Treatment for neurological disorders

Abstract

To provide therapeutic agents more effective in the treatment of neurological disorders comprising a culture supernatant of mesenchymal stem cells.SOLUTION: The invention provides a therapeutic agent for a neurological disorder, where the therapeutic agent comprises a culture supernatant of a mesenchymal stem cell and / or a cell capable of differentiating to a mesenchymal stem cell, and the agent is used in combination with a stimulation to the nerve of a patient, where the stimulation is preferentially applied to one or more sites selected from the group consisting of a nerve injury site, a site around a nerve injury and a site that compensates the function of the nerve injury site.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for neurological disorders. [Background technology]

[0002] Mesenchymal stem cells and their culture supernatants are used as therapeutic agents for various diseases.

[0003] Patent Documents 1 and 2 describe that the combined administration of mesenchymal stem cells and rehabilitation can improve the therapeutic effects of neurological disorders and the like.

[0004] Patent Document 3 describes the treatment of damaged areas using a stem cell culture supernatant obtained by serum-free culture of dental pulp stem cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2017 / 188457 [Patent Document 2] Special Publication No. 2013-508013 [Patent Document 3] Japanese Patent No. 6296622 Summary of the Invention [Problem to be solved by the invention]

[0006] However, there is a need for further improvements in the treatment of neurological disorders.

[0007] The present invention has been made in view of the above circumstances, and aims to provide a therapeutic agent comprising a culture supernatant of mesenchymal stem cells, which is more effective in treating nerve disorders. [Means for solving the problem]

[0008] The present inventors discovered that the above-mentioned problems can be solved by administering a culture supernatant of mesenchymal stem cells and / or cells that can differentiate into mesenchymal stem cells in combination with stimulating the patient's nerves, and by adjusting the timing and site of the stimulation, and thus completed the present invention. More specifically, the present invention provides the following.

[0009] (1) A therapeutic agent for a neurological disorder, the therapeutic agent is a therapeutic agent containing a culture supernatant of mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells, and is used in combination with stimulating the nerves of a patient; The stimulation is preferentially applied to one or more areas selected from the group consisting of a nerve damaged area, a periphery of the nerve damaged area, and an area compensating for the function of the nerve damaged area. Therapeutic agent.

[0010] (2) A therapeutic agent for nasal administration for nerve disorders, the therapeutic agent is a therapeutic agent containing a culture supernatant of mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells, and is used in combination with stimulating the nerves of a patient; The stimulation is performed from before administration of the therapeutic agent to 16 hours after administration. Therapeutic agent.

[0011] (3) A therapeutic agent for intravenous administration for nerve disorders, the therapeutic agent is a therapeutic agent containing a culture supernatant of mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells, and is used in combination with stimulating the nerves of a patient; The stimulation is performed from before administration of the therapeutic agent to 3 hours after administration. Therapeutic agent.

[0012] (4) A therapeutic agent for subcutaneous administration for nerve disorders, the therapeutic agent is a therapeutic agent containing a culture supernatant of mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells, and is used in combination with stimulating the nerves of a patient; The stimulation is performed from before administration of the therapeutic agent to 16 hours after administration. Therapeutic agent.

[0013] (5) The therapeutic agent according to any one of (1) to (4), wherein the stimulation is one or more selected from the group consisting of motor stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, and higher brain function stimulation.

[0014] (6) The therapeutic agent according to any one of (1) to (5), wherein the culture supernatant is undiluted or concentrated in terms of soluble solid content.

[0015] (7) The therapeutic agent according to any one of (1) to (5), wherein the culture supernatant is a lyophilized product that is dissolved at the time of administration. [Effects of the Invention]

[0016] According to the present invention, a therapeutic agent containing a culture supernatant of mesenchymal stem cells is provided, which is more effective in treating nerve disorders. DETAILED DESCRIPTION OF THE INVENTION

[0017] Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited to this.

[0018] <Therapeutic agent> The therapeutic agent for nerve disorders of the present invention (hereinafter also referred to as "therapeutic agent of the present invention") is a preparation containing a culture supernatant of mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells, and is used in combination with stimulating the nerves of a patient, and its usage modes include the following four modes. The therapeutic agent of the present invention may have any one of the following four aspects, or may have a combination of aspect 1 and any one of aspects 2 to 4. (Aspect 1) An aspect in which stimulation is preferentially applied to one or more areas selected from the group consisting of the damaged nerve area, the area surrounding the damaged nerve area, and areas compensating for the function of the damaged nerve area. (Aspect 2) When the formulation is a therapeutic agent for nasal administration, the stimulation is carried out from before administration of the therapeutic agent up to 16 hours after administration. (Aspect 3) In the case where the formulation is a therapeutic agent for intravenous administration, the stimulation is carried out from before administration of the therapeutic agent up to 3 hours after administration. (Aspect 4) In the case where the formulation is a therapeutic agent for subcutaneous administration, the stimulation is carried out from before administration of the therapeutic agent up to 16 hours after administration.

[0019] It has been known that stem cell culture supernatants can be useful in the treatment of neurological disorders and the like (for example, Patent Document 3). However, as a result of the inventors' investigations, it was found that the therapeutic effect is particularly enhanced by combining the administration of culture supernatant of mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells with stimulation of the patient's nerves.

[0020] Furthermore, the inventor has made the surprising discovery that the therapeutic effect is particularly high when the stimulation applied to the patient's nerves is preferentially applied to one or more areas selected from the group consisting of the damaged nerve area, the area surrounding the damaged nerve area, and areas that compensate for the function of the damaged nerve area.

[0021] Furthermore, the present inventors have made the unexpected discovery that the therapeutic effect can be further enhanced by adjusting the timing of stimulation depending on the administration route of the culture supernatant.

[0022] The reason why the therapeutic effect of nerve damage is enhanced by the present invention is presumably because, by adjusting the site of stimulation and / or the timing of stimulation as described above, the active ingredients contained in the culture supernatant are more likely to migrate to the affected area (such as the area of ​​nerve damage), thereby enhancing the effect of the culture supernatant.

[0023] In the present invention, the term "neuropathy" refers to any disorder that impairs the nerve itself or its function, and the underlying disease, the site of nerve damage, etc. are not particularly limited.

[0024] The disease causing the neuropathy is not particularly limited and may be any disease that causes the neuropathy, such as cerebrovascular disease, brain tumor, encephalitis, dementia, neurodegenerative disease, spinal cord injury, myelitis, herniated disc, and other central nervous system diseases and peripheral nerve disorders.

[0025] In the present invention, "treatment of neurological disorders" means alleviating or completely curing various symptoms associated with neurological disorders (movement disorders, dysarthria, dysphagia, higher brain dysfunction, dementia, aphasia, Parkinson's syndrome, ataxia, sensory disorders, pain, chills, numbness, hot flashes, etc.). Whether or not the treatment of neurological disorders has been effective is evaluated based on known standards and methods such as the National Institutes of Health Stroke Scale (NIHSS), modified Rankin scale (mRS), ASIA Impairment Scale (AIS), Frankel classification, Stroke Impairment Assessment (SIAS), Brunnstrom stage (BRS), Fugl-Meyer Assessment (FMA), manual muscle testing (MMT), Fugl-Meyer Assessment (FMA), Standard Linear Aphasia Test (SLTA), Watson-Abdominal Brain Aphasia Test, token test, and Mini-Mental State Examination (MMSE).

