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 nerve 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 improved 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 have found that the above-mentioned problems can be solved by administering a culture supernatant of mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells and stimulating the patient's nerves, and by adjusting the timing of the stimulation and the site to which the stimulation is applied, and have completed the present invention. More specifically, the present invention provides the following.

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

[0010] (2) A therapeutic agent for nasal administration for a nerve disorder, comprising: The therapeutic agent is a therapeutic agent containing a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells, and is used in combination with stimulation of 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 a nerve disorder, comprising: The therapeutic agent is a therapeutic agent containing a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells, and is used in combination with stimulation of 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, comprising: The therapeutic agent is a therapeutic agent containing a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells, and is used in combination with stimulation of 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 which is dissolved at the time of administration. Effect of the Invention

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

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

[0018] <Treatment 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 area of ​​nerve damage, the area surrounding the area of ​​nerve damage, and areas compensating for the function of the area of ​​nerve damage. (Mode 2) When the formulation is a therapeutic agent for nasal administration, the stimulation is performed 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 previously known that stem cell culture supernatants can be useful for treating neurological disorders and the like (eg, Patent Document 3). However, as a result of the inventors' investigations, it was found that the therapeutic effect is particularly enhanced by combining 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 present inventor has made the unexpected discovery that the therapeutic effect is particularly high when stimulation is applied to the patient's nerves preferentially to one or more areas selected from the group consisting of the area of ​​nerve damage, the area surrounding the area of ​​nerve damage, and areas compensating for the function of the area of ​​nerve damage.

[0021] Furthermore, the present inventors have unexpectedly discovered 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 to which stimulation is applied and / or the timing of application of the stimulation as described above, the active ingredients, etc. contained in the culture supernatant are more easily transferred 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 causative 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 neuropathy.

[0025] In the present invention, "treatment of neurological disorders" means alleviating or completely curing various symptoms associated with neurological disorders (movement disorders, speech disorders, swallowing disorders, 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), WAB Aphasia Test, token test, and Mini-Mental State Examination (MMSE).

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

[0027] The composition 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 capable of differentiating into mesenchymal stem cells (hereinafter also referred to as "culture supernatant in 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 capable of differentiating 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 capable of differentiating into mesenchymal stem cells, the mixing ratio thereof is not particularly limited and is appropriately adjusted depending on the therapeutic effect to be obtained, etc.

[0029] The mesenchymal stem cells (MSCs) used in the present invention are not particularly limited with respect to the preparation method, tissue from which they are derived, etc., so 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, the term "cells capable of differentiating into mesenchymal stem cells" refers to cells capable of differentiating into mesenchymal stem cells by normal division and proliferation. Such cells have both the ability to self-replicate 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] The mesenchymal stem cells and cells that can be differentiated into mesenchymal stem cells may be derived from cells of the patient to which they are to be administered (autologous cells), or may be derived from cells other than the patient (allogeneic cells).

[0033] The mesenchymal stem cells may be cells induced to differentiate from ES cells, cells induced to differentiate from induced pluripotent stem cells (such as iPS cells), established cell lines, Muse cells (Multi-lineage differentiating Stress Euduring Cells), and the like.

[0034] As mesenchymal stem cells, cells that maintain 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 expression of various markers satisfies any of the following criteria. At least one of the following is positive: CD73, CD90, CD105, and CD200. At least one of the following is negative: CD19, CD34, CD45, CD74, CD79α, and HLA-DR.

[0036] 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 is no particular limitation on the mesenchymal stem cells, and any method known for preparing mesenchymal stem cells can be used. A preferred preparation method is the method described in Japanese Patent No. 4061487. This method includes the steps of adding fresh bone marrow cells to a culture dish and allowing the cells to adhere to and grow on the culture dish, and allowing a portion of the obtained cells to grow again on the culture dish.

[0039] The culture supernatant in 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] In 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 necessary from the resulting culture to obtain the culture supernatant of the present invention. Such processing may include commonly known steps such as removal of cells (eg, by filtration), concentration, freezing, drying, dilution, and the like.

[0043] The culture supernatant in 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 effects of the present invention are more easily achieved when the total amount of the culture supernatant is smaller (the higher the concentration of the soluble solids). 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 solids 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 solids content. Although not particularly limited, the soluble solid content may be 0.02 to 200 mg / mL.

