Pharmaceutical composition for preventing or treating hemorrhagic brain diseases, comprising, as active ingredient, BDNF gene-introduced mesenchymal stem cells or extracellular vesicles derived therefrom

Abstract

According to the present invention, hemorrhagic brain diseases, which are one of intractable diseases, can be treated using: a pharmaceutical composition for preventing or treating hemorrhagic brain diseases, comprising, as an active ingredient, BDNF gene-introduced mesenchymal stem cells or extracellular vesicles derived therefrom; and a pharmaceutical preparation for preventing or treating hemorrhagic brain diseases, comprising the composition.

Description

A pharmaceutical composition for the prevention or treatment of hemorrhagic encephalopathy comprising, as an active ingredient, mesenchymal stem cells into which the BDNF gene has been introduced or extracellular vesicles derived therefrom.

[0001] One example of the present invention is,

[0002] The present invention relates to a pharmaceutical composition for the prevention or treatment of hemorrhagic brain disease comprising, as an active ingredient, mesenchymal stem cells into which the BDNF (Brain Derived Neurotrophic Factor) gene has been introduced or extracellular vesicles derived therefrom, and a pharmaceutical preparation for the prevention or treatment of hemorrhagic brain disease comprising said composition.

[0003]

[0004] Hemorrhagic encephalopathy refers to a series of conditions caused by the rupture of blood vessels within the brain, leading to blood leakage. These conditions primarily include intraventricular hemorrhage, periventricular hemorrhage, intracerebral hemorrhage, and subarachnoid hemorrhage. Hemorrhagic encephalopathy interferes with normal brain function and, in severe cases, can lead to complications such as nerve damage, hydrocephalus, and cerebral infarction.

[0005] Intraventricular hemorrhage (IVH) refers to a condition in which bleeding occurs within the ventricles of the brain. The ventricles are spaces in the brain that contain cerebrospinal fluid (CSF); when blood flows into these spaces, pressure within the brain increases, which can lead to nerve cell damage, compression of brain tissue, and a decline in brain function.

[0006] Periventricular hemorrhage refers to bleeding occurring in the periventricular white matter region surrounding the ventricles, resulting from the rupture of weak blood vessels in the vicinity. The severity of neurological damage can vary depending on the amount and location of the bleeding, and in severe cases, it can lead to long-term brain damage and delayed neurodevelopment.

[0007] Intracerebral hemorrhage refers to bleeding that occurs within the brain parenchyma, or brain tissue. This type of bleeding is primarily caused by various factors such as hypertension, trauma, ruptured cerebral aneurysms, and vascular malformations, and can result in severe neurological deficits by causing direct damage to brain tissue. Symptoms of intracerebral hemorrhage vary depending on the location and size of the bleeding, and in severe cases, it can lead to a critical condition that may result in death.

[0008] Subarachnoid hemorrhage refers to a condition in which blood leaks into the subarachnoid space, the area between the arachnoid membrane and the pia mater, two of the three meninges surrounding the brain. While the rupture of a cerebral aneurysm is the most common cause, trauma, vascular malformations, and hemorrhagic diseases can also induce subarachnoid hemorrhage. Subarachnoid hemorrhage is accompanied by severe headaches, vomiting, decreased consciousness, and seizures, and is a life-threatening emergency that requires immediate treatment.

[0009] Hemorrhagic brain disease varies from mild to severe depending on the degree of bleeding; in severe cases, it can cause long-term complications such as neurological damage, hydrocephalus, cerebral palsy, and developmental delay. However, as there is currently no definitive preventive or curative method, it remains one of the most intractable diseases.

[0010]

[0011] Brain-Derived Neurotrophic Factor (BDNF) is a protein that plays an important role in the nervous system and is involved in the survival, growth, differentiation, maintenance, and synaptic plasticity of nerve cells (neurons). BDNF is produced in large quantities, particularly in the hippocampus, cerebral cortex, and basal forebrain, and these regions play important roles in learning, memory, and emotion regulation.

[0012] BDNF has the ability to promote the survival and recovery of neurons, and thus can exhibit a protective effect against neuronal damage caused by hemorrhage. In addition, BDNF has the potential to aid in the recovery of the nervous system, and can contribute to regenerating brain tissue damaged by hemorrhagic brain disease and promoting functional recovery.

[0013]

[0014] All cells must exchange information with their surrounding environment and other cells for survival, and to facilitate this information exchange, cells secrete various substances outside the cell, such as cytokines, hormones, and soluble factors like neurotransmitters. Recently, extracellular vesicles, also known as exosomes, have been attracting attention as a new type of intercellular information exchange mechanism.

[0015] Extracellular vesicles (EVs) refer to microscopic particles ranging in size from several nanometers to several micrometers that are secreted from cells or cells into which genetic material has been introduced, and are surrounded by a lipid bilayer. Recently, various studies have been conducted on the important functions of extracellular vesicles.

[0016] The secretion of extracellular vesicles is a phenomenon that is evolutionarily conserved across all biological kingdoms, ranging from bacteria and archaea to eukaryotes.

[0017] In particular, extracellular vesicles containing DNA, RNA, functional proteins, and antigens are utilized as a new mode of cell communication. Extracellular vesicles are known to contain specific genetic material and bioactive factors depending on the properties and state of the cell from which they originate. Since extracellular vesicles are fundamentally derived from cells, they are biocompatible unlike other nanoparticles. Furthermore, because they can encapsulate or label drugs or biologically active ingredients on their interior or surface, there are ongoing attempts to utilize them as raw materials for drug delivery systems, cosmetics, and pharmaceuticals.

[0018]

[0019] Accordingly, the researchers aimed to develop mesenchymal stem cells that overexpress BDNF for the treatment of hemorrhagic brain disease, and after research, completed the present invention.

[0020]

[0021] Accordingly, the present invention

[0022] The purpose is to provide a pharmaceutical composition for the prevention or treatment of hemorrhagic brain disease comprising, as an active ingredient, mesenchymal stem cells into which the BDNF (Brain Derived Neurotrophic Factor) gene has been introduced or extracellular vesicles derived therefrom.

[0023]

[0024] In addition, the present invention

[0025] The purpose is to provide a pharmaceutical preparation for the prevention or treatment of hemorrhagic encephalopathy comprising the above-mentioned pharmaceutical composition.