[0026] In the present invention, the term "patient" refers to any living organism that has developed a neurological disorder, including, for example, mammals such as humans, monkeys, cows, horses, pigs, dogs, and cats, birds, reptiles, and any other pet animals.

[0027] The constitution of the therapeutic agent of the present invention will be described in detail below.

[0028] (Mesenchymal stem cells, culture supernatant of cells that can differentiate into mesenchymal stem cells) The therapeutic agent of the present invention contains a culture supernatant of mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells (hereinafter also referred to as "culture supernatant of the present invention"). The therapeutic agent of the present invention may contain both a culture supernatant of mesenchymal stem cells and a culture supernatant of cells that can differentiate into mesenchymal stem cells, or may contain either one of them. When the therapeutic agent of the present invention contains both a culture supernatant of mesenchymal stem cells and a culture supernatant of cells that can differentiate into mesenchymal stem cells, the mixing ratio thereof is not particularly limited and can be adjusted appropriately depending on the therapeutic effect to be obtained, etc.

[0029] The mesenchymal stem cells (MSCs) used in the present invention are not particularly limited in terms of the preparation method or the tissue from which they are derived, as long as they are somatic stem cells derived from the mesenchyme and have the ability to self-renew and differentiate.

[0030] In the present invention, "cells capable of differentiating into mesenchymal stem cells" refers to cells that can differentiate into mesenchymal stem cells through normal division and proliferation. Such cells have both the ability to self-renew and the ability to differentiate into various cells. Examples of cells that can differentiate into mesenchymal stem cells include IPS cells (Induced Pluriopotent Stem Cells) and ES cells (Embryonic Stem Cells).

[0031] Mesenchymal stem cells and cells that can differentiate into mesenchymal stem cells may be isolated from bone marrow, fat, dental pulp, blood (peripheral blood, umbilical cord blood, etc.), placenta, umbilical cord, and other tissues in the body.

[0032] Mesenchymal stem cells and cells that can differentiate into mesenchymal stem cells may be derived from cells of the patient to be administered (autologous cells), or may be derived from cells other than the patient (allogeneic cells).

[0033] Mesenchymal stem cells may be cells induced to differentiate from ES cells, cells induced to differentiate from induced pluripotent stem cells (iPS cells, etc.), established cell lines, Muse cells (Multi-lineage differentiating Stress Euduring Cells), etc.

[0034] As mesenchymal stem cells, cells that remain in an undifferentiated state and are negative for differentiation markers (such as CD24) are usually used.

[0035] The mesenchymal stem cells may be those in which the expression of various markers satisfies any of the following criteria. At least one selected from CD73, CD90, CD105, and CD200 is positive. Negative for at least one of CD19, CD34, CD45, CD74, CD79α, and HLA-DR.

[0036] The mesenchymal stem cells are preferably positive for two or more of CD73, CD90, CD105, and CD200, and negative for four or more of CD19, CD34, CD45, CD74, CD79α, and HLA-DR, and more preferably positive for CD73, CD90, CD105, and CD200, and negative for CD19, CD34, CD45, CD74, CD79α, and HLA-DR.

[0037] Examples of mesenchymal stem cells include mesenchymal stem cells that have been reported to be applicable to the treatment of various diseases, such as the mesenchymal stem cells described in International Publication No. 2017 / 188457, International Publication No. 2009 / 002503, and Published Japanese Translation of PCT International Publication No. 2013-508013.

[0038] There are no particular limitations on the mesenchymal stem cells that can be prepared, and any method known to be used for preparing mesenchymal stem cells can be used. A preferred preparation method is the method described in Japanese Patent No. 4061487. This method includes steps of adding fresh bone marrow cells to a culture dish, allowing them to adhere to and proliferate on the dish, and then proliferating a portion of the obtained cells again on the culture dish.

[0039] The culture supernatant of the present invention is obtained by culturing the above-mentioned mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells.

[0040] When obtaining the culture supernatant of the present invention, the medium and culture conditions used are not particularly limited, and can be appropriately selected depending on the type of mesenchymal stem cells or cells that can be differentiated into mesenchymal stem cells.

[0041] Media used for culturing mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells include DMEM medium, RPMI1640 medium, HamF12 medium, and combinations thereof. The medium may contain components used in stem cell culture (various types of serum, bovine serum albumin, antibiotics, vitamins, minerals, etc.).

[0042] After culturing mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells, the cells are appropriately treated as needed from the resulting culture to obtain the culture supernatant of the present invention. Such treatments include commonly known steps such as removal of cells (by filtration, etc.), concentration, freezing, drying, dilution, etc.

[0043] The culture supernatant of the present invention is preferably one in which cells have been removed from a culture of mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells.

[0044] The present inventors have found that, when the amount of soluble solids in the culture supernatant (the content of active ingredients (proteins, etc.)) is the same, the smaller the total amount of the culture supernatant (the higher the concentration of soluble solids), the more likely it is that the effects of the present invention will be achieved. Therefore, the culture supernatant in the present invention is preferably one that has not been diluted (with a medium, physiological saline, etc.) in terms of soluble solid content after culturing mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells, or one that has been concentrated (by ultrafiltration, etc.) in terms of soluble solid content. Although not particularly limited, the amount of soluble solids may be 0.02 to 200 mg / mL.

[0045] Commercially available kits may be used to isolate and culture mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells.

[0046] In the therapeutic agent of the present invention, the dose and frequency of administration of the culture supernatant of the present invention can be adjusted appropriately depending on the effect to be obtained and the condition of the patient to be administered (age, body weight, severity of symptoms, etc.).

[0047] The time required for each administration of the therapeutic agent of the present invention can be adjusted depending on the administration method of the culture supernatant of the present invention, the number of administrations, etc.

[0048] The frequency of the combination of administration of the culture supernatant and stimulation according to the present invention is not particularly limited, and each may be performed once or repeatedly two or more times. When repeated two or more times, the combination may be performed over a period of several weeks (e.g., one week) to several months (e.g., 36 months). The number of administrations of the culture supernatant and the number of stimulations may be the same or different.

[0049] (Form of therapeutic agent) The therapeutic agent of the present invention may contain conventionally known ingredients depending on the administration method and the like.

[0050] The therapeutic agent of the present invention may be any of a therapeutic agent for nasal administration, a therapeutic agent for intravenous administration, and a therapeutic agent for subcutaneous administration.

[0051] When the therapeutic agent of the present invention is a therapeutic agent for nasal administration, a vehicle (physiological buffer solution, sterilized water, physiological saline, glucose solution, medium) or a component known to be added to a therapeutic agent for nasal administration (emulsifier, surfactant, stabilizer, etc.) may be added as needed together with the culture supernatant of the present invention.

[0052] When the therapeutic agent of the present invention is a therapeutic agent for intravenous administration, a vehicle (physiological buffer solution, sterilized water, physiological saline, glucose solution, medium) or a component known to be added to a therapeutic agent for intravenous administration (emulsifier, surfactant, stabilizer, etc.) may be added, if necessary, together with the culture supernatant of the present invention. When the therapeutic agent of the present invention is a therapeutic agent for intravenous administration, it is usually administered via injection or drip infusion.