[0045] Mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells may be isolated and cultured using commercially available kits.

[0046] In the therapeutic agent of the present invention, the dosage and frequency of administration of the culture supernatant of the present invention can be appropriately adjusted 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, and the like.

[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 may be repeated 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 medium (physiological buffer solution, sterile water, physiological saline, glucose solution, medium) and components known to be added to therapeutic agents for nasal administration (emulsifier, surfactant, stabilizer, etc.) may be added, if necessary, 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 medium (physiological buffer solution, sterile 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 an 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 medium (physiological buffer solution, sterile 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.

[0054] The method of preserving the therapeutic agent of the present invention is not particularly limited, and examples thereof include freezing, freeze-drying, refrigerated storage, etc. The frozen therapeutic agent is used for treatment by thawing. The freeze-dried therapeutic agent is used for treatment by dissolving in a medium (physiological buffer solution, sterile water, physiological saline, glucose solution, culture medium).

[0055] The therapeutic agent of the present invention preferably contains a solution in which the lyophilized culture supernatant of the present invention is dissolved. Examples of the solvent for the solution include physiological buffer solution, sterile water, physiological saline, glucose solution, and culture medium.

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

[0057] From the viewpoint of convenience, etc., 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) 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. (Requirement 2) When the preparation is a therapeutic agent for nasal administration, the stimulation is performed from before administration of the therapeutic agent to 16 hours after administration. (Requirement 3) When the preparation is a therapeutic agent for intravenous administration, the 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, the stimulation is performed from before administration of the therapeutic agent until 16 hours after administration.

[0059] When the administered culture supernatant of the present invention remains in or around an affected area (such as a nerve damage site in the brain), if 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 the therapeutic effect can be efficiently enhanced. Specific methods for achieving this are the above requirements 1 to 4.

[0060] When the therapeutic agent of the present invention is used in an embodiment that satisfies the above requirement 1, stimulation (such as motor stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, and higher brain function stimulation, which will be described later) is administered at any timing (preferably at a timing that satisfies any of requirements 2 to 4). By providing stimulation so as to satisfy the above requirement 1), the blood flow and metabolic rate of the affected area (such as the site of nerve damage) are preferentially increased compared to other nerve regions, and therefore the amount of the culture supernatant of the present invention that migrates to the affected area can be increased.

[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 of the therapeutic agent specified by these requirements. By providing a stimulus so as to satisfy any one of the above requirements 2 to 4, the amount of the culture supernatant of the present invention that transfers to the affected area (such as the brain) can be increased by increasing the blood flow to the affected area or the metabolic rate of the affected area at a timing when the remaining amount of the culture supernatant of the present invention in the blood or cerebrospinal fluid is large, thereby making it possible to increase the amount of the culture supernatant of the present invention that transfers to the affected area.

[0062] In the above requirements 2 to 4, "the stimulation is applied before the administration of the therapeutic agent" means that the start of the stimulation is before the start of the administration of the therapeutic agent. In the above requirements 2 to 4, "the stimulation is performed within n hours after administration of the therapeutic agent" means that the start of the stimulation is prior to the point n hours after the start of 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 to up to 10 hours after administration. In such a case, from the viewpoint of particularly facilitating the effect of the present invention, after the culture supernatant of the present invention is administered intranasally, the stimulation is preferably carried out within 3 hours after administration of the therapeutic agent.

[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 after 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, the reason why it is preferable to apply stimulation immediately after to one hour after nasal administration is as follows. The culture supernatant of the present invention usually increases in blood and cerebrospinal fluid concentrations immediately after administration, then rapidly decreases in blood and cerebrospinal fluid, respectively, within about 24 hours and about 12 hours, and reaches an undetectable level in the affected area (brain, etc.) 24 hours after administration. In the case of intranasal administration, in addition to via the blood, there is also an earlier and more direct route by which the culture supernatant passes into the cerebrospinal fluid, i.e., the culture supernatant passes through the nasal mucosa epithelial cells, reaches the cerebrospinal fluid around the olfactory nerve bundle, and then passes into the cerebrospinal fluid in the subarachnoid space. Through such a route, the cerebrospinal fluid concentration of the culture supernatant increases to several tens of times the blood concentration, for example, 15 to 30 minutes after administration. Since there is no substance transfer barrier 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 the brain tissue. Therefore, since the amount of drug in the extracellular fluid of brain tissue is highest immediately after intranasal administration to one hour later, 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 making the effects of the present invention more easily obtainable, the culture supernatant of the present invention may be administered intranasally before sleep (e.g., 0 to 3 hours before sleep), and stimulation may be performed after sleep (e.g., immediately after awakening to 5 hours after awakening). It is believed that the blood and cerebrospinal fluid concentrations of the culture supernatant (supernatant) increase when the subject sleeps. In addition, sleep stimulation facilitates resynthesis of neural circuits. For these reasons, the effects of the present invention can be more efficiently achieved by combining sleep stimulation after nasal administration.