[0026]

[0027] In addition, the present invention

[0028] The purpose is to provide the above pharmaceutical composition for the prevention or treatment of hemorrhagic brain disease.

[0029]

[0030] In addition, the present invention

[0031] The purpose is to provide a method for preventing or treating hemorrhagic encephalopathy, comprising the step of administering the above pharmaceutical composition to an individual in need thereof.

[0032]

[0033] In order to achieve the above objective,

[0034] The present invention

[0035] A pharmaceutical composition for the prevention or treatment of hemorrhagic brain disease is provided, comprising as an active ingredient mesenchymal stem cells into which the BDNF (Brain-Derived Neurotrophic Factor) gene has been introduced or extracellular vesicles derived therefrom.

[0036] In one embodiment of the present invention, the BDNF gene may be introduced by a viral vector.

[0037] In another embodiment of the present invention, the mesenchymal stem cells may be derived from the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta.

[0038] In another embodiment of the present invention, the hemorrhagic brain disease may be one or more selected from the group consisting of intraventricular hemorrhage, periventricular hemorrhage, intracerebral hemorrhage, and subarachnoid hemorrhage.

[0039] In another embodiment of the present invention, the pharmaceutical composition may inhibit hydrocephalus.

[0040] In another embodiment of the present invention, the pharmaceutical composition may reduce the expression level of GFAP (glial fibrillary acidic protein) in the periventricular region.

[0041] In another embodiment of the present invention, the pharmaceutical composition may reduce the expression level of inflammatory cytokines.

[0042] In another embodiment of the present invention, the inflammatory cytokine may be one or more selected from the group consisting of IL-1α (Interleukin-1alpha) and IL-6 (Interleukin-6).

[0043] In another embodiment of the present invention, the pharmaceutical composition may improve cognitive decline caused by hemorrhagic brain injury.

[0044]

[0045] In addition, the present invention

[0046] A pharmaceutical preparation for the prevention or treatment of hemorrhagic brain disease comprising the above pharmaceutical composition is provided.

[0047] In one embodiment of the present invention, the pharmaceutical formulation may further comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0048] In another embodiment of the present invention, the pharmaceutical formulation may be an injectable formulation, an infusion formulation, a spray formulation, a liquid formulation, or a patch formulation.

[0049] In another embodiment of the present invention, the hemorrhagic brain disease may be one or more selected from the group consisting of intraventricular hemorrhage, periventricular hemorrhage, cerebral parenchymal hemorrhage, and subarachnoid hemorrhage.

[0050] In another embodiment of the present invention, the pharmaceutical preparation may inhibit ventricular dilation.

[0051] In another embodiment of the present invention, the pharmaceutical formulation may reduce the level of GFAP expression in the periventricular region.

[0052] In another embodiment of the present invention, the pharmaceutical formulation may reduce the expression level of inflammatory cytokines.

[0053] In another embodiment of the present invention, the inflammatory cytokine may be one or more selected from the group consisting of IL-1α and IL-6.

[0054] In another embodiment of the present invention, the pharmaceutical formulation may improve cognitive decline caused by hemorrhagic brain injury.

[0055]

[0056] In addition, the present invention

[0057] The above pharmaceutical composition is used for the prevention or treatment of hemorrhagic brain disease.

[0058]

[0059] In addition, the present invention

[0060] A method for preventing or treating hemorrhagic brain disease is provided, comprising the step of administering the above pharmaceutical composition to an individual in need thereof.

[0061]

[0062] A pharmaceutical composition for the prevention or treatment of hemorrhagic brain disease comprising a mesenchymal stem cell into which the BDNF gene of the present invention has been introduced or an extracellular vesicle derived therefrom as an active ingredient, and a pharmaceutical preparation for the prevention or treatment of hemorrhagic brain disease comprising said composition, can treat hemorrhagic brain disease, which is one of the intractable diseases.

[0063]

[0064] The effects of the present invention are not limited to those mentioned above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

[0065]

[0066] Figure 1 shows the results of fluorescence-activated cell sorting (FACS) performed to confirm whether mesenchymal stem cells (BDNF-MSCs) introduced with the BDNF gene still retain the characteristics of mesenchymal stem cells.

[0067] Figure 2 shows the results of an enzyme-linked immunosorbent assay (ELISA) performed to evaluate the BDNF expression levels in mesenchymal stem cells into which BDNF was introduced.

[0068] Figure 3 shows the results of a nanoparticle tracking analysis (NTA) performed to confirm whether the isolation of extracellular vesicles derived from BDNF-MSC was successful.

[0069] Figure 4 shows the results of the CCK-8 assay (Cell Counting Kit-8 assay) performed to evaluate the therapeutic effect of mesenchymal stem cells (BDNF MSCs) into which the BDNF gene was introduced.

[0070] Figure 5 shows the results of a CCK-8 assay performed to evaluate the therapeutic effect of extracellular vesicles (BDNF MSC-EV) derived from mesenchymal stem cells into which the BDNF gene was introduced.

[0071] Figure 6 shows the MRI results and quantitative analysis results of measuring the volume ratio of ventricle to whole brain for each experimental group at 1 day and 4 weeks after inducing intraventricular hemorrhage.

[0072] Figures 7A to 7C show the results of evaluating the therapeutic effect of BDNF MSC-EV on hemorrhagic encephalopathy using an in vivo disease model of hemorrhagic encephalopathy. Specifically, Figure 7A shows the results of immunohistochemistry performed to measure GFAP levels in the periventricular region, Figure 7B shows the results of ELISA measuring inflammatory cytokines (IL-1α and IL-6) in cerebrospinal fluid, and Figure 7C shows the results of measuring latency (sec) reflecting memory function by performing a passive avoidance test.

[0073]

[0074] The present invention will be described in detail below.

[0075] The present invention

[0076] A pharmaceutical composition for the prevention or treatment of hemorrhagic brain disease is provided, comprising as an active ingredient mesenchymal stem cells into which the BDNF (Brain-Derived Neurotrophic Factor) gene has been introduced or extracellular vesicles derived therefrom.

[0077] As used herein, the term "BDNF (Braind Derived Neurotrophic Factor)" refers to a brain-derived neurotrophic factor, that is, a protein that plays an important role in the nervous system and is involved in the survival, growth, differentiation, maintenance, and synaptic plasticity of nerve cells (neurons). BDNF is produced in large quantities, particularly in the hippocampus, cerebral cortex, and basal forebrain of the brain, and these regions play an important role in learning, memory, and emotional regulation.