[0053] When the therapeutic agent of the present invention is a therapeutic agent for subcutaneous administration, a vehicle (physiological buffer solution, sterile water, physiological saline, glucose solution, medium) or components known to be added to therapeutic agents for intravenous administration (emulsifiers, surfactants, stabilizers, etc.) may be added together with the culture supernatant of the present invention, as needed.

[0054] The method for storing the therapeutic agent of the present invention is not particularly limited, and examples include cryopreservation, freeze-drying, and refrigerated storage. A cryopreserved therapeutic agent is used for treatment by thawing. A freeze-dried therapeutic agent is used for treatment by dissolving it in a medium (physiological buffer solution, sterilized water, physiological saline, glucose solution, culture medium).

[0055] The therapeutic agent of the present invention preferably contains a solution prepared by dissolving the lyophilized culture supernatant of the present invention. Examples of the solvent for the solution include physiological buffer solution, sterilized water, physiological saline, glucose solution, and culture medium.

[0056] The therapeutic agent of the present invention may be divided into containers (vials, etc.) each containing a single dose or multiple doses.

[0057] From the viewpoint of convenience, the culture supernatant of the present invention is preferably stored frozen in a state in which it is aliquoted into containers for each dose.

[0058] (Stimulation) The therapeutic agent of the present invention is used in combination with stimulation of the patient's nerves, and the stimulation satisfies one of the following four requirements, or a combination of requirement 1 and any of requirements 2 to 4. (Requirement 1) The stimulation is preferentially applied to one or more areas selected from the group consisting of the damaged nerve area, the area surrounding the damaged nerve area, and areas compensating for the function of the damaged nerve area. (Requirement 2) If the formulation is a therapeutic agent for nasal administration, the stimulation is carried out from before administration of the therapeutic agent up to 16 hours after administration. (Requirement 3) When the preparation is a therapeutic agent for intravenous administration, stimulation is performed from before administration of the therapeutic agent until 3 hours after administration. (Requirement 4) When the preparation is a therapeutic agent for subcutaneous administration, stimulation is performed from before administration of the therapeutic agent until 16 hours after administration.

[0059] If the administered culture supernatant of the present invention remains in or around the affected area (such as a nerve damage site in the brain) and the blood flow to the affected area increases or the metabolic rate of the affected area increases, the culture supernatant of the present invention will migrate to the affected area and efficiently enhance the therapeutic effect. Specific methods for achieving this are the above-mentioned requirements 1 to 4.

[0060] When the therapeutic agent of the present invention is used in a manner that satisfies the above-mentioned requirement 1, stimulation (such as motor stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, and higher brain function stimulation, as described below) is administered at any timing (preferably at a timing that satisfies any of requirements 2 to 4). By applying stimulation so as to satisfy the above requirement 1), the blood flow and metabolic rate in the affected area (such as the site of nerve damage) increase preferentially compared to other nerve regions, thereby increasing the amount of the culture supernatant of the present invention that migrates to the affected area.

[0061] When the therapeutic agent of the present invention is used in a manner that satisfies any of the above requirements 2 to 4, stimulation (whole body exercise, local exercise, motor stimulation described below, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, higher brain function stimulation, etc.) is administered within the administration period specified by these requirements of the therapeutic agent. By providing stimulation so as to satisfy any one of the above requirements 2 to 4, the amount of the culture supernatant of the present invention that migrates to the affected area (brain, etc.) can be increased by increasing the blood flow rate to the affected area or the metabolic rate at the affected area when the amount of the culture supernatant of the present invention remaining in the blood or cerebrospinal fluid is high.

[0062] In the above requirements 2 to 4, "the stimulation is performed before the administration of the therapeutic agent" means that the stimulation starts before the administration of the therapeutic agent starts. In the above requirements 2 to 4, "the stimulation is performed within n hours after the administration of the therapeutic agent" means that the stimulation begins before n hours have elapsed since the start of the administration of the therapeutic agent.

[0063] In regard to requirement 2 above, from the viewpoint of facilitating the effect of the present invention, the stimulation is preferably carried out from before administration of the therapeutic agent until 10 hours after administration. In such cases, from the viewpoint of particularly facilitating the effects of the present invention, it is preferable that the stimulation be carried out within 3 hours after the administration of the therapeutic agent after the culture supernatant of the present invention is administered intranasally.

[0064] In the above requirement 2, from the viewpoint of easily achieving the effects of the present invention, it is preferable to apply stimulation immediately to 1 hour after nasal administration, more preferably 5 minutes to 1 hour after nasal administration, and most preferably 30 minutes to 1 hour after nasal administration.

[0065] In the above requirement 2, it is preferable to apply stimulation immediately after to one hour after nasal administration for the following reasons. The culture supernatant of the present invention usually increases in blood and cerebrospinal fluid concentrations immediately after administration, then rapidly decreases in blood over about 24 hours and in cerebrospinal fluid over about 12 hours, reaching an undetectable level in the affected area (brain, etc.) 24 hours after administration. In the case of nasal administration, in addition to the bloodstream, culture supernatant can also be transferred to the cerebrospinal fluid via an earlier, more direct route, in which the administered culture supernatant penetrates the nasal mucosal epithelial cells, reaches the cerebrospinal fluid around the olfactory nerve bundle, and then transfers to the cerebrospinal fluid in the subarachnoid space. Through this route, the cerebrospinal fluid concentration of the culture supernatant increases to several tens of times that of the blood concentration, for example, within 15 to 30 minutes after administration. Since there is no barrier for substance transfer between the cerebrospinal fluid and brain tissue, the amount of drug (culture supernatant) in the cerebrospinal fluid can correspond to the amount of drug (culture supernatant) in the extracellular fluid of brain tissue. Therefore, since the amount of drug in the extracellular fluid of brain tissue is highest immediately to one hour after nasal administration, the effects of the present invention can be achieved more efficiently by providing stimulation at this time.

[0066] In another embodiment of the above requirement 2, from the viewpoint of facilitating the exertion of the effects of the present invention, the culture supernatant of the present invention may be administered intranasally before sleep (e.g., 0 to 3 hours before sleep), and the stimulation may be performed after sleep (e.g., immediately after awakening to 5 hours after awakening). It is believed that sleep increases the blood and cerebrospinal fluid concentrations of the culture supernatant. Furthermore, sleep stimulation facilitates resynthesis of neural circuits. Therefore, the effects of the present invention can be more efficiently achieved by combining sleep stimulation with intranasal administration.

[0067] Regarding requirement 2, regardless of the start of stimulation, the subject may be kept in a supine position for at least 15 to 30 minutes after administration to facilitate retention of the culture supernatant in the nasal cavity after nasal administration. Also, the subject may be put to sleep for a short period (e.g., within 1 hour) immediately after administration.

[0068] In the above requirement 2, particularly preferred embodiments are as follows. Immediately after nasal administration of the culture supernatant of the present invention, any stimulus (preferably magnetic stimulation in a supine position) is applied, for example, for 1 to 20 minutes. During the application of the stimulus, the subject may transition to a sleep state. After the application of the stimulus, another arbitrary stimulus (preferably exercise stimulus) may be further applied. If the subject transitions to sleep during stimulation, it is preferable to administer the drug intranasally before falling asleep and to fall asleep as quickly as possible (for example, within one hour after administration). In the above embodiment, nasal administration may be performed once or twice or more times, for example, before transitioning to a sleep state or immediately before application of a stimulus.

