentially achieve a higher level of functional recovery.

[0057] "Filling" means filling all or part of the intracerebral hematoma cavity with the composition of the present invention and components used in combination with the composition of the present invention.

[0058] "Injection" means pouring the composition of the present invention and components used in combination with the composition of the present invention into the intracerebral hematoma cavity in order to fill all or part of the intracerebral hematoma cavity. "Injecting A and B into C" means injecting A and B into C in any order. This can be any of the following: (1) mixing A and B and injecting them into C, (2) injecting A and B into C simultaneously, (3) injecting A into C and then injecting B into C, (4) injecting B into C and then injecting A into C.

[0059] "Combining A and B" means having A and B coexist in the hematoma cavity within the brain, which is the affected area. Specifically, this can be done in any of the following ways: (1) mixing A and B before injecting them into the affected area, (2) injecting A and B into the affected area simultaneously, (3) injecting A into the affected area, followed by injecting B into the affected area, or (4) injecting B into the affected area, followed by injecting A into the affected area.

[0060] "Using A and B in combination" means using A and B in such a way that they coexist in the hematoma cavity within the brain, which is the affected area. Specifically, this can be done in any of the following ways: (1) mixing A and B and then injecting them into the affected area; (2) injecting A and B into the affected area simultaneously; (3) injecting A into the affected area, then injecting B; (4) injecting B into the affected area, then injecting A into the affected area. Note that "combination of A and B" simply refers to the combination of A and B.

[0061] The term "cerebral hemorrhage" encompasses the acute, subacute, and chronic phases, but the compositions of the present invention are preferably used for the treatment of cerebral hemorrhage in the acute and subacute phases, and particularly preferably in the acute phase. The "acute phase" is the period from immediately after the onset of cerebral hemorrhage, for example, within one week, two weeks, or three weeks. The "subacute phase" is the period from one week, two weeks, or three weeks after the onset of cerebral hemorrhage, and for example, within one month. The "chronic phase" is the period from one month, two months, three months, six months, or twelve months after the onset of cerebral hemorrhage.

[0062] The site of cerebral hemorrhage to which the composition of the present invention can be applied is not particularly limited, and examples include subcortical hemorrhage, subthalamic hemorrhage, brainstem hemorrhage, cerebellar hemorrhage, putaminal hemorrhage, pontine hemorrhage, caudate nucleus hemorrhage, lobar hemorrhage, intraventricular hemorrhage, mixed-type hemorrhage in which multiple of these occur, and hemorrhagic cerebral infarction.

[0063] The causes of cerebral hemorrhage are not particularly limited and include, for example, hypertensive cerebral hemorrhage, cerebral amyloid angiopathy, arteriovenous malformations, rupture of cerebral aneurysms, occlusion of the circle of Willis (including moyamoya disease), bleeding from brain tumors, inflammation of cerebral arteries or veins, hemorrhagic diathesis, drug use such as anticoagulants, and trauma.

[0064] "Treating a cerebral hemorrhage" refers, for example, to "treatment after the onset of a cerebral hemorrhage," and more specifically, to "promoting the recovery of functions lost due to a cerebral hemorrhage." "Promoting the recovery of functions" does not necessarily mean that functions will be completely restored to the state before the onset of a cerebral hemorrhage, but rather that they will be partially restored compared to the functions after the onset of a cerebral hemorrhage. In other words, "methods for treating cerebral hemorrhage" in this specification may be rephrased as "methods for treatment after the onset of a cerebral hemorrhage," promoting the recovery of functions after the onset of a cerebral hemorrhage," or "methods for treating intracerebral hematoma cavities," etc.

[0065] The composition of the present invention may be in liquid form when applied to the target intracerebral hematoma cavity. Furthermore, the composition of the present invention may remain in liquid form even after being mixed with components used in combination with the composition of the present invention. Moreover, the composition of the present invention may maintain its liquid form even after being applied to the target intracerebral hematoma cavity. Thus, the composition of the present invention may be fluid both during and after application to the target.