[0078] BDNF has the ability to promote the survival and recovery of neurons, and thus can exhibit a protective effect against neuronal damage caused by hemorrhage. In addition, BDNF has the potential to aid in the recovery of the nervous system, and can contribute to regenerating brain tissue damaged by hemorrhagic brain disease and promoting functional recovery.

[0079] As used herein, the term "Mesenchymal Stem Cell (MSC)" refers to stem cells present in cartilage, bone tissue, adipose tissue, bone marrow stroma, etc., that have differentiated from the mesoderm formed by the division of a fertilized egg. In this case, "Mesenchymal Stem Cells" may be mesenchymal stem cells derived from the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, placenta, and other tissues, but are not limited thereto.

[0080] The term "extracellular vesicle (EV)" as used in this specification refers to a substance surrounded by a lipid bilayer, consisting of particles ranging in size from several nanometers to several micrometers that are secreted from a cell or a cell into which genetic material has been introduced. Recently, various studies have been conducted on the important functions of extracellular vesicles.

[0081] The secretion of extracellular vesicles is a phenomenon that is evolutionarily conserved across all biological kingdoms, ranging from bacteria and archaea to eukaryotes.

[0082] In particular, extracellular vesicles containing DNA, RNA, functional proteins, and antigens are utilized as a new mode of cell communication. Extracellular vesicles are known to contain specific genetic material and bioactive factors depending on the properties and state of the cell from which they originate. Since extracellular vesicles are fundamentally derived from cells, they are biocompatible unlike other nanoparticles. Furthermore, because they can encapsulate or label drugs or biologically active ingredients on their interior or surface, there are ongoing attempts to utilize them as raw materials for drug delivery systems, cosmetics, and pharmaceuticals.

[0083] As used herein, the term "hemorrhagic encephalopathy" refers to a series of diseases caused by the rupture of blood vessels within the brain, resulting in blood leakage. These diseases primarily include cerebral hemorrhage, subarachnoid hemorrhage, and intraventricular hemorrhage (IVH). Hemorrhagic encephalopathy interferes with the normal functioning of the brain and, in severe cases, can cause complications such as nerve damage, hydrocephalus, and cerebral infarction.

[0084] Hemorrhagic brain disease varies from mild to severe depending on the degree of bleeding; in severe cases, it can cause long-term complications such as neurological damage, hydrocephalus, cerebral palsy, and developmental delay. However, as there is currently no definitive preventive or curative method, it remains one of the most intractable diseases.

[0085] As used herein, the term "prevention" refers to any act of suppressing symptoms caused by hemorrhagic encephalopathy in an individual or delaying the onset of the disease by administering a pharmaceutical composition according to the present invention.

[0086] As used herein, the term "treatment" refers to any act in which symptoms caused by a hemorrhagic encephalopathy in an individual are improved or beneficially altered by the administration of a pharmaceutical composition according to the present invention. The intended therapeutic effects include the prevention of the onset or recurrence of the disease, the alleviation of symptoms, the reduction of all direct or indirect pathological consequences associated with the disease, the prevention of metastasis, the reduction of the rate of disease progression, the alleviation or temporary relief of the disease state, and the improvement of the disease state or prognosis. For the purposes of the present invention, the above treatment may be interpreted to include all acts of improving said symptoms by administering the pharmaceutical composition of the present invention to a patient with the onset of the disease, but is not specifically limited thereto.

[0087] As used herein, the term "administration" means introducing a pharmaceutical composition of the present invention to a subject by any appropriate method, by providing a composition of the present invention to the subject. The route of administration may be administered via various oral or parenteral routes as long as it can reach the target tissue.

[0088] The pharmaceutical composition of the present invention may be appropriately administered to an individual according to the conventional methods, routes of administration, and dosages used in the art, depending on the purpose or need. Examples of routes of administration may be oral, parenteral, subcutaneous, intraperitoneal, pulmonary, and intranasal, and parenteral injection includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.

[0089] In addition, appropriate dosage and frequency of administration may be selected according to methods known in the art, and the amount and frequency of administration of the pharmaceutical composition of the present invention actually administered may be appropriately determined by various factors such as the type of condition to be treated, route of administration, sex, health status, diet, age and body weight of the subject, and severity of the disease.

[0090] As used herein, the term "individual" refers to a subject requiring treatment for a disease, and more specifically, to all animals including humans who are likely to develop or have already developed the disease. It may refer to mammals such as humans or non-human primates, mice, dogs, cats, horses, and cattle, but is not limited thereto.

[0091] Additionally, the above pharmaceutical composition may be provided as a pharmaceutical composition or pharmaceutical preparation comprising an active ingredient alone or comprising one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0092] Specifically, the term “pharmaceuticalally acceptable carrier” as used herein is a substance commonly used in formulations and may be a colloidal suspension, powder, saline, lipid, liposome, microsphere, or nanosphere. It may also include, but is not limited to, saline, sterile water, Ringer’s solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc., and may further include other conventional additives such as antioxidants and buffers as needed. These may form a complex with a transport means or be associated with it, and may be transported in vivo using a carrier system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, absorption enhancers, or fatty acids.

[0093] Additionally, the term “pharmaceuticalally acceptable excipient” as used herein means any substance that is used as a carrier or vehicle for the delivery of a pharmaceutical composition according to the present invention, or is added to a formulation to improve handling or stability, or to facilitate the preparation of a dosage unit formulation into a dosage form such as a capsule or tablet suitable for oral administration. Excipients may include, but are not limited to, diluents, disintegrants, binders, adhesives, humectants, lubricants, glidants, flavoring agents, surfactants, and inclusion compounds.

[0094] When the above pharmaceutical composition is formulated, it may be prepared using excipients or diluents such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, bulking agents, binders, humectants, disintegrants, and surfactants that are commonly used. Regarding suitable pharmaceutically acceptable carriers and formulations, each component may be preferably formulated using the methods disclosed in Remington's Pharmaceutical Sciences.

[0095] Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients.

[0096] Liquid preparations for oral administration include suspensions, internal solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavoring agents, and preservatives, may be included.