[0067] In the above requirement 2, regardless of the start of stimulation, in order to make it easier for the culture supernatant to remain in the nasal cavity after nasal administration, the subject may be kept in a supine position for at least 15 to 30 minutes after administration. Also, the subject may be put into a sleep state for a short period of time (for example, within 1 hour) immediately after administration.

[0068] In the above requirement 2, particularly preferred embodiments are as follows. Immediately after the culture supernatant of the present invention is administered intranasally, an arbitrary stimulus (preferably, a magnetic stimulus in a lying position) is applied, for example, for 1 to 20 minutes. The subject may enter a sleep state during the application of the stimulus. After the application of the stimulus, another arbitrary stimulus (preferably, an exercise stimulus) may be further applied. If the patient is to transition to a sleep state during stimulation, it is preferable to administer the drug intranasally before falling asleep and to fall asleep as soon as possible (for example, within one hour after administration). In the above embodiment, intranasal administration may be performed once or more than once, for example, before entering a sleep state or immediately before application of a stimulus.

[0069] In the above requirement 3, from the viewpoint of facilitating the effect of the present invention, the stimulation is preferably performed 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 performed 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, the term "stimulation" refers to 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 appropriately adjusted 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 nerve of a patient" means that the stimulation is given to the whole or part of the patient's body, thereby causing an arbitrary reaction in the nerve. However, a mode in which stimulation is given to any tissue adjacent to the nerve is not excluded. Stimulation to a patient may be given at only one site on the nerve, or at multiple sites.

[0073] The stimulation of the present invention may be performed 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 may be simultaneously applied by one stimulation application means. For example, a voluntary movement-assisting electrical stimulation device may simultaneously apply motor stimulation, sensory stimulation (somatosensory stimulation), and electrical stimulation to a patient. If a robot-assisted training device (e.g., "Robot Suit HAL" (trademark)) is used in combination, motor stimulation and sensory stimulation may simultaneously be applied to a patient.

[0075] [Motor stimulation] In the present invention, the term "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, etc.). Examples of motor stimulation include those that combine neuromuscular facilitation techniques (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 those that combine not only motor stimulation but also sensory stimulation, electrical stimulation, and magnetic stimulation. In addition, by using a robot-assisted training device (for example, "Robot Suit HAL" (trademark)), repetitive motor and sensory stimulation can be given to the patient simultaneously.

[0076] For example, a preferred stimulation to be given to a patient with swallowing disorder 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] A motor stimulus is a stimulus to a motor neural pathway. Motor nerve 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, tectospinal tract, and corticobulbar tract. In addition, the motor nerve pathway synapses with lower motor neurons, and their axons extend as peripheral nerves and synapse with extrapyramidal muscle fibers, causing the target muscle to contract and produce 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 (sight, hearing, touch, 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, it includes sensations obtained from the skin, mucous membrane, 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 touch pressure, acupuncture, heat, weights, vibration, and the like.

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

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

[0083] Examples of heat treatments include the use of moxibustion (burning moxa), hot packs, and hydrotherapy.

[0084] As for the weight, a weight band for performing mild resistance exercise used in rehabilitation may be used.

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

[0086] In the present invention, the term "auditory stimulation" refers to a stimulation provided by a sound. The sound is not particularly limited, but may include a human voice, 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 characters, pictures, and images).

[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, excretion, grooming, bathing, reading, painting, etc.), prism adaptation, etc.