[0066] "Having fluidity" means having the property of changing its form into an amorphous shape, and does not necessarily mean that it is constantly flowing, like a solution. As a guideline, it is desirable that the composition of the present invention has fluidity such that it can be sealed in a syringe and injected into the hematoma cavity of the target brain. Furthermore, it is desirable that the composition of the present invention has fluidity such that it can be injected into the hematoma cavity of the target brain using a syringe fitted with a 14G to 26G injection needle after being left to stand at 20°C for 1 hour, and more preferably it is desirable that it can be injected with a 21G injection needle. If the first composition of the present invention is provided in a solid state such as a freeze-dried product, it can be made into a fluid composition as described above using a solvent before application to the target.

[0067] In particular, monovalent metal salts of alginate are metal ion compounds with two or more valent ions (CaCl ２ MgCl ２ CaSO ４ BaCl ２It is known that when crosslinked with such substances, it hardens into a gel-like state. However, the first composition of the present invention is preferably fluid, liquid or non-gel-like, and preferably does not contain a crosslinking agent, at least when applied to the target.

[0068] Furthermore, it is preferable that the composition of the present invention does not contain fibrin glue. This is because monovalent metal alginate salts or biomaterials may function as hemostatic agents.

[0069] The method of applying the composition of the present invention is not particularly limited, but one example is to expose the affected area by a known surgical technique and then inject the composition of the present invention into the intracerebral hematoma cavity using a syringe, filling instrument, etc., under direct visualization or under a microscope. More specifically, for example, the first composition of the present invention and mesenchymal stem cells, or the second composition of the present invention and a biomaterial, are mixed immediately before or during surgery. At this time, it is desirable that the cell density of the mesenchymal stem cells be within the range described above. In this way, the first composition of the present invention and mesenchymal stem cells, or the second composition of the present invention and a biomaterial, can be combined and injected into the intracerebral hematoma cavity after hematoma removal, immediately before or during surgery.

[0070] 4. Kit The present invention provides a kit for the treatment of cerebral hemorrhage comprising mesenchymal stem cells and a monovalent metal alginate salt. The present invention also provides a kit for the treatment of cerebral hemorrhage comprising mesenchymal stem cells and a biomaterial. The kit for the treatment of cerebral hemorrhage of the present invention may also be referred to as a kit for filling intracerebral hematoma cavities. The "mesenchymal stem cells," "monovalent metal alginate salt," and "biomaterial" that constitute the kit are as described above. The monovalent metal alginate salt or biomaterial included in the kit of the present invention may be in a solid state or a liquid state. In the solid state, it may be a freeze-dried body or a freeze-dried powder, and in the liquid state, it may be liquid or non-gel.

[0071] The mesenchymal stem cells included in the kit of the present invention may be a suspension recovered from a culture medium or a frozen suspension. The mesenchymal stem cell suspension may contain a solution suitable for cell preservation.

[0072] When the monovalent metal alginate salt and / or biomaterial is in a solid state, it is desirable that the kit include a solvent for dissolution. The solvent for dissolution is not particularly limited as long as it is a solvent that can be applied to living organisms, and examples include purified water, distilled water, deionized water, Milli-Q water, physiological saline, and phosphate-buffered saline (PBS). These are preferably sterilized and preferably treated with low endotoxin. When the mesenchymal stem cells are frozen in a suspension, the kit may also include a solvent for dissolution. The solvent for dissolution is not particularly limited as long as it is a solvent that can be applied to living organisms, and examples include water for injection, physiological saline, or cell preservation solution. These are preferably sterilized and preferably treated with low endotoxin.

[0073] The kit of the present invention may further include a syringe, a needle, instructions for use, etc.

[0074] Specific examples of the kit include a kit containing (1) a vial or tube or other container containing a monovalent metal alginate salt or biomaterial in a solid or liquid state, (2) a vial or tube or other container containing a mesenchymal stem cell in a liquid or frozen state, and (3) a container containing a solvent for dissolution, all in one or separate boxes. Another embodiment is a kit in which a solution of monovalent metal alginate or biomaterial is sealed in a pre-filled syringe and can be used immediately.