[0097] Parenteral preparations may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Witepsol, macrogol, tween 61, cacao butter, laurin fat, glycero-gelatin, etc. may be used as the base for suppositories, and known excipients or diluents may be used when manufactured in the form of ophthalmic formulations.

[0098] In addition, the above pharmaceutical composition may be administered alone or in combination with other therapeutic agents. That is, the above pharmaceutical composition may be administered in conjunction with a known composition or other agent having a preventive or therapeutic effect on the said disease, and may be administered simultaneously, separately, or sequentially, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0099] The above pharmaceutical composition may be administered orally or parenterally, and when administered parenterally, the injection method may be selected as topical, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intraosseous injection, intracardiac injection, intrathecal injection, transdermal injection, intranasal injection, intracanal injection, local injection, sublingual injection, intrarectal injection, or intrahoracic injection.

[0100] The above pharmaceutical composition is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level may be determined based on factors including the patient's type of disease and severity of disease, age, sex, condition, body weight, absorption of active ingredient in the body, activity of the drug, inactivation rate and excretion rate, sensitivity to the drug, timing of administration, route of administration and clearance rate, duration of treatment, concomitant medications, and other factors well known in the medical field. In addition, the dosage amount varies depending on the patient's condition and body weight, severity of disease, dosage form, route of administration, and time of administration, but can be appropriately selected by a person skilled in the art.

[0101] Specifically, the above pharmaceutical composition may be administered at a dose of 0.001 to 1000 mg / kg / day, more specifically at a dose of 0.1 to 100 mg / kg / day. The administration may be administered once a day or divided into several doses. For example, it may be administered every other day or once a week. However, since the dosage may be increased or decreased depending on the route of administration, severity of obesity, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0102]

[0103] In one embodiment of the present invention, the BDNF gene may be introduced by a viral vector.

[0104] As used herein, the term "viral vector" refers to a gene delivery technology that utilizes a virus to deliver external genes to specific cells or tissues. Viral vectors generally deliver genes by utilizing the natural infectious properties of viruses. Viruses possess the ability to penetrate cells, replicate the genes within them, and propagate; a viral vector is a modified virus designed to carry a specific gene and deliver it to a desired cell by leveraging this property. Such viral vectors can be utilized in various fields, including gene therapy, genetic engineering, and vaccine development.

[0105] The virus vector of the present invention may be a lentivirus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, etc., but is not limited thereto.

[0106] As used herein, the term "Lentivirus" refers to a type of retrovirus characterized by a long incubation period in mammalian species; it is used as a gene delivery vector because it has low pathogenicity and possesses the ability to safely deliver and stably maintain genes.

[0107]

[0108] In another embodiment of the present invention, the mesenchymal stem cells may be derived from the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta.

[0109] As used in this specification, the term "umbilical cord" means the baby's umbilical cord.

[0110] As used in this specification, the term "cord blood" refers to blood collected from the umbilical vein connecting the placenta and the fetus. Cord blood is a byproduct that occurs naturally during childbirth and is much easier to collect than general mesenchymal tissues such as bone marrow, which require multiple surgeries. Furthermore, compared to bone marrow transplantation, the cord blood banking industry is active and infrastructure is already established, making it easier to find donors. In addition, cord blood-derived cells do not express HLA-DR (class II), which is the most important cause of rejection in tissue or organ transplants; therefore, they can avoid or minimize immune reactions such as rejection that were problematic in conventional transplant surgeries.

[0111] As used herein, the term "bone marrow" refers to a flexible tissue located within the bone cavity, which is also a hematopoietic organ responsible for producing most of an adult's blood. Bone marrow is classified into red bone marrow and white bone marrow based on the proportion of constituent cells; red bone marrow is composed mostly of hematopoietic cells, while white bone marrow is composed mostly of adipose tissue.

[0112] As used herein, the term "fat" refers to one of the three major nutrients, along with carbohydrates and proteins. Fat is composed primarily of carbon and hydrogen atoms, making it hydrophobic; it dissolves well in organic solvents but does not dissolve well in water. Fat is a type of lipid, along with phospholipids and cholesterol.

[0113] As used in this specification, the term "muscle" refers collectively to tendons and flesh, and is an organ responsible for movement in animals. Furthermore, in terms of function, it is classified into voluntary skeletal muscle and involuntary visceral muscle, and structurally into striated muscle and smooth muscle. Muscles are classified into three types—skeletal muscle, cardiac muscle, and smooth muscle—based on their form, cellular signaling pathways, the manner in which contractile force changes, contraction patterns (periodic or differential), and the role played by the nervous system in muscle function.

[0114] As used in this specification, the term "nerve" refers to an organ in which an organism detects and responds to its surrounding environment and stimuli. It constitutes the nervous system, particularly the peripheral nervous system, and consists of multiple axons of nerve cells bundled together. Since a single nerve is mainly composed of axons originating from nerve cells in adjacent areas, it mostly transmits only specific information.

[0115] As used herein, the term “skin” refers to the largest tissue in the integumentary system, which is composed of a number of epithelial tissues that protect the muscles and organs within the body. When exposed to the external environment, the skin plays a very important role in protecting the body from pathogens, and other major functions of the skin include insulation, thermoregulation, sensory functions, and the synthesis of vitamin D and protection of vitamin B folates.

[0116] As used herein, the term "amnion" refers to a membrane covering an embryo, which covers the outer part of the embryo within the uterus. The inside of the amnion is filled with a fluid called amniotic fluid, which expands the membrane to form a sac called the amniotic sac, creating an environment where the embryo can be protected as it grows.

[0117] As used herein, the term "placenta" refers to a reproductive organ in mammalian development that transfers nutrients from the mother to the fetus and fetal waste to the mother. The placenta is formed through the differentiation and division of the embryonic trophoblast implanted in the maternal endometrium, the resulting differentiation of endometrial basal cells and endocytes, and the growth of spiral arteries.

[0118]

[0119] In another embodiment of the present invention, the hemorrhagic brain disease may be one or more selected from the group consisting of intraventricular hemorrhage, periventricular hemorrhage, intracerebral hemorrhage, and subarachnoid hemorrhage.

[0120] As used herein, the term "intraventricular hemorrhage (IVH)" refers to a condition in which bleeding occurs within the ventricles of the brain. The ventricles are spaces in the brain containing cerebrospinal fluid (CSF); when blood flows into these spaces, the pressure within the brain increases, which can lead to nerve cell damage, compression of brain tissue, and a decline in brain function.