[0069] In regard to requirement 3 above, from the viewpoint of facilitating the effect of the present invention, the stimulation is preferably carried out from before administration of the therapeutic agent to up to one hour after administration.

[0070] In the above requirement 4, from the viewpoint of easily achieving the effects of the present invention, the stimulation is preferably carried out from before administration of the therapeutic agent to 10 hours after administration, more preferably 4 hours after administration, and even more preferably 3 hours after administration.

[0071] In the present invention, "stimulation" means something that brings about a physiological change (at least one, preferably two or more, of electrical change, change in blood flow, change in metabolic rate, etc.) at the site to which the stimulation is applied. The strength of the stimulation applied in the present invention can be adjusted appropriately depending on the effect to be obtained and the condition of the patient to be administered (age, body weight, severity of symptoms, etc.).

[0072] In the present invention, "stimulating a patient's nerve" means that stimulation is applied to the whole or part of the patient's body to induce a desired reaction in the nerve. However, this does not exclude the application of stimulation to any tissue adjacent to the nerve. Stimulation of the patient may be applied to only one site on the nerve or to multiple sites.

[0073] The stimulation of the present invention may be applied at one or more points before, during, or after administration of the therapeutic agent of the present invention.

[0074] The type of stimulation given to the patient is not particularly limited as long as it can stimulate the nerves. From the viewpoint of easily enhancing the therapeutic effect of the present invention, the stimulation is preferably one or more selected from the group consisting of motor stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, and higher brain function stimulation. One or more of these stimuli can be simultaneously applied by a single stimulation device. For example, a voluntary movement-assisted electrical stimulation device can simultaneously apply motor stimulation, sensory stimulation (somatosensory stimulation), and electrical stimulation to a patient. When used in conjunction with a robot-assisted training device (e.g., "Robot Suit HAL" (trademark)), motor stimulation and sensory stimulation can simultaneously be applied to a patient.

[0075] [Motor stimulation] In the present invention, "exercise stimulation" refers to exercise stimulation targeted at the affected area. However, exercise stimulation targeted at the affected area does not exclude exercise stimulation involving whole-body exercise (such as exercise using a treadmill). Examples of motor stimulation include combining neuromuscular facilitation methods (PNF, Brunnstrom, Bobath, etc.) to increase the amount of stimulation to the target nerve pathway, concentrated repetitive motor stimulation such as the Kawahira method (repetitive facilitation therapy) and arm basis training, forced motor stimulation such as non-paralyzed side restraint motor therapy (CI therapy: Constraint-Induced Movement Therapy), and a combination of sensory stimulation, electrical stimulation, and magnetic stimulation in addition to motor stimulation. Furthermore, by using a robot-assisted training device (for example, "Robot Suit HAL" (trademark)), it is possible to simultaneously provide the patient with repetitive motor and sensory stimulation.

[0076] For example, a preferred stimulation to be given to a patient with swallowing disorders is a combination of any exercise stimulation and swallowing training. For patients with speech disorders, a combination of optional motor stimulation and speech training is a preferred stimulation.

[0077] Motor stimulation is stimulation of motor nerve pathways. Motor neural pathways are pathways that transmit motor information from upper motor neurons (originating in the primary motor cortex or brainstem) to lower motor neurons. These pathways include the lateral corticospinal tract, rubrospinal tract, reticulospinal tract, vestibulospinal tract, tectomy tract, and corticobulbar tract. In addition, the motor nerve pathway synapses with lower motor neurons, and its axon extends as a peripheral nerve, synapsing with extrapyramidal muscle fibers to contract the target muscle and cause movement. The primary motor cortex is regulated by the premotor cortex, supplementary motor area, cingulate motor area, thalamus, primary somatosensory cortex, superior parietal lobule, etc., and the motor and sensory pathways constantly regulate each other while working.

[0078] [Sensory stimulation] In the present invention, the term "sensory stimulation" refers to stimulation of any of the senses (visual, auditory, tactile, etc.) associated with nerve damage. From the viewpoint of easily achieving the effects of the present invention, the sensory stimulation in the present invention is preferably a somatosensory stimulation, an auditory stimulation, or a visual stimulation.

[0079] In the present invention, the term "somatosensory stimulation" refers collectively to cutaneous sensation, deep sensation, and visceral sensation, specifically including sensations obtained from the skin, mucous membranes, joints, muscles, tendons, etc. (pain sensation, temperature (low to high temperature) sensation, tactile sensation, etc.).

[0080] The means for providing somatosensory stimulation to a patient is not particularly limited, but examples include tactile pressure, acupuncture, heat, weights, vibration, and the like.

[0081] Examples of tactile pressure include rehabilitation of nerve-damaged areas (hands, feet, etc.) (for example, massage performed while visually checking the damaged area).

[0082] Acupuncture and moxibustion include methods using needles and moxibustion (burning moxa).

[0083] Heat treatments include moxibustion (burning moxa), hot packs, and hydrotherapy.

[0084] As for the weight, a weight band is used for performing mild resistance exercises used in rehabilitation.

[0085] As the vibration, a method using vibration can be mentioned.

[0086] In the present invention, "auditory stimulation" refers to stimulation provided by sound. Examples of sound include, but are not limited to, human voices, any music (a certain rhythmic rhythm, etc.), etc.

[0087] The means for providing the auditory stimulation to the patient is not particularly limited, but may be rhythmic auditory stimulation (RAS) or the like.

[0088] In the present invention, the term "visual stimulus" refers to a stimulus provided by visual information. The visual information is not particularly limited, but may include any information present in space (such as letters, pictures, and videos).

[0089] Means of providing visual stimulation to patients include, but are not limited to, functional training (visual search tasks, visual scanning training, etc.), activities of daily living (eating, dressing, toileting, grooming, bathing, reading, painting, etc.), prism adaptation, etc.

[0090] Sensory stimulation is usually stimulation of the somatic senses (sensations obtained from the skin, mucous membranes, joints, muscles, tendons, etc.). Somatic sensation can be broadly divided into four modalities (pain, temperature, touch, and deep (proprioception) sensation), and specialized sensory receptors, nerve fibers, conduction pathways, etc. are used for each reception. The sensory neural pathways through which somatosensory signals reach the cerebral cortex have been elucidated, and the following pathways are known, for example: Deep sensation and fine tactile sensation: Passes through the dorsal column-medial ciliary tract system (receptor → first-order neuron (enters the spinal cord, ascends the ipsilateral dorsal column, and terminates in the ipsilateral dorsal column nucleus of the medulla oblongata) → second-order neuron (crosses over, ascends the contralateral medial lemniscus, and terminates in the contralateral thalamic VPL) → tertiary neuron (contralateral cerebral cortical somatosensory cortex)). Thermal and pain sensations and gross tactile sensations: travel through the spinothalamic tract (receptor → first-order neuron (enters the spinal cord and terminates in the dorsal horn of the spinal cord) → second-order neuron (crosses over, ascends the contralateral anterior funiculus, ascends the contralateral spinothalamic tract, and terminates in the contralateral thalamic VPL) → tertiary neuron (contralateral cerebral cortical somatosensory area).

[0091] [Electrical stimulation] In the present invention, "electrical stimulation" refers to stimulation applied electrically using an electric current. For example, an example is stimulation in which an electrode is attached to the affected area and neural circuits are excited by electricity (low frequency, medium frequency, high frequency, interference wave, etc.).