[0075] 5. Treatment Methods The present invention provides a method for treating cerebral hemorrhage using the compositions of the present invention. More specifically, the method involves injecting the first composition of the present invention into the hematoma cavity in the brain of a target patient in combination with mesenchymal stem cells to promote the recovery of functions lost due to cerebral hemorrhage. The second composition of the present invention involves injecting the hematoma cavity in the brain of a target patient in combination with a biomaterial to promote the recovery of functions lost due to cerebral hemorrhage. In these treatment methods, "mesenchymal stem cells," "monovalent metal alginate," and "biomaterial" are as described above.

[0076] The procedure for using the composition of the present invention includes aspirating and removing a hematoma that has formed in the brain, washing the hematoma cavity with physiological saline, and injecting the composition of the present invention into the hematoma cavity in a predetermined combination. As an example, the procedure for using the composition of the present invention in endoscopic hematoma removal surgery may be as follows: (1) Insert an endoscope into the hematoma cavity of the brain and aspirate and remove as much of the hematoma as possible through the endoscope port. (2) After sufficient removal of the hematoma, wash the hematoma cavity with physiological saline and confirm that there is no bleeding from the hematoma removal cavity or the wall surrounding the cavity. (3) Inject the composition of the present invention into the hematoma cavity in a predetermined combination. (4) Cover the burr hole with a plate or the like and suture the wound to close it.

[0077] As an example, the procedure for using the composition of the present invention in craniotomy and hematoma removal surgery may be as follows: (1) After craniotomy, the hematoma is aspirated and removed under a microscope. (2) After sufficient removal of the hematoma, the hematoma cavity is washed with physiological saline solution and it is confirmed that there is no bleeding from the hematoma removal cavity or the walls surrounding the cavity. (3) The composition of the present invention is injected into the hematoma cavity in a predetermined combination. (4) The craniotomy is covered with a bone flap or plate, and the dura mater and wound are sutured closed.

[0078] In the treatment method of the present invention, known surgical techniques can be used, except for injecting the composition of the present invention into the target intracerebral hematoma cavity in a predetermined combination. Furthermore, the treatment method of the present invention may be appropriately combined with other methods for treating cerebral hemorrhage (including medical and surgical methods) and various therapeutic drugs.

[0079] Furthermore, before, simultaneously with, or after injecting the composition of the present invention into the intracerebral hematoma cavity, concomitant agents such as antibiotics including streptomycin, penicillin, tobramycin, amikacin, gentamicin, neomycin, and amphotericin B; anti-inflammatory drugs including aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen; proteases, corticosteroids, and HMG-CoA reductase inhibitors such as simvastatin and lovastatin may be added. These agents may be mixed with the composition of the present invention and may be administered orally or parenterally. In addition, muscle relaxants, opioid analgesics, neuropathic pain relievers, etc., may be administered orally or parenterally as needed.

[0080] The present invention will now be described in more detail by illustrating some examples, but the present invention is not limited to these examples.

[0081] 1. Preparation of experimental animals Nine-week-old male Sprague-Dawley rats (260-300 g) were obtained from CLEA Japan Co., Ltd. The rats were housed in a controlled environment with a temperature of 25°C, humidity of 50%, and a 12-hour light-dark cycle, and were given free access to food and water. These rats were randomly divided into the following groups: Group 1: Cerebral hemorrhage group treated with 0.9 w / v% saline (n=15), sometimes labeled "Saline" in the figure. Group 2: Cerebral hemorrhage group treated with 2 w / v% sodium alginate (ALG) solution (n=15), sometimes labeled "ALG" in the figure. Group 3: 1.0 × 10 6 20 μL of physiological saline containing 5.0 × 10¹ mesenchymal stem cells ７ The group treated with cerebral hemorrhage (cells / mL) (n=8), sometimes referred to as "REC" in the figure. Group 4: 1.0 × 10 6 20 μL of w / v% sodium alginate solution containing 2 mesenchymal stem cells (5.0 × 10⁶) ７The group treated with cerebral hemorrhage (n=15), sometimes referred to as "REC + ALG" in the figure, was a low-endotoxin sodium alginate (Kimika, AL500). The endotoxin content of the sodium alginate was less than 50 EU / g. Human bone marrow-derived mesenchymal stem cells (REC, Fujifilm Wako Pure Chemical Industries, Ltd., 387-16591) were used as mesenchymal stem cells. 2 The mesenchymal stem cells were cultured in 25 mL of low-glucose Dulbecco-modified Eagle medium (DMEM, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in a flask. The medium was further supplemented with 20% fetal bovine serum (FBS), 0.01 mol / L HEPES, 100 U / mL penicillin G, 100 μg / mL streptomycin, and 20 ng / mL bFGF, respectively. The mesenchymal stem cells were cultured in an incubator containing 5% CO2 at 37°C, with the medium changed every 3 days. The mesenchymal stem cells were passaged when they reached 80% confluence, and passages of 3 to 4 passages were used in the administration study.