[0121] As used herein, the term "periventricular hemorrhage" refers to bleeding occurring in the periventricular white matter region surrounding the ventricles, resulting from the rupture of weak blood vessels around the ventricles. The degree of neurological damage caused by periventricular hemorrhage can vary depending on the amount and location of the bleeding, and in severe cases, it can lead to long-term brain damage and delayed neurodevelopment.

[0122] As used herein, the term "intracerebral hemorrhage" refers to bleeding that occurs within the parenchyma of the brain, that is, within the brain tissue. This bleeding is primarily caused by various factors such as hypertension, trauma, rupture of cerebral aneurysms, and vascular malformations, and can result in severe neurological deficits by causing direct damage to brain tissue. Symptoms of intracerebral hemorrhage vary depending on the location and size of the bleeding, and in severe cases, it can lead to a critical condition that may result in death.

[0123] As used herein, the term "subarachnoid hemorrhage" refers to a condition in which blood leaks into the space between the arachnoid membrane and the pia mater—two of the three meninges surrounding the brain. The rupture of a cerebral aneurysm is the most common cause, but other factors such as trauma, vascular malformations, and hemorrhagic diseases can also induce subarachnoid hemorrhage. Subarachnoid hemorrhage is accompanied by severe headache, vomiting, decreased consciousness, and seizures, and is a life-threatening emergency that requires immediate treatment.

[0124]

[0125] In another embodiment of the present invention, the pharmaceutical composition may inhibit hydrocephalus.

[0126] As used herein, the term "hydrocephalus" refers to a condition in which the ventricles are abnormally expanded due to circulatory abnormalities or impaired absorption of cerebrospinal fluid (CSF) present within the ventricles. Under normal circumstances, CSF is produced, circulated, and absorbed along the ventricular system, maintaining constant intracranial pressure. However, if the flow of CSF is blocked or its absorption is inhibited due to various causes such as intraventricular hemorrhage, infection, tumors, or trauma, intraventricular pressure rises and the ventricular space expands; this leads to compression of brain tissue and causes structural or functional damage. In children, hydrocephalus may manifest as cranial expansion or increased head circumference, while in adults, it can cause symptoms such as headaches, gait disturbances, and cognitive decline.

[0127] In the treatment of hemorrhagic brain diseases, hydrocephalus is considered one of the major pathological consequences of intraventricular hemorrhage (IVH) in newborns. In particular, intraventricular hemorrhage occurring in premature infants can block the ventricular system or disrupt cerebrospinal fluid circulation, inducing secondary hydrocephalus, which leads to damage to peripheral brain tissue, reactive gliosis, inflammatory responses, and cognitive decline. The technology of the present invention aims to inhibit the progression of hydrocephalus and prevent or improve structural and functional brain damage resulting therefrom by administering mesenchymal stem cell-derived extracellular vesicles (BDNF MSC-EV) into the ventricles. Therefore, hydrocephalus is a pathological indicator that serves as a direct criterion for judging the therapeutic effect.

[0128]

[0129] In another embodiment of the present invention, the pharmaceutical composition may reduce the expression level of GFAP (glial fibrillary acidic protein) in the periventricular region.

[0130] As used herein, the term "GFAP (glial fibrillary acidic protein)" refers to a cytoskeletal protein specifically expressed in astrocytes and belongs to the intermediate filament protein family. GFAP is involved in various physiological functions such as the development of the nervous system, maintenance of homeostasis, stabilization of cell structure, and reactive glial cell responses. Generally, GFAP is widely used as a marker to confirm the presence of astrocytes in central nervous system tissues such as the brain or spinal cord, and is utilized as an indicator to determine the activation status or degree of pathological response of astrocytes through histological or molecular biological analysis.

[0131] In the present invention, GFAP (glial fibrillary acidic protein) is used as a key biomarker for reactive gliosis induced by hemorrhagic brain disease, particularly intraventricular hemorrhage in newborns. When periventricular tissue is damaged by hemorrhage or inflammation, astrocytes proliferate or become activated reactively, and the expression level of GFAP increases during this process. It has been confirmed that the BDNF gene-introduced mesenchymal stem cell-derived extracellular vesicles (BDNF MSC-EV) according to the present invention act on the site of brain injury to inhibit the abnormal activation of astrocytes, thereby reducing the expression level of GFAP. This is important experimental evidence demonstrating the improvement of the pathological state of brain tissue and neuroprotective effects.

[0132]

[0133] In another embodiment of the present invention, the pharmaceutical composition may reduce the expression level of inflammatory cytokines.

[0134] As used herein, the term "inflammatory cytokine" refers to a family of signaling proteins secreted by immune or non-immune cells that induce, regulate, or amplify inflammatory responses. Inflammatory cytokines are involved in both innate and adaptive immune responses, and are overexpressed or dysregulated under various pathological conditions such as pathogen infection, tissue damage, tumor formation, and autoimmune diseases. Major inflammatory cytokines include Interleukin-1 alpha (IL-1α), Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), and Tumor Necrosis Factor-alpha (TNF-α), which contribute to increased vascular permeability at the site of inflammation, promotion of leukocyte influx, fever response, and activation of cytotoxicity.

[0135] In the present invention, inflammatory cytokines are considered pathological markers of secondary neuroinflammatory responses caused by hemorrhagic brain disease, particularly intraventricular hemorrhage. When neonatal intraventricular hemorrhage occurs, inflammatory cytokines are secreted from damaged blood vessels and brain cells, inducing an inflammatory response in the ventricles and surrounding neural tissues, which leads to additional neuronal damage and glial cell responses. When the BDNF gene-introduced mesenchymal stem cell-derived extracellular vesicles (BDNF MSC-EV) according to the present invention are administered into the ventricles, they exhibit the effect of suppressing such inflammatory responses and reducing the expression levels of inflammatory cytokines. In particular, the reduction in the expression of IL-1α and IL-6 was confirmed through the experimental results of the present invention, which serves as important pharmacological evidence suggesting that extracellular vesicle-based therapeutic agents contribute to brain tissue protection through a neuroinflammation regulatory mechanism.

[0136]

[0137] In another embodiment of the present invention, the inflammatory cytokine may be one or more selected from the group consisting of IL-1α (Interleukin-1alpha) and IL-6 (Interleukin-6).