[0090] Sensory stimuli are usually stimuli to 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 sensation. The sensory neural pathways through which somatosensory sensation reaches the cerebral cortex have been elucidated. For example, the following pathways are known: Deep sensation / fine tactile sensation: Passing through the dorsal columns-medial ciliary tract system (receptor → primary neuron (enters the spinal cord, ascends the ipsilateral dorsal columns, and terminates in the ipsilateral dorsal column nucleus of the medulla oblongata) → secondary neuron (crosses over and ascends the contralateral medial lemniscus, terminating in the contralateral thalamic VPL) → tertiary neuron (contralateral cerebral cortical somatosensory cortex). Thermo-pain sensation and gross tactile sensation: travels through the spinothalamic tract (receptor → first-order neuron (enters the spinal cord and terminates in the posterior horn of the spinal cord) → second-order neuron (crosses over and 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, the term "electrical stimulation" refers to stimulation given electrically using an electric current. For example, the stimulation includes attaching an electrode to an affected area and exciting a neural circuit with electricity (low frequency, medium frequency, high frequency, interference wave, etc.).

[0092] The means for applying electrical stimulation to a patient is not particularly limited, but may include a method using a voluntary movement-assisted electrical stimulation device (IVES, etc.) or a method conventionally known as current stimulation therapy (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 usually 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" refers to 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, and 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 in order to control its functions) may be used. In addition, while the classic rTMS method such as low-frequency rTMS (1 Hz or less) which acts on inhibition and high-frequency rTMS (5 Hz or more) which acts on excitability are representative, "theta burst stimulation (TBS)" which performs burst stimulation consisting of three consecutive stimuli of 50 Hz at a frequency of 5 Hz may also be used. In intermittent TBS (iTBS), theta burst stimulation (three consecutive stimuli of 50 Hz at 5 Hz) is performed for 2 seconds and then rested for 8 seconds to increase the excitability of the motor cortex. In addition, continuous TBS (cTBS) suppresses the excitability of the motor cortex by continuously performing theta burst stimulation (three consecutive stimuli of 50 Hz at 5 Hz). In other words, TBS has an inhibitory effect when performed continuously and an excitatory effect when performed intermittently. TBS can be performed with a weaker stimulation intensity than low-frequency or high-frequency rTMS, and has a longer duration of action.

[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 the patient with language stimulation 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 involves 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 function" is a general term for mental (psychological) functions including cognitive processes (perception, memory, learning, thinking, judgment, etc.) and feelings (emotions) 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, and the like.

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

[0102] [Other stimuli] In the present invention, the "stimulus" includes not only the above but also any stimulus that can bring about a physiological change in a patient. For example, when the stimulation is performed from before administration of the therapeutic agent to up 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 the degree of a physiological change in the patient.

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

[0105] The 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 performed once or multiple times at any time within the period specified in the present invention, although it is not excluded that the stimulation may be performed after the period specified in the present invention.

[0109] When the stimulation is applied 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 stimulation until the blood flow and metabolism to the affected area (such as the nerve damage site) increase and the time for which the increase in blood flow and metabolism is maintained may differ. Therefore, it is preferable to adjust the timing of administration of the therapeutic agent so that it overlaps with the time when the blood flow and metabolism of the affected area are higher. Specifically, when the stimulation is sensory or linguistic stimulation, the time from the application of the stimulation to the increase in blood flow to the affected area or the increase in metabolic rate tends to be longer than when the stimulation is electrical or magnetic stimulation. Therefore, when the stimulation is a sensory or linguistic stimulation, it is preferable to set the time period between the application of the stimulation and the administration of the therapeutic agent to be longer, or to set the time period between the administration of the therapeutic agent and the application of the stimulation to be shorter. 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 areas compensating for the function of the area of ​​nerve damage, it does not exclude stimulation being applied to areas other than these areas.

[0112] In the present invention, "preferentially applied (to a specified area)" means that when stimulation begins, 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 damage, spinal cord damage, etc.) has occurred. Usually, the nerve damage site is the cause of nerve disorder.

[0114] In the present invention, "the vicinity of the nerve damage site" means not the nerve damage site itself but the area surrounding the nerve damage site (for example, the area surrounding the nerve damage site or the area adjacent to the nerve damage site).

[0115] In the present invention, "area compensating for the function of damaged nerve area" means an area that works to compensate for the function of damaged nerve area (for example, when the primary motor cortex is damaged, the right parietal lobe in the peripheral area of ​​the affected side and the primary motor cortex, premotor cortex, supplementary motor area, etc. on the healthy side).