[0082] 2. Preparation of Acute Cerebral Hemorrhage Model Rats General anesthesia was induced using 5% isoflurane in 70% N2O and 30% O2 gas, followed by maintenance anesthesia using 1.5–2% isoflurane in 70% N2O and 30% O2 gas. 100 μL of autologous blood was collected from the right femoral artery. The rats were then fixed in a prone position in a small animal stereotactic device (Model 900; David Kopf Instruments), and the skull was exposed through a midline skin incision. A 23-gauge Hamilton syringe needle was inserted into the right internal capsule through a burr hole prepared with a dental drill (coordinates: 2.0 mm posterior, 3.7 mm lateral, and 6.0 mm ventral to the dura mater). Subsequently, 100 μL of autologous blood was injected into the right internal capsule over 10 minutes using a double injection method with a microinjection pump (Model KDS-310; Muromachi Machinery Co., Ltd.). To prevent reflux, the needle was left in place for an additional 7 minutes. After recovery from anesthesia, the Bederson score was assessed, and rats with a score of less than 2 were excluded. Two hours after cerebral hemorrhage, alteplase (0.5 mg / kg) was injected into the intracerebral hematoma, and 30 minutes after the alteplase injection, the intracerebral hematoma was removed. After hematoma removal, saline was injected into the intracerebral hematoma cavity in Group 1, sodium alginate in Group 2, mesenchymal stem cells and saline in Group 3, and mesenchymal stem cells and sodium alginate in Group 4. Finally, the skin wound was sutured.

[0083] 3. Assessment of Neurological and Motor Functions The following assessments were performed using the prepared acute cerebral hemorrhage model rats. Statistical analysis was performed using Graph Pad Prism, ver 8.0. Comparisons between groups were examined using two-way ANOVA followed by Dunnet's multicomparison test, with p<0.05 considered statistically significant.

[0084] 3.1 Modified Neurological Severity Score (mNSS) Neurological function was assessed using the mNSS before and on days 1, 3, 7, 14, 21, and 28 after cerebral hemorrhage. The mNSS ranges from 0 (normal) to 18 (most severe impairment), and the tests consist of the tail suspension test (0–3 points), gait test (0–3 points), sensory test (0–2 points), beam balance test (0–6 points), and reflex deficit / abnormal movement (0–4 points). A 2 cm wide square rod was used for the beam balance test. As shown in Figure 1, Group 4 showed significant normalization compared to Groups 1, 2, and / or 3 on days 3, 7, 14, 21, and 28 after cerebral hemorrhage.

[0085] 3.2 Rotarot Test Motor function was evaluated using a Rotarot treadmill (Model MK-630B; Muromachi Machinery Co., Ltd.) on days 1, 3, 7, 14, 21, and 28 before and after cerebral hemorrhage. The Rotarot treadmill was set to an acceleration mode of 4 to 25 rpm per minute. Each rat received 3 days of training before the start of the experiment. The maximum time the rats stayed on the Rotarot was recorded, and a total of 5 measurements were taken. The three highest values ​​were used to statistically calculate the mean. As shown in Figure 2, Group 4 showed a significant improvement in fall time compared to Group 1 and / or Group 2 on days 3, 7, and 14 after cerebral hemorrhage.