[0138] As used herein, the term "IL-1α (Interleukin-1alpha)" refers to a representative inflammatory cytokine belonging to the interleukin-1 family, a protein that plays a crucial role in innate immune responses. IL-1α is primarily produced by damaged cells or stimulated immune cells (such as macrophages and monocytes) and is rapidly expressed during the early stages of inflammation induction to amplify the immune response. IL-1α induces various physiological changes, such as the activation of vascular endothelial cells, the promotion of leukocyte migration to the site of inflammation, and the induction of fever, and functions as a major initiator of inflammatory responses alongside IL-1β. IL-1α is also considered a "danger signal" of the immune system, as it is not only present within cells but is also released extracellularly when cells undergo necrosis or damage, transmitting strong inflammatory signals to surrounding tissues.

[0139] As used herein, the term "IL-6 (Interleukin-6)" refers to a multifunctional inflammatory cytokine involved in immune responses, the regulation of inflammation, hematopoiesis, and tissue damage repair processes. It is primarily secreted by macrophages, fibroblasts, vascular endothelial cells, and T cells, and is rapidly expressed in response to various stimuli such as infection, tissue damage, and tumors. IL-6 acts on hepatocytes to induce the production of acute-phase response proteins such as C-reactive protein (CRP), and influences the entire immune system, including B cell differentiation and antibody production, regulation of T cell differentiation, and activation of the hematopoietic system. In addition, IL-6 contributes to maintaining or chronicizing inflammatory states and plays a pathogenic role in various pathophysiological conditions, such as autoimmune diseases, neuroinflammation, and tumors.

[0140]

[0141] In another embodiment of the present invention, the pharmaceutical composition may improve cognitive decline caused by hemorrhagic brain injury.

[0142]

[0143] In addition, the present invention

[0144] A pharmaceutical preparation for the prevention or treatment of hemorrhagic brain disease comprising the above pharmaceutical composition is provided.

[0145] The above terms "pharmaceutical composition," "hemorrhagic encephalopathy," "prevention," "treatment," etc. may fall within the scope described above.

[0146]

[0147] In one embodiment of the present invention, the pharmaceutical formulation may further comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0148] The above terms, such as "pharmaceuticalally acceptable carrier," "pharmaceuticalally acceptable excipient," "pharmaceutically acceptable diluent," etc., may be within the scope described above.

[0149]

[0150] In another embodiment of the present invention, the pharmaceutical formulation may be an injectable formulation, an infusion formulation, a spray formulation, a liquid formulation, or a patch formulation.

[0151] As used herein, the term "Injection Formulation" refers to a pharmaceutical formulation intended for direct administration into body tissues or organs, such as subcutaneous, intramuscular, or intravenous, and is provided as a solution, suspension, emulsion, or a solid sterile preparation to be dissolved or suspended in a solvent at use. Since the drug is delivered directly into the body, the injection formulation can exhibit rapid absorption and immediate therapeutic effects, and is particularly suitable for patients in emergency situations or those for whom oral administration is difficult. This formulation offers the advantages of ensuring drug stability, maximizing bioavailability, and enabling precise dosage control.

[0152] As used herein, the term “Infusion Formulation” refers to a pharmaceutical formulation designed to deliver a drug into the body at a constant rate and administered intravenously, subcutaneously, or via other routes. Infusion formulations are designed to deliver the drug slowly into the bloodstream to maintain a long-term therapeutic effect and are used for the treatment of acute and chronic diseases. They also include pharmaceutical formulations designed to be inserted into the body through the urethra, anus, vagina, etc., and to release their therapeutic effect after dissolving due to body heat or secretions. Infusion formulations are generally provided in liquid form and are designed to maximize the stability and bioavailability of the drug.

[0153] As used herein, the term "spray formulation" refers to a pharmaceutical formulation designed to apply a drug internally or topically by spraying it in the form of a fine mist using a device. Spray formulations are designed to ensure that the drug is evenly dispersed and effectively delivered to specific areas, and are primarily used for localized areas such as the respiratory tract, skin, and mucous membranes. Formulations that utilize a device to spray the drug as a mist are included, and are designed to deliver the drug directly to the lungs, nose, skin, etc., to exert a therapeutic effect. Spray formulations are provided in liquid form and may contain appropriate solvents and additives to maximize drug stability and delivery efficiency.

[0154] As used herein, the term "Liquid Formulation" refers to a pharmaceutical form in which a drug is provided in a liquid form, meaning a formulation containing an active ingredient in liquid form. Liquid formulations are provided by dissolving or suspending a drug in a solvent such as water, alcohol, or oil, and can be used in various ways, such as oral administration, topical application, injection, or inhalation. Liquid formulations have a rapid absorption rate and are easy to administer, making them suitable for patients who have difficulty swallowing solid formulations, such as children or the elderly. Additionally, additives or stabilizers may be included to maintain drug stability and maximize bioavailability.

[0155] As used herein, the term "Patch Formulation" refers to a formulation designed to provide a continuous therapeutic effect when applied to the skin. Patch formulations have the advantage of a lower risk of side effects, such as gastrointestinal or liver damage associated with oral medications, and can be administered to patients for whom oral administration is difficult. Furthermore, because they are absorbed directly into the bloodstream through the skin without undergoing hepatic metabolism, they can produce the same therapeutic effect without side effects at lower doses compared to oral formulations. Patch formulations are primarily used in the treatment of chronic diseases, pain management, and hormone therapy, and various polymer-based technologies are applied to control drug concentration and release rates.

[0156]

[0157] In another embodiment of the present invention, the hemorrhagic brain disease may be one or more selected from the group consisting of intraventricular hemorrhage, periventricular hemorrhage, cerebral parenchymal hemorrhage, and subarachnoid hemorrhage.

[0158] The above terms "intraventricular hemorrhage," "periventricular hemorrhage," "parenchymal hemorrhage," "subarachnoid hemorrhage," etc., may fall within the aforementioned range.

[0159]

[0160] In another embodiment of the present invention, the pharmaceutical preparation may inhibit ventricular dilation.

[0161] The above term "ventricular dilation" may be within the aforementioned range.

[0162]

[0163] In another embodiment of the present invention, the pharmaceutical formulation may reduce the level of GFAP expression in the periventricular region.

[0164] The above term "GFAP" may be within the aforementioned range.