[0092] The means for administering electrical stimulation to a patient is not particularly limited, but may include a method using a voluntary movement-assisted electrical stimulation device (such as IVES) or a method conventionally known as electrical current stimulation therapy (such as transcutaneous electrical nerve stimulation (TENS) method, functional electrical stimulation (FES) method, therapeutic electrical stimulation (TES) method, transcranial direct current stimulation (tDCS) method, deep brain stimulation (DBS) method, etc.).

[0093] Electrical stimulation typically involves attaching electrodes to the paralyzed limbs and repeatedly exciting neural circuits by stimulating them with low, medium, high, or interference waves. By exciting neural circuits with electrical stimulation, it is possible to increase the pain threshold as explained by gate control theory, or to lower the motor threshold, making it easier to move paralyzed limbs.

[0094] [Magnetic stimulation] In the present invention, "magnetic stimulation" means stimulation that is applied magnetically using a static magnet or an electromagnet.

[0095] The means for applying magnetic stimulation to a patient is not particularly limited, but conventionally known methods of magnetic stimulation (e.g., transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS) which stimulates with a weak electric current, and deep brain stimulation (DBS) which involves inserting electrodes deep into the brain to continuously apply electrical stimulation to the nervous system and control its functions) may be used. While classic rTMS techniques, such as low-frequency rTMS (1 Hz or less), which has an inhibitory effect, and high-frequency rTMS (5 Hz or more), which has an excitatory effect, are typically used, "theta burst stimulation (TBS)," which consists of three 50 Hz bursts delivered at a frequency of 5 Hz, can also be used. Intermittent TBS (iTBS) involves pulse stimulation of theta bursts (5 Hz, three 50 Hz bursts) for 2 seconds followed by an 8-second pause, which increases motor cortex excitability. Continuous TBS (cTBS) involves continuous theta burst stimulation (5 Hz, three 50 Hz bursts) which suppresses motor cortex excitability. Continuous TBS exerts an inhibitory effect, whereas intermittent TBS exerts an excitatory effect. TBS can be delivered at a lower intensity than low-frequency or high-frequency rTMS, and its effects are longer-lasting.

[0096] [Language stimulation] In the present invention, the term "linguistic stimulus" refers to a stimulus provided by communication via language.

[0097] The means for providing language stimulation to a patient is not particularly limited, but examples include encouraging the patient to read, write, draw, listen, speak, repeat, calculate, and the like.

[0098] Language stimulation usually refers to stimulation of areas of the brain related to language (Broca's area, Wernicke's area, left angular gyrus (Brodmann's area 39), left supramarginal gyrus (Brodmann's area 40), cerebellum, thalamus, basal ganglia, etc.).

[0099] [Higher brain function stimulation] In the present invention, "higher brain functions" is a general term for mental (psychological) functions including cognitive processes (perception, memory, learning, thinking, judgment, etc.) and emotions (feelings) of actions. In the present invention, "stimulation of higher brain functions" means memory training, attention training, executive function training, and social behavior training.

[0100] The means for providing higher brain function stimulation to a patient is not particularly limited, but examples include encouraging the patient to undergo memory training, attention training, executive function training, social behavior training, etc.

[0101] When the stimulation is aimed at the frontal lobe function, etc., higher brain function stimulation usually refers to stimulation at one of the stages in a pyramidal hierarchy, from the lowest level onwards: arousal → inhibition / activation → attention and concentration → information processing → memory → executive function / logical thinking ("Comprehensive Rehabilitation, May 2006 issue (Igaku-Shoin)").

[0102] [Other stimuli] In addition to the above, the "stimulus" in the present invention includes any stimulus that can bring about a physiological change in a patient. For example, in an embodiment in which stimulation is provided from before administration of a therapeutic agent to 6 hours after administration, the stimulation may be whole-body exercise or the like.

[0103] [Confirmation and evaluation of stimuli] In the present invention, whether or not a stimulus has been applied to a patient and the strength of the stimulus are determined based on the presence or absence and degree of physiological changes in the patient.

[0104] Physiological changes that serve as indicators of the application of stimulation include electrical changes, changes in blood flow, changes in metabolic rate (metabolic rate of oxygen, etc.), etc. Generally, the stronger the applied stimulation, the greater the changes in these physiological changes. For example, the stronger the applied stimulation, the greater the increase in cerebral blood flow and metabolic rate.

[0105] Electrical changes are identified by non-invasive brain function measurement methods such as scalp electroencephalography (EEG) and magnetoencephalography (MEG).

[0106] Changes in blood flow, for example, in the brain, are identified by non-invasive brain function measurement methods such as functional magnetic resonance imaging (FMRI) and single photon emission CT (SPECT).

[0107] Changes in cerebral blood flow and oxygen metabolism are identified by optical topography, PET (Positron Emission Tomography) examinations, and the like.

[0108] [Timing of stimulus application] The stimulation may be carried out once or multiple times at any time within the period specified in the present invention, although this does not exclude the stimulation being carried out at a time after the period specified in the present invention.

[0109] When the stimulation is performed before the administration of a therapeutic agent, the timing of the stimulation can be appropriately set depending on the type of stimulation, the condition of the patient, and the like.

[0110] Depending on the type of stimulation, the time from application of stimulation until the blood flow and metabolic rate to the affected area (such as the nerve damage site) increase and the duration for which the increase in blood flow and metabolic rate is maintained may vary. Therefore, it is preferable to adjust the timing of administration of the therapeutic agent so that it coincides with the time when the blood flow and metabolic rate in the affected area are higher. Specifically, when the stimulation is sensory or verbal stimulation, the time from application of the stimulation to an increase in blood flow to the affected area or an increase in metabolic rate tends to be longer than when the stimulation is electrical or magnetic stimulation. Therefore, when the stimulus is a sensory stimulus or a verbal stimulus, it is preferable to set the time between the application of the stimulus and the administration of the therapeutic agent to be long, or to set the time between the administration of the therapeutic agent and the application of the stimulus to be short. On the other hand, when the stimulation is electrical or magnetic stimulation, it is preferable to set the time between the application of the stimulation and the administration of the therapeutic agent to be short, and it is also possible to set the time between the administration of the therapeutic agent and the application of the stimulation to be long.

[0111] [Area to be stimulated] When stimulation is preferentially applied to one or more selected from the group consisting of the area of ​​nerve damage, the area surrounding the area of ​​nerve damage, and an area compensating for the function of the area of ​​nerve damage, it does not exclude the stimulation being applied to areas other than these areas.

[0112] In the present invention, "preferentially applied (to a specified area)" means that when starting to apply stimulation, stimulation is first applied to at least one of the damaged nerve area, the area surrounding the damaged nerve area, and an area compensating for the function of the damaged nerve area.

[0113] In the present invention, the term "nerve damage site" refers to the site itself where nerve damage (atrophy, nerve block, transection, severance, defect, brain injury, spinal cord injury, etc.) has occurred. Usually, the nerve damage site is the cause of nerve disorders.

[0114] In the present invention, "the area surrounding the nerve damage site" does not mean the nerve damage site itself, but rather the area surrounding the nerve damage site (for example, the area surrounding the nerve damage site or the area close to the nerve damage site).