[0086] 3.3 Elevated Body Swing Test (EBST) The EBST was used to assess motor impairment. The rats were grasped by the base of their tails, lifted off the ground, and their heads were kept 1 cm above the ground. Head deviation was observed, and one deviation was counted when the head tilted 10 degrees or more in either direction. The test was performed a total of 20 times, and the percentage of deviations was calculated. In normal rats, the number of deviations to both sides was approximately the same. A deviation of 75% in one direction was used as the criterion for motor impairment. As shown in Figure 3, Group 4 showed significant normalization compared to Group 1 on days 3, 7, and 14 after cerebral hemorrhage.

[0087] 3.4 Open Field Test Anxiety-like behavior was assessed using the open field test. A 100 cm × 100 cm circular black field was used. Rats were placed in the center of the field, and their activity was recorded for 5 minutes using a video tracking system. The total distance the rats moved (a) and the time spent in the central area (b) were recorded. As shown in Figure 4, Group 4 showed significant normalization compared to Group 1 and / or Group 2 at 7, 14, 21, and 28 days after cerebral hemorrhage.

[0088] 4. Histological Evaluation The following evaluations were performed using the prepared acute cerebral hemorrhage model rats. Statistical analysis was performed using Graph Pad Prism, ver 8.0. Comparisons between groups were examined using one-way ANOVA followed by Tukey's multicomparison test, with p<0.05 considered statistically significant.

[0089] 4.1 H&E staining To evaluate brain atrophy, brain tissue was collected 28 days after cerebral hemorrhage. Rats were anesthetized with 5% isoflurane in 70% N2O and 30% O2 gas, perfused transcardially with chilled saline, and fixed with 4% paraformaldehyde (PFA). After decapitation, the brain was carefully collected and fixed with 4% PFA for 24 hours. The brain was then sliced ​​into 2 mm thick coronal sections and embedded in paraffin. Finally, it was cut into 4 μm thick sections using a manual microtome (Leica RM2125 RTS, Leica Biosystems). Hematoxylin and eosin (H&E) staining was performed to evaluate brain atrophy. Coronal fragments of the brain were located 2 mm posterior to the bregma, which indicates the maximum diameter of most cerebral hemorrhages, and were immersed in hematoxylin solution (Mutoh Co., Ltd.) for 5 minutes and eosin solution (Mutoh Co., Ltd.) for 4 minutes. For each slide, ipsilateral and contralateral parenchymal regions were measured using ImageJ software (ImageJ 1.54g, NIH). Parenchymal region and brain atrophy rate were calculated using the following formulas. Figure 5 shows the hematoma cavity volume (a) and brain atrophy rate (b) for each group. Parenchymal region (mm 2) = Hemispheric area - (Hematoma area + Ventricular area) Brain atrophy rate (%) = [(Contralateral parenchymal area - Ipsilateral parenchymal area) / Contralateral parenchymal area] × 100

[0090] As shown in Figure 5, Group 4 (indicated as REC+ALG in the figure) showed a tendency for reduced hematoma cavity volume and significantly suppressed brain atrophy compared to the other groups. This indicates that brain atrophy is suppressed when brain hemorrhage is treated with a sodium alginate solution containing mesenchymal stem cells.