[0165]

[0166] In another embodiment of the present invention, the pharmaceutical formulation may reduce the expression level of inflammatory cytokines.

[0167] The above term "inflammatory cytokine" may be within the aforementioned range.

[0168]

[0169] In another embodiment of the present invention, the inflammatory cytokine may be one or more selected from the group consisting of IL-1α and IL-6.

[0170] The above terms "IL-1α" and "IL-6" may be within the aforementioned range.

[0171]

[0172] In another embodiment of the present invention, the pharmaceutical formulation may improve cognitive decline caused by hemorrhagic brain injury.

[0173]

[0174] In addition, the present invention

[0175] The above pharmaceutical composition is used for the prevention or treatment of hemorrhagic brain disease.

[0176]

[0177] In addition, the present invention

[0178] A method for preventing or treating hemorrhagic brain disease is provided, comprising the step of administering the above pharmaceutical composition to an individual in need thereof.

[0179]

[0180] To aid in understanding the present invention, it will be explained in more detail below through examples. However, the following examples are merely illustrative for explaining the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the technical field to which the invention belongs.

[0181]

[0182] [Example]

[0183] Example 1. Production of BDNF gene-introduced mesenchymal stem cells

[0184] BDNF-overexpressing stem cells (BDNF-MSCs) were produced by transfecting human Wharton's jelly-derived mesenchymal stem cells with a retrovirus vector into which the gene sequence of Brain Derived Neurotrophic Factor (BDNF) was inserted.

[0185] We aimed to confirm that mesenchymal stem cell characteristics were well maintained even after transduction through the analysis of mesenchymal stem cell characteristics using Fluorescence-Activated Cell Sorting (FACS).

[0186] As a result, as shown on the left side of Figure 1, it was confirmed that the positive markers of mesenchymal stem cells (MSC positive markers), CD90, CD73, and CD105, showed high expression levels. As shown on the right side of Figure 1, when measuring the expression levels of the negative markers of mesenchymal stem cells (MSC negative markers), CD14, CD45, and HLA-DR, the gray and red areas of each graph overlapped significantly, indicating that these negative markers were not expressed in BDNF-MSCs. In each graph, the gray area represents the negative control, and the red area represents the positive marker cells for the corresponding marker.

[0187] This result indicates that the original characteristics of mesenchymal stem cells are well maintained even after the introduction of the BDNF gene.

[0188]

[0189] Example 2. Evaluation of BDNF expression levels in mesenchymal stem cells with introduced BDNF gene

[0190] To compare and evaluate the expression levels of human brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells with the BDNF gene introduced (BDNF-MSC) and control non-transduced mesenchymal stem cells (naive MSC), the concentration of BDNF protein in the culture medium of BDNF-MSC and naive MSC was measured and compared using an enzyme-linked immunosorbent assay (ELISA).

[0191] As a result, as shown in Figure 2, the control group (naive MSC) hardly expressed BDNF, but BDNF-MSC lot #1 expressed more than 1,000 pg / ml and BDNF-MSC lot #2 expressed more than 2,500 pg / ml, confirming that BDNF was expressed at very high levels.

[0192] This result indicates that the BDNF gene was successfully introduced and protein expression was effectively achieved.

[0193]

[0194] Example 3. Isolation of extracellular vesicles derived from BDNF-MSC

[0195] After obtaining culture medium of mesenchymal stem cells (BDNF-MSC) into which the BDNF gene was introduced, extracellular vesicles (EVs) were isolated using tangential flow filtration (TFF), and the size, concentration, and purity of the isolated extracellular vesicles were determined using nanoparticle tracking analysis (NTA) and protein quantification methods.

[0196] As a result, as shown in Figure 3, it was confirmed that the main size of the isolated extracellular vesicles was within the size range of typical extracellular vesicles, which is 100 nm to 150 nm.

[0197] This NTA analysis result indicates that high-purity extracellular vesicles were successfully isolated.

[0198]

[0199] Example 4. Evaluation of the therapeutic effects of BDNF-MSC and BDNF-MSC-EV on hemorrhagic encephalopathy using an in vitro disease model

[0200] An in vitro disease model of hemorrhagic brain disease was established by inducing hemorrhagic neuronal damage in primary cultured rat neuronal cells by exposing them to 40 U thrombin for 24 hours.

[0201] To evaluate the therapeutic effects of BDNF gene-introduced mesenchymal stem cells (BDNF MSCs) and extracellular vesicles derived from BDNF gene-introduced mesenchymal stem cells (BDNF MSC-EVs) on hemorrhagic brain disease 24 hours after neuronal damage induction, cell viability was analyzed using the CCK-8 assay (Cell Counting Kit-8 assay). The normal control group was not treated, and the thrombin control group was treated with the vehicle (DPBS).

[0202] (1) Therapeutic effects of BDNF-MSC

[0203] To evaluate the therapeutic effect of mesenchymal stem cells (BDNF MSCs) into which the BDNF gene was introduced, BDNF MSCs were treated to the experimental group (Thrombin + BDNF MSC lot #1, #2).

[0204] As a result, as shown in Figure 4, neuronal survival rates in the disease control group (Thrombin control) were significantly reduced to approximately 40% compared to the normal control group, but neuronal survival rates in the experimental groups (Thrombin + BDNF MSC lot #1, #2) recovered to 60% and 80%, respectively, confirming that they have a significant neuroprotective effect. In particular, it was confirmed that BDNF-MSC lot #2 has a more significant neuroprotective effect than BDNF-MSC lot #1.

[0205] (2) Therapeutic effects of BDNF MSC-EV

[0206] To evaluate the therapeutic effect of extracellular vesicles derived from mesenchymal stem cells into which the BDNF gene was introduced (BDNF MSC-EV), the experimental group (Thrombin + BDNF MSC-EV) was treated with BDNF MSC-EV.

[0207] As a result, as shown in Figure 5, compared to the normal control, the neuronal survival rate in the disease control group (Thrombin control) was significantly reduced to about 60%, but the neuronal survival rate in the group treated with non-transduced control MSC, naive MSC-EV (Thrombin + naive MSC-EV), recovered to about 70%, showing a neuroprotective effect, and the neuronal survival rate in the experimental group (Thrombin + BDNF MSC-EV) recovered to about 80%, confirming that it has a significantly increased neuroprotective effect.