[0115] In the present invention, "areas that compensate for the function of damaged nerve areas" means areas that work to compensate for the function of damaged nerve areas (for example, if the primary motor cortex is damaged, the right parietal lobe in the periphery of the affected side and the primary motor cortex, premotor cortex, supplementary motor area, etc. on the healthy side).

[0116] Examples of areas to which stimulation is applied to a patient include the head (brain, etc.), face, eyes, ears, mouth, upper limbs, lower limbs, trunk, articulatory organs, and swallowing organs.

[0117] The areas to which stimulation can be applied and the methods for applying stimulation are exemplified below for each type of stimulation.

[0118] [Example of exercise stimulation] When nerve damage occurs in the right primary motor cortex (the area controlling the fingers), the "damaged nerve area" is the right primary motor cortex (the area controlling the fingers). If the right primary motor cortex (the area controlling the fingers) suffers nerve damage, the "area surrounding the nerve damage" refers to the right primary motor cortex (other than the area controlling the fingers) that was not damaged, such as the premotor cortex, supplementary motor area, and right primary sensory cortex. When the right primary motor cortex (the area that controls the fingers) is damaged by nerves, the "areas that compensate for the function of the damaged nerve area" are the right parietal lobe, the left primary motor cortex, the premotor cortex, the supplementary motor area, etc.

[0119] If nerve damage occurs in the right primary motor cortex (the area controlling the fingers), the damaged nerve area can be stimulated preferentially by trying to move the paralyzed fingers of the left hand. If nerve damage occurs in the right primary motor cortex (the area controlling the fingers), by trying to move areas close to the paralyzed fingers of the left hand (uninjured fingers of the left hand, left wrist, etc.), it is possible to preferentially stimulate the area around the nerve damage. If nerve damage occurs to the right primary motor cortex (the area controlling the fingers), various tasks and gross movements using the paralyzed fingers of the left hand can be encouraged to stimulate preferentially the areas that compensate for the function of the nerve damage.

[0120] [Example of providing sensory stimulation] If the right primary sensory cortex (tertiary neurons) is nerve damaged, the "damaged area" is the right primary sensory cortex. When nerve damage occurs to the right primary sensory cortex (tertiary neurons), the "surroundings of the nerve damage area" refers to the right primary sensory cortex to the right primary motor cortex, etc., that are spared from damage. When the right primary sensory cortex (tertiary neurons) is damaged, the "areas that compensate for the function of the damaged area" are the left primary sensory cortex, the secondary somatosensory cortex that connects to the primary sensory cortex, the parietal association cortex, the motor cortex, the visual cortex, etc.

[0121] When nerve damage occurs in the right primary sensory cortex (tertiary neurons), sensory stimulation can be given to the affected area with sensory impairment to give priority to stimulation of the nerve damage area. If nerve damage occurs in the right primary sensory cortex (tertiary neurons), stimulation of an area close to the area of ​​sensory impairment can be given preferential stimulation to the area surrounding the nerve damage. In the case of nerve damage to the right primary sensory cortex (tertiary neurons), by having the patient visually confirm when applying sensory stimulation to the area with sensory impairment or the surrounding area, or by applying the same level of sensory stimulation to the healthy side at the same time, it is possible to give preferential stimulation to the area that compensates for the function of the nerve damage.

[0122] [Example of electrical stimulation] If the second finger on the right side has nerve damage, the "damaged area" is the second finger on the right side. If the second finger on the right side has nerve damage, the "area surrounding the nerve damage" refers to the first, third, fourth, and fifth fingers on the right side that were spared from nerve damage. If the right second finger has nerve damage, the "areas that compensate for the function of the nerve damage" include the right wrist, forearm, upper arm, and shoulder.

[0123] If the second finger on the right side has nerve damage, electrical stimulation can be given to the damaged area of ​​the nerve by giving electrical stimulation to the second finger on the right side, which is the damaged area of ​​the nerve. If the second finger on the right side has nerve damage, electrical stimulation can be given to the first, third, fourth, or fifth finger on the right side, which is close to the nerve-damaged second finger, to preferentially stimulate the area around the nerve damage. If the right second finger has nerve damage, electrical stimulation can be given to the right wrist, forearm, upper arm, or shoulder, allowing preferential stimulation to be given to the area that compensates for the function of the nerve damage.

[0124] [Example of magnetic stimulation] If the right primary motor cortex (the area controlling the fingers) is nerve damaged, the "damaged area of ​​the nerve" is the right primary motor cortex (the area controlling the fingers). If the right primary motor cortex (the area controlling the fingers) suffers nerve damage, the "area surrounding the nerve damage" refers to the right primary motor cortex (other than the area controlling the fingers) that was not damaged, such as the premotor cortex, supplementary motor area, and right primary sensory cortex. When the right primary motor cortex (the area that controls the fingers) is damaged by nerves, the "areas that compensate for the function of the damaged nerve area" are the right parietal lobe, the left primary motor cortex, the premotor cortex, the supplementary motor area, etc.

[0125] If the right primary motor cortex (the area controlling the fingers) is damaged, it is possible to preferentially stimulate the damaged area by applying excitatory magnetic stimulation (high-frequency rTMS (5 Hz or higher) or intermittent TBS, etc.) to the right primary motor cortex (the area controlling the fingers). If the right primary motor cortex (the area controlling the fingers) is damaged, it is possible to preferentially stimulate the area around the damaged nerve by applying excitatory magnetic stimulation to the right primary motor cortex (other than the area controlling the fingers), the premotor cortex, the supplementary motor area, and the right primary sensory cortex, which are areas close to the damaged nerve. When nerve damage occurs to the right primary motor cortex (the area controlling the fingers), applying excitatory magnetic stimulation to the right parietal lobe allows preferential stimulation to be given to the area that compensates for the function of the nerve damage. In addition, by applying inhibitory magnetic stimulation (low-frequency rTMS (below 1 Hz) or continuous TBS) to the left primary motor cortex, the inhibition of activity from the left cerebral cortex to the right cerebral cortex (interhemispheric inhibition) is reduced, which ultimately releases the damaged nerve area (right primary motor cortex (area controlling the fingers)), peripheral areas (right primary motor cortex (area other than the area controlling the fingers) - premotor cortex, supplementary motor area, right primary sensory area), and compensatory areas (right parietal lobe) from inhibition, thereby increasing their blood flow and excitability.

[0126] [Example of providing verbal stimulation] When nerve damage occurs in Wernicke's area, the "area of ​​nerve damage" is Wernicke's area. When nerve damage occurs in Wernicke's area, the "area surrounding the nerve damage" refers to the Wernicke's area and Broca's area that were spared from nerve damage, as well as the language circuit including the conduction pathway (arcuate fasciculus) connecting the two. When Wernicke's area is damaged, the areas that "compensate for the function of the damaged area" include the left angular gyrus (Brodmann's area 39), left supramarginal gyrus (Brodmann's area 40), cerebellum, thalamus, and basal ganglia.

[0127] When Wernicke's area is nerve damaged, providing sensory language stimulation can provide preferential stimulation to the nerve damaged area. When Wernicke's area is damaged, stimulation can be preferentially given to the area surrounding the damaged nerve by having the patient repeat words or by providing motor speech stimulation. When Wernicke's area is damaged, tasks such as phonology, vocabulary, grammar, reading comprehension, and calculations can be given to stimulate preferentially the areas that compensate for the function of the damaged area.