[0091] 4.2 Immunohistochemical staining and apoptosis assays Local inflammation was evaluated using coronal sections of the brain on days 3 and 28 after intracerebral hemorrhage (n=8 in each group). Microglia were evaluated using anti-Iba1 antibody (1:1500, room temperature, 1 hour, 019-19741, Wako, Japan). Macrophages were evaluated using anti-CD68 antibody (1:1000, room temperature, 1 hour, MCA341GA, Bio-Rad). Neutrophil infiltration was evaluated using anti-myeloperoxidase antibody (1:1000, room temperature, 1 hour, ab208670, Abcam). In all cases, Histofine® Simple Stain Rat MAX PO (Nichirei Bioscience Co., Ltd.) was applied for 1 hour after the primary antibody reaction. Subsequently, the DAB Substitute Kit (Nichirei Bioscience Co., Ltd.) was used according to the manufacturer's instructions. For the apoptosis assay, the kit (ApopTag® Fluorescein In Situ Apoptosis Detection Kit, MilliporeSigma) was used according to the instructions. Brain sections were deparaffinized three times with 100% dimethylbenzene, and then treated with ethanol solutions of different concentrations (100% twice, 95%, and 70%). Subsequently, they were treated with Proteinase K (1:10, 21627, Millipore, CA, USA) for 15 minutes, and then with TdT Enzyme (3:10, S7110, Millipore, CA, USA) for 60 minutes at 37°C. Finally, they were incubated with anti-Digoxigenin (3:10, S7110, Millipore, CA, USA) at room temperature for 30 minutes. Immunohistochemical staining and apoptosis assay images were both obtained from around the hematoma cavity. Six non-overlapping regions were randomly selected, and cells showing a positive signal were semi-quantitatively counted using software (BZ-X Analyzer, Keyence, Osaka, Japan). Figure 6 shows the number of Iba1-positive cells for each group on day 3 (a) and day 28 (b) post-cerebral hemorrhage. Figure 7 shows the CD68-positive cell areas for each group on day 3 (a) and day 28 (b) post-cerebral hemorrhage. Furthermore, Figure 8 shows the number of MPO-positive cells for each group on day 3 post-cerebral hemorrhage.In addition, Figure 9 shows the number of TUNEL-positive cells in each group three days after cerebral hemorrhage.

[0092] As shown in Figures 6, 7, and 8, Group 4 (indicated as REC+ALG in the figures) showed suppression of microglia activation (Figure 6), macrophage accumulation (Figure 7), and neutrophil infiltration (Figure 8) compared to the other groups. This indicates that in cerebral hemorrhage, treatment with a sodium alginate solution containing mesenchymal stem cells suppressed inflammation around the hematoma cavity from the third day after cerebral hemorrhage, a period of high inflammation. Furthermore, as shown in Figure 9, apoptosis was suppressed in Group 4 (indicated as REC+ALG in the figures) compared to the other groups. This suggests that in cerebral hemorrhage, treatment with a sodium alginate solution containing mesenchymal stem cells may provide neuroprotection from the third day after cerebral hemorrhage, a period when neuronal cell death due to nerve damage is induced.

[0093] 4.3 Evaluation of Neurotrophic Factor (BDNF) Production Rats used were collected 1, 3, and 7 days post-surgery and perfused with 200 ml of cold saline via transcardiac perfusion. The brain was excised, and coronal sections including the internal capsule were cut to a thickness of 4 mm to prepare the sample. Right brain tissue was placed in a ShakeMan3 homogenizer (BMS-SMN03, BMS, Japan). 1 ml of RIPA lysis buffer for homogenization (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was added and mixed with the right brain tissue to prepare a homogenate (300 rpm for 30 seconds). The homogenate was centrifuged twice at 12,000 × g for 10 minutes, and the supernatant was collected for analysis. The total protein content of the sample was evaluated using a protein sample kit (Thermo Scientific USA, Cat#23227). A separate fraction of the supernatant was used to evaluate neurotrophic factor (BDNF) production. BDNF production was evaluated using a detection kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. Corrected BDNF was calculated using the following formula: Corrected BDNF = BDNF concentration / Total protein concentration

[0094] As shown in Figure 10, Group 4 (indicated as REC+ALG in the figure) showed an enhanced effect on BDNF production compared to the other groups, suggesting that treatment with a sodium alginate solution containing mesenchymal stem cells may provide neuroprotection in cases of cerebral hemorrhage.

[0095] 5. Physical Properties Evaluation 5.1 Viscosity The apparent viscosity of sodium alginate (Kimika Co., Ltd., AL500) used in the examples was measured according to the viscosity measurement method of the Japanese Pharmacopoeia (16th edition) under the following conditions. Samples: 1 w / v% solution and 2 w / v% solution prepared using MilliQ water. Measuring instrument: Cone plate rotational viscometer (Viscose and viscoelasticity measuring device Rheostress RS600 (Thermo Haake GmbH), sensor: 35 / 1) Rotation speed: 1 rpm (1 w / v% solution), 0.5 rpm (2 w / v% solution) Reading time: 2 minutes measurement, average value from 1 minute to 2 minutes (1 w / v% solution), 2.5 minutes measurement, average value from 0.5 minutes to 2.5 minutes (2 w / v% solution) Test value: Average value of 3 measurements Measuring temperature: 20°C The results showed that the apparent viscosity was 300 mPa·s to 500 mPa·s for the 1 w / v% solution and 3000 mPa·s to 5000 mPa·s for the 2 w / v% solution.