[0208]

[0209] These results suggest that BDNF-MSC or extracellular vesicles derived therefrom are effective in alleviating damage to brain neurons and restoring cell viability.

[0210]

[0211] Example 5. Evaluation of the therapeutic effect of BDNF MSC-EV on hemorrhagic encephalopathy using an in vivo model of hemorrhagic encephalopathy

[0212] Intraventricular hemorrhagic brain injury was induced in 4-day-old neonates (SD rats) by injecting 200 µl of blood collected from the maternal rat caudate vein into both ventricles. Two days after inducing intraventricular hemorrhage, naive MSC-EV (ctrl MSC-EV) or BDNF MSC-EV (2 x 10 8 particles in 0.01 cc) were administered into the ventricles.

[0213] After 4 weeks, a passive avoidance test was performed, and on the same day, the animals were sacrificed to analyze the extent of brain damage by obtaining brain tissue and cerebrospinal fluid through MRI-based ventricular volume measurement, GFAP immunohistochemistry, inflammatory cytokine analysis (IL-1α, IL-6), and measurement of memory function indicators (latency).

[0214] As a result, as shown in Figure 6, in an in vivo model of intraventricular hemorrhage newborn rat, ventricular dilation (hydrocephalus) was observed similarly in all groups 1 day after induction of intraventricular hemorrhage prior to administration of extracellular vesicle agents, confirming that brain damage including hydrocephalus was induced. As a result of measuring ventricular volume via in vivo MRI performed 4 weeks later, it was confirmed that ventricular dilation did not significantly decrease in the ctrl MSC-EV administration group, but significantly decreased in the BDNF MSC-EV administration group.

[0215] In addition, GFAP (glial fibrillary acidic protein; a marker of reactive gliosis) levels were measured by immunohistochemistry in the periventricular region. As shown in Figure 7A, it was confirmed that the levels significantly increased in the intraventricular hemorrhage control group (IVH ctrl) compared to the normal control group (Normal ctrl), did not significantly decrease in the naive MSC-EV administration group (IVH + ctrl MSC-EV), while they significantly decreased in the BDNF MSC-EV administration group (IVH + BDNF MSC-EV).

[0216] In addition, when inflammatory cytokines (IL-1α and IL-6) were measured in cerebrospinal fluid (CSF) using ELISA, as shown in Figure 7B, it was confirmed that the expression of inflammatory cytokines was significantly increased in the intraventricular hemorrhage control group (IVH ctrl) compared to the normal control group (Normal ctrl), while it was significantly decreased in the naive MSC-EV administration group (IVH + ctrl MSC-EV), and in the BDNF MSC-EV administration group (IVH + BDNF MSC-EV), the expression of inflammatory cytokines was significantly decreased not only compared to the intraventricular hemorrhage control group but also compared to the naive MSC-EV administration group.

[0217] In addition, a passive avoidance test, a behavioral assessment evaluating memory retention, was performed to measure the latency (sec) reflecting memory function. As shown in Figure 7C, the latency was significantly reduced in the intraventricular hemorrhage control group (IVH ctrl) compared to the normal control group (Normal ctrl), while it was significantly improved in the naive MSC-EV administration group (IVH + ctrl MSC-EV). Furthermore, it was confirmed that the BDNF MSC-EV administration group (IVH + BDNF MSC-EV) showed significant improvement not only compared to the intraventricular hemorrhage control group but also compared to the naive MSC-EV administration group.

[0218]

[0219] These results suggest that BDNF MSC-EV exhibits a multifaceted therapeutic effect in an in vivo model of hemorrhagic encephalopathy, improving not only structural brain damage but also inflammatory responses and cognitive decline.

[0220]

[0221] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for the prevention or treatment of hemorrhagic brain disease comprising, as an active ingredient, mesenchymal stem cells into which the BDNF (Brain-Derived Neurotrophic Factor) gene has been introduced or extracellular vesicles derived therefrom.

2. In Claim 1, A pharmaceutical composition characterized in that the above BDNF gene is introduced by a viral vector.

3. In Claim 1, A pharmaceutical composition characterized in that the above mesenchymal stem cells are derived from the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta.

4. In Claim 1, A pharmaceutical composition characterized in that the above-mentioned hemorrhagic brain disease is one or more selected from the group consisting of intraventricular hemorrhage, periventricular hemorrhage, intrarebral hemorrhage, and subarachnoid hemorrhage.

5. In Claim 1, The above pharmaceutical composition is characterized by inhibiting hydrocephalus.

6. In Claim 1, The above pharmaceutical composition is characterized by reducing the expression level of GFAP (glial fibrillary acidic protein) in the periventricular region.

7. In Claim 1, The above pharmaceutical composition is characterized by reducing the expression level of inflammatory cytokines.

8. In Claim 7, A pharmaceutical composition characterized in that the above-mentioned inflammatory cytokine is one or more selected from the group consisting of IL-1α (Interleukin-1alpha) and IL-6 (Interleukin-6).

9. In Claim 1, The above pharmaceutical composition is characterized by improving cognitive decline caused by hemorrhagic brain injury.

10. A pharmaceutical preparation for the prevention or treatment of hemorrhagic encephalopathy comprising the pharmaceutical composition of any one of claims 1 to 9.

11. In Claim 10, The above pharmaceutical formulation is characterized by further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

12. In Claim 10, The above pharmaceutical preparation is characterized by being injectable, injectable, sprayable, liquid, or patch-type.

13. In Claim 10, A pharmaceutical preparation characterized in that the above-mentioned hemorrhagic brain disease is one or more selected from the group consisting of intraventricular hemorrhage, periventricular hemorrhage, parenchymal hemorrhage, and subarachnoid hemorrhage.

14. In Claim 10, The above pharmaceutical preparation is characterized by inhibiting ventricular dilation.

15. In Claim 10, The above pharmaceutical preparation is characterized by reducing the level of GFAP expression in the periventricular region.

16. In Claim 10, The above pharmaceutical preparation is characterized by reducing the expression level of inflammatory cytokines.

17. In Claim 16, A pharmaceutical preparation characterized in that the above-mentioned inflammatory cytokine is one or more selected from the group consisting of IL-1α and IL-6.

18. In Claim 10, The above pharmaceutical preparation is characterized by improving cognitive decline caused by hemorrhagic brain injury.

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