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

[0117] The sites to which stimulation can be applied and the methods of 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 (part that controls the fingers), the "nerve damaged area" is the right primary motor cortex (part that controls the fingers). When the right primary motor cortex (the area that controls the fingers) suffers nerve damage, the "area surrounding the nerve damage" refers to the undamaged right primary motor cortex (other than the area that controls the fingers) - the premotor cortex, supplementary motor area, right primary sensory cortex, etc. In the case of nerve damage to the right primary motor cortex (the area that controls the fingers), the "areas that compensate for the function of the damaged nerve area" are the right parietal lobe, the left primary motor cortex / premotor cortex, supplementary motor area, etc.

[0119] If there is nerve damage to the right primary motor cortex (controlling the fingers), trying to move the paralyzed fingers of the left hand can prioritize stimulation of the damaged nerve area. If the right primary motor cortex (controlling the fingers) suffers from nerve damage, by attempting to move areas close to the paralyzed left fingers (the uninjured fingers of the left hand, the left wrist, etc.), it is possible to preferentially stimulate the area around the damaged nerves. In the event of nerve damage to the right primary motor cortex (the area controlling the fingers), by encouraging various tasks and gross movements using the paralyzed fingers of the left hand, it is possible to preferentially stimulate the areas that compensate for the function of the damaged nerve area.

[0120] [Examples of sensory stimulation] When nerve damage occurs to the right primary sensory cortex (tertiary neurons), the "nerve damaged area" is the right primary sensory cortex. When the right primary sensory cortex (tertiary neurons) is subjected to nerve damage, the "area surrounding the nerve damage" refers to the right primary sensory cortex to the right primary motor cortex, etc., which were spared from damage. When the right primary sensory cortex (tertiary neurons) suffers nerve damage, the "areas that compensate for the function of the damaged nerve 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), it is possible to preferentially stimulate the nerve-damaged area by applying sensory stimulation to the affected area with sensory impairment. When nerve damage occurs in the right primary sensory cortex (tertiary neurons), stimulation can be given to an area adjacent to the area of ​​sensory impairment, allowing preferential stimulation of 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 sensory impaired area or the surrounding area, or by simultaneously applying the same degree of sensory stimulation to the healthy side, it is possible to preferentially stimulate the area that compensates for the function of the nerve damaged area.

[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. In the case where the second finger on the right side has nerve damage, the "areas that compensate for the function of the damaged nerve area" are the right wrist, forearm, upper arm, shoulder, etc.

[0123] If the second finger on the right side has nerve damage, the damaged area can be stimulated preferentially by applying electrical stimulation to the second finger on the right side, which has the damaged nerve. If the second finger on the right side has nerve damage, the area around the nerve damage can be preferentially stimulated by applying electrical stimulation to the first, third, fourth, or fifth finger on the right side, which are adjacent to the nerve-damaged second finger. If nerve damage occurs in the second finger on the right side, electrical stimulation can be applied 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 damaged nerve.

[0124] [Example of magnetic stimulation] When nerve damage occurs to the right primary motor cortex (the area that controls the fingers), the "area that is nerve damaged" is the right primary motor cortex (the area that controls the fingers). When the right primary motor cortex (the area that controls the fingers) suffers nerve damage, the "area surrounding the nerve damage" refers to the undamaged right primary motor cortex (other than the area that controls the fingers) - the premotor cortex, supplementary motor area, right primary sensory cortex, etc. In the case of nerve damage to the right primary motor cortex (the area that controls the fingers), the "areas that compensate for the function of the damaged nerve area" are the right parietal lobe, the left primary motor cortex / premotor cortex, supplementary motor area, etc.

[0125] If the right primary motor cortex (controlling the fingers) suffers from nerve damage, 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 (controlling the fingers). In the event of nerve damage to the right primary motor cortex (area controlling the fingers), it is possible to preferentially stimulate the area surrounding the nerve damage by applying excitatory magnetic stimulation to the areas adjacent to the nerve damage, such as the right primary motor cortex (other than the area controlling the fingers) - the premotor cortex, supplementary motor area, and right primary sensory cortex. In the event of nerve damage to the right primary motor cortex (the area controlling the fingers), by applying excitatory magnetic stimulation to the right parietal lobe, it is possible to preferentially stimulate the area that compensates for the function of the damaged nerve area. 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 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 language stimulation] When nerve damage occurs in Wernicke's area, the "area of ​​nerve damage" is Wernicke's area. When Wernicke's area suffers nerve damage, the "area surrounding the nerve damage" refers to the Wernicke's area and Broca's area that were spared nerve damage, as well as the language circuit including the conductive pathway (arcuate fasciculus) connecting the two. When Wernicke's area suffers nerve damage, the "areas that compensate for the function of the damaged area" are the left angular gyrus (Brodmann's area 39), left supramarginal gyrus (Brodmann's area 40), cerebellum, thalamus, basal ganglia, etc.