[0096] 5.2 Molecular Weight The absolute molecular weight of sodium alginate (Kimika Co., Ltd., AL500) used in the examples was measured under the following conditions.

[0097] [Measurement conditions (measurement of refractive index increment (dn / dc))] Suggested refractometer: Optilab T-rEX Measurement wavelength: 658 nm Measurement temperature: 40°C Solvent: 200 mM sodium nitrate aqueous solution Sample concentration: 0.5 to 2.5 mg / mL (5 concentrations) As a result of the measurement, the refractive index increment (dn / dc) (mL / g) of the sample was 0.153 to 0.158.

[0098] [Pretreatment method] The sample was dissolved by adding the eluent, and the solution was filtered through a 0.45 μm membrane filter to be used as the measurement solution. [Measurement conditions (SEC-MALS method, absolute molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm I.D. x 300 mm x 3) Eluent: 200 mM sodium nitrate aqueous solution Flow rate: 1.0 mL / min Concentration: 0.5 mg / mL Detector: RI detector, light scattering detector (MALS) Column temperature: 40°C Injection volume: 200 μL The measurement results showed that the absolute molecular weight ranged from 140,000 to 280,000.

[0099] 6. In-virto evaluation of enhanced neurotrophic factor (BDNF) production and cell viability. First passages of RECs were used in cell experiments. 3 ml of culture medium was added to each well of a 6-well plate, followed by 20 μl of 2% (w / v) alginate (ALG) solution or 20 μl of physiological saline. RECs were 4.5 × 10⁶ per well. 4 Cells were seeded at the specified cell density in 6-well plates (Corning Inc.). Cells were cultured for 7 days in an incubator at 37°C and 5% CO2. The culture medium was not changed. The culture supernatant was collected and BDNF was evaluated using an ELISA kit. The number of viable cells was Luna TM Evaluation was performed using the FL Dual Fluorescence Cell Counter (Logos Biosystems, Korea).

[0100] As shown in Figure 11, it was found that the addition of alginate significantly improved BDNF production and viability compared to REC alone. This suggests that sodium alginate solution containing mesenchymal stem cells may provide neuroprotection in cerebral hemorrhage.

Claims

A composition for filling intracerebral hematoma cavities containing a monovalent metal alginate salt, which is used in combination with mesenchymal stem cells. 　 The composition according to claim 1, wherein the monovalent metal salt of alginate is low in endotoxin. 　 The composition according to claim 1, wherein the monovalent metal salt of alginate is sodium alginate. 　 The composition according to claim 1, wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, skeletal muscle, umbilical cord, dental pulp, dermis, or peripheral blood. 　 The composition according to claim 1, wherein the mesenchymal stem cells are of allogeneic origin. 　 The cell density of mesenchymal stem cells, after being combined with the composition for filling intracerebral hematoma cavities, is 1.0 × 10⁻⁶. ４ The composition according to claim 1, used to have a cell count of 1 / mL or more. 　 The composition according to claim 1, used in a method for treating a target cerebral hemorrhage. 　 The composition according to claim 7, wherein the cerebral hemorrhage is acute cerebral hemorrhage. 　 The composition according to claim 7, wherein the cerebral hemorrhage comprises one or more selected from the group consisting of subcortical hemorrhage, subthalamic hemorrhage, brainstem hemorrhage, cerebellar hemorrhage, and putaminal hemorrhage. 　 A kit for treating cerebral hemorrhage containing mesenchymal stem cells and monovalent metal alginate. 　 The kit according to claim 10, wherein the monovalent metal salt of alginate is in a freeze-dried state or a liquid state. 　 A composition for filling intracerebral hematoma cavities, containing mesenchymal stem cells, which is used in combination with a biomaterial.

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