[0128] [Example of higher brain function stimulation] When executive dysfunction is caused by nerve damage, the "area of ​​nerve damage" is the part of the brain that controls executive function. When executive dysfunction occurs due to nerve damage, the "area around the nerve damage" refers to the part of the brain that controls memory and information processing, which are at the base of executive function. When executive dysfunction occurs due to nerve damage, the "area that compensates for the function of the damaged nerve" is the part of the brain that controls attention, concentration, inhibition, motivation, alertness, etc., which is located even lower than memory and information processing.

[0129] When executive dysfunction occurs due to nerve damage, executive function training can be performed to provide preferential stimulation to the damaged nerve area. When executive dysfunction occurs due to nerve damage, training of memory and information processing, which are at the lower level of executive function, can provide preferential stimulation to the area surrounding the nerve damage. When executive dysfunction occurs due to nerve damage, training for attention, concentration, inhibition, motivation, and alertness, which are at a level lower than memory and information processing, can provide preferential stimulation to areas that compensate for the function of the nerve damage. [Example]

[0130] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0131] <Preparation of mesenchymal stem cell culture supernatant> Mesenchymal stem cells derived from each tissue were cultured and the culture supernatant was collected by the following method.

[0132] All of the following cultures were carried out for 3 weeks in an incubator at a temperature of 37°C and a CO2 concentration of 5%.

[0133] (1) Cultivation of bone marrow-derived mesenchymal stem cells Using "KBM ADSC-2" (manufactured by Kohjin Bio Co., Ltd.), bone marrow-derived mesenchymal stem cells were isolated from human bone marrow tissue, and the obtained mesenchymal stem cells were cultured. From the resulting culture, cells were removed using a 0.1 to 0.22 μm PVDF filter to obtain a culture supernatant of bone marrow-derived mesenchymal stem cells (hereinafter also referred to as "culture supernatant-1"). The resulting culture supernatant had a soluble solid content of 2 mg / ml.

[0134] (2) Cultivation of adipose-derived mesenchymal stem cells Using the same method as in "(1) Culture of bone marrow-derived mesenchymal stem cells" above, mesenchymal stem cells obtained from human adipose tissue were used to obtain a culture supernatant of adipose-derived mesenchymal stem cells. Cells were removed from the resulting culture using a 0.1 to 0.22 μm PVDF filter to obtain a culture supernatant of bone marrow-derived mesenchymal stem cells (hereinafter also referred to as "culture supernatant-2"). The resulting culture supernatant had a soluble solid content of 2 mg / ml.

[0135] (3) Cultivation of dental pulp-derived mesenchymal stem cells Using the same method as in "(1) Culture of bone marrow-derived mesenchymal stem cells" above, mesenchymal stem cells obtained from human dental pulp tissue were used to obtain a culture supernatant of dental pulp-derived mesenchymal stem cells. Cells were removed from the resulting culture using a 0.1 to 0.22 μm PVDF filter to obtain a culture supernatant of bone marrow-derived mesenchymal stem cells (hereinafter also referred to as "culture supernatant-3"). The resulting culture supernatant had a soluble solid content of 2 mg / ml.

[0136] (4) Cultivation of umbilical cord-derived mesenchymal stem cells In the same manner as in "(1) Culture of bone marrow-derived mesenchymal stem cells" above, mesenchymal stem cells obtained from human umbilical cord tissue were used to obtain a culture supernatant of umbilical cord-derived mesenchymal stem cells. Cells were removed from the resulting culture using a 0.1 to 0.22 μm PVDF filter to obtain a culture supernatant of bone marrow-derived mesenchymal stem cells (hereinafter also referred to as "culture supernatant-4"). The resulting culture supernatant had a soluble solid content of 2 mg / ml.

[0137] <Treatment for patients> The culture supernatant prepared above was administered to patients with the following symptoms of neurological disorders by either intranasal administration, intravenous administration, or subcutaneous administration. In addition, stimulation (one of the following: motor stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, and higher brain function stimulation) was administered.

[0138] (Administration of culture supernatant) The culture supernatant was administered to each patient by the following methods.

[0139] The timing of administration of the culture supernatant was varied for each patient depending on the administration method. Details of the timing of administration of the culture supernatant are shown in the leftmost column of each of Tables 1 to 15. The phrase "n hours before administration" means that the start of stimulation was n hours before the start of administration of the culture supernatant. "Immediately after administration" means that the stimulation was initiated immediately after administration of the culture supernatant (any time within 15 minutes after administration of the culture supernatant). "n hours after administration" means that the start of stimulation was n hours after the start of administration of the culture supernatant.

[0140] The type of culture supernatant administered (any of culture supernatants-1 to 5) is shown in the "culture supernatant" section of Tables 1 to 15.

[0141] (1) Nasal administration Each patient received 1 ml of culture supernatant intranasally using a standard nasal dropper, administered daily for 14 weeks. Nasal administration was performed in two cases, immediately after the start of administration, 30 minutes after administration, 3 hours, 10 hours, and 20 hours after administration, with and without a one-hour sleep period immediately after administration.

[0142] (2) Intravenous administration Each patient received 5 ml of culture supernatant via intravenous infusion over 1 hour, every 7 days for 4 weeks.

[0143] (3) Subcutaneous administration Each patient received 2 ml of culture supernatant on the extensor aspect of the upper arm every 7 days for 4 weeks.

[0144] (Providing stimulation) Details of the stimulation given to each patient are as follows:

[0145] [Motor stimulation] Repetitive facilitation therapy was administered to a group of cerebral hemorrhage patients in their 40s and 50s to stimulate movement. In this example, the areas to be stimulated were set to the nerve damage area (left fingers, left foot to lower leg), the area surrounding the nerve damage (left wrist to forearm, left knee to thigh), and the area compensating for the function of the nerve damage area (left elbow to shoulder, left hip). The duration of each stimulation session was set to 30 minutes for each site. In addition, a group of patients in their 40s and 50s with similar symptoms were asked to perform whole-body exercise (exercise on a treadmill for 30 minutes per session) and were administered the culture supernatant in the same manner as above.

[0146] [Sensory stimulation] For a group of cerebral hemorrhage patients in their 40s and 50s, sensory stimulation was given to the upper and lower limbs of patients with sensory impairments by cooling the paralyzed limbs with ice packs, while also providing auditory stimulation (vocal prompts), and instructing them to visually confirm the position of their upper and lower limbs. In this example, the areas to be stimulated were set to the nerve damage area (left fingers, left foot to lower leg), the area surrounding the nerve damage (left wrist to forearm, left knee to thigh), and the area compensating for the function of the nerve damage area (left elbow to shoulder, left hip). The duration of each stimulation session was set to 30 minutes for each site. The above sensory stimulation was performed on the affected upper and lower limbs. In addition, a group of patients in their 40s and 50s with similar symptoms underwent sensory stimulation (30 minutes per session) on the healthy upper and lower limbs, and the culture supernatant was administered in the same manner as above.

[0147] [Electrical stimulation] For a group of cerebral hemorrhage patients in their 40s and 50s, low-frequency stimulation was applied to the upper and lower limbs of paralyzed patients via electrodes attached to the limbs of patients with paralysis. In this example, the areas to be stimulated were set to the nerve damage area (left fingers, left foot to lower leg), the area surrounding the nerve damage (left wrist to forearm, left knee to thigh), and the area compensating for the function of the nerve damage area (left elbow to shoulder, left hip). The duration of each stimulation session was set to 20 minutes for each site. The above electrical stimulation was applied to the affected upper and lower limbs. In addition, a group of patients in their 40s and 50s with similar symptoms received electrical stimulation (20 minutes per session) on the healthy upper and lower limbs, and the culture supernatant was administered in the same manner as above.

[0148] [Magnetic stimulation] Using a transcranial magnetic stimulation (TMS) device (CRTechnology), magnetic stimulation was administered to the heads of patients in their 40s and 50s with cerebral hemorrhage. Specifically, intermittent TBS (iTBS; 1 burst of 50 Hz, 3 stimuli) was administered at 80% of motor threshold intensity, with a 5 Hz pulse interval of 200 ms. Stimulation was administered for 2 seconds followed by an 8-second pause, for a total of 2,000 pulses. In this example, the stimulation areas were set to the nerve damage area (the area controlled by the fingers of the affected primary motor field), the area surrounding the nerve damage (other than the area controlled by the fingers of the affected primary motor field), and the area compensating for the function of the nerve damage area (right parietal lobe). The stimulation time per session was set to 2000 pulses per site, approximately 11 minutes. The above magnetic stimulation was applied to the affected temporal region. In addition, a group of patients in their 40s and 50s with similar symptoms underwent placebo magnetic stimulation of the affected area of ​​the head (2000 pulses per session, approximately 11 minutes), which only emitted sound, and were administered the culture supernatant in the same manner as above. Note that the placebo magnetic stimulation of the affected area of ​​the head did not actually deliver magnetic stimulation to the patients.

[0149] [Language stimulation] We conducted language stimulation for patients with aphasia in their 70s and 80s who had suffered from cerebral hemorrhage. The language stimulation consisted of communication tasks (reading, writing, listening, speaking, repetition, and calculation). The stimulation time was set to 60 minutes.

[0150] [Higher brain function stimulation] Among cerebral hemorrhage patients in their 70s and 80s, patients with higher brain function disorders (attention disorders) were given higher brain function stimulation. As higher brain function stimulation, attention process training was performed as attention training. The stimulation time was set to 60 minutes.

[0151] [Reference exam] For reference, among a group of cerebral hemorrhage patients in their 70s and 80s who had aphasia, they were asked to passively listen to the radio or watch television for one hour.

[0152] <Treatment effect for neurological disorders> After each treatment, the patient was evaluated for the effectiveness of treatment for each symptom using the following indexes, and the evaluation results were classified according to the following criteria. The results are shown in Table 1.

[0153] In each evaluation criterion, "after the longest test time has elapsed since administration of the culture supernatant" means the time point at which the longest test time has elapsed since administration by each method. For example, in the case of intranasal administration, "after the longest test time has elapsed since administration of the culture supernatant" means "20 hours after administration."

[0154] (evaluation) Paralysis and sensory impairment: SIAS (Stroke Impairment Assessment), FMA (Fugl-Meyer assessment) Cerebral infarction: NIHSS (National Institutes of Health Stroke Scale) Aphasia: Standardized Aphasia Assessment (SLTA) Higher brain function: MMSE (Mini-Mental State Examination)

[0155] (Evaluation criteria - exercise stimulation) ◎: A significant therapeutic effect was observed compared with the results of whole-body exercise (longest test time after administration of culture supernatant). ○: A therapeutic effect was observed compared with the results of whole-body exercise (the longest test time after administration of the culture supernatant). △: A slight therapeutic effect was observed compared to the results of whole-body exercise (the longest test time after administration of the culture supernatant).

[0156] (Evaluation criteria - sensory stimulation, electrical stimulation) ◎: A significant therapeutic effect was observed compared with the results of stimulation of the healthy side (longest test time after administration of culture supernatant). ○: A therapeutic effect was observed compared with the results of stimulation of the healthy side (longest test time after administration of the culture supernatant). △: A slight therapeutic effect was observed compared to the results of stimulation of the healthy side (longest test time after administration of the culture supernatant).

[0157] (Evaluation Criteria - Magnetic Stimulation) ◎: A significant therapeutic effect was observed compared to the results of placebo-treated magnetic stimulation on the affected side (the longest test time after administration of the culture supernatant). ○: A therapeutic effect was observed compared with the results of placebo-treated magnetic stimulation on the affected side (the longest test time after administration of the culture supernatant). △: A slight therapeutic effect was observed compared to the results of placebo-treated affected side magnetic stimulation (longest test time after administration of culture supernatant).

[0158] (Evaluation criteria - language stimulation, higher brain function stimulation, reference test) ◎: A significant therapeutic effect was observed compared to before administration of the culture supernatant and stimulation. ○: A therapeutic effect was observed compared to before administration of the culture supernatant and stimulation. △: A slight therapeutic effect was observed compared to before administration of the culture supernatant and stimulation. ×: No change was observed compared to before administration of the culture supernatant and stimulation.

[0159] [Table 1]

[0160] [Table 2]

[0161] [Table 3]

[0162] [Table 4]

[0163] [Table 5]

[0164] [Table 6]

[0165] [Table 7]

[0166] [Table 8]

[0167] [Table 9]

[0168] [Table 10]

[0169] [Table 11]

[0170] [Table 12]

[0171] [Table 13]

[0172] [Table 14]

[0173] [Table 15]

[0174] As described above, when the therapeutic agent was used in a manner that satisfied the requirements of the present invention, significant improvement in the symptoms of neuropathy was observed.

[0175] In each administration method, the culture supernatant obtained in the above <Preparation of mesenchymal stem cell culture supernatant> was diluted approximately 3 to 10 times with the medium used for cell culture, and administered with the same soluble solid content. As described above, improvement in the symptoms of neuropathy was observed. However, the effect was greatest when the culture supernatant was not diluted, and when diluted, the effect was greater at lower dilution rates.

[0176] When intranasal administration was performed, the therapeutic effect tended to be higher when stimulation was given 5 to 30 minutes after intranasal administration.

[0177] Furthermore, when intranasal administration was performed, regardless of the type of culture supernatant, the therapeutic effect was particularly high when magnetic stimulation was applied for 30 minutes while the subject was in a supine position 5 minutes after intranasal administration of the culture supernatant, followed immediately by motor stimulation, although this data is not shown.

[0178] When administered intranasally, even if the interval between administration and stimulation was the same, the effect tended to be greater when the patient was asleep than when there was no sleep interval. In such cases, falling asleep within one hour of nasal administration tended to be more effective than falling asleep over more than one hour.

Claims

1. A therapeutic agent for a neurological disorder, The therapeutic agent is a therapeutic agent containing a culture supernatant, and is used in combination with stimulating the nerves of a patient; the culture supernatant is a culture supernatant of mesenchymal stem cells, The stimulation is one or more selected from the group consisting of motor stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, and higher brain function stimulation; The stimulation is preferentially applied to one or more areas selected from the group consisting of a nerve damaged area, a periphery of the nerve damaged area, and an area compensating for the function of the nerve damaged area. Therapeutic agent.

2. The therapeutic agent according to claim 1 , wherein the culture supernatant is undiluted or concentrated in terms of soluble solid content.

3. The therapeutic agent according to claim 1 or 2, wherein the culture supernatant is a lyophilized product that is dissolved at the time of administration.

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