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

[0128] [Example of stimulation of higher brain functions] 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 area" is the part of the brain that is at a level lower than memory and information processing and controls attention, concentration, inhibition, motivation, alertness, etc.

[0129] When executive dysfunction occurs due to nerve damage, executive function training can be performed to preferentially stimulate the damaged area of ​​the nerve. When executive dysfunction occurs due to nerve damage, it is possible to preferentially stimulate the area surrounding the nerve damage by performing memory and information processing training, which is at the lower level of executive function. 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 be used to preferentially stimulate the areas that compensate for the functions of the damaged nerves. EXAMPLES

[0130] The present invention will be described 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 according to the following method.

[0132] All of the following cultures were carried out for three weeks in an incubator at 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 In a similar manner to the above "(1) Culture of bone marrow-derived mesenchymal stem cells," a culture supernatant of adipose-derived mesenchymal stem cells was obtained using mesenchymal stem cells obtained from human adipose tissue. The 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 mesenchymal stem cells derived from dental pulp In a similar manner to the above "(1) Culture of bone marrow-derived mesenchymal stem cells," a culture supernatant of dental pulp-derived mesenchymal stem cells was obtained using mesenchymal stem cells obtained from human dental pulp tissue. The 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) Culture of umbilical cord-derived mesenchymal stem cells In a similar manner to the above "(1) Culture of bone marrow-derived mesenchymal stem cells," a culture supernatant of umbilical cord-derived mesenchymal stem cells was obtained using mesenchymal stem cells obtained from human umbilical cord tissue. The 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 various symptoms of neuropathy described below. Each patient was administered intranasally, intravenously, or subcutaneously. In addition, stimulation was administered (one of the following: motor stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, or higher brain function stimulation).

[0138] (Administration of culture supernatant) The culture supernatant was administered to each patient by each of 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 term "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 started 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 as shown in the section "culture supernatant" in each of Tables 1 to 15.

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

[0142] (2) Intravenous administration Each patient received 5 ml of culture supernatant intravenously 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 Stimulus) Details of the stimulation given to each patient are as follows:

[0145] [Motor stimulation] Repetitive facilitation therapy was administered to provide motor stimulation to a group of cerebral hemorrhage patients in their 40s and 50s. In this example, the areas to be stimulated were set to the damaged nerve area (left fingers, left foot to lower leg), the area surrounding the damaged nerve area (left wrist to forearm, left knee to thigh), and the areas compensating for the function of the damaged nerve area (left elbow to shoulder, left hip joint). The duration of each stimulation session was set at 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 each time) 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 an ice pack while also providing auditory stimulation (by speaking), and instructing them to visually confirm the position of the upper and lower limbs. In this example, the areas to be stimulated were set to the damaged nerve area (left fingers, left foot to lower leg), the area surrounding the damaged nerve area (left wrist to forearm, left knee to thigh), and the areas compensating for the function of the damaged nerve area (left elbow to shoulder, left hip joint). The duration of each stimulation session was set at 30 minutes for each site. The above sensory stimulation was applied to 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] In 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 whose limbs were paralyzed. In this example, the areas to be stimulated were set to the damaged nerve area (left fingers, left foot to lower leg), the area surrounding the damaged nerve area (left wrist to forearm, left knee to thigh), and the areas compensating for the function of the damaged nerve area (left elbow to shoulder, left hip joint). The stimulation time for each site was set to 20 minutes. 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 TMS device (CRTechnology), magnetic stimulation was applied to the heads of cerebral hemorrhage patients in their 40s and 50s. Specifically, intermittent TBS (iTBS; 1 burst 50Hz, 3 stimuli) was applied at 5Hz (time interval 200ms) at 80% of motor threshold intensity, with stimulation for 2 seconds and rest for 8 seconds, for a total of 2000 pulses. In this example, the areas where stimulation was applied were set to the damaged nerve area (the area controlled by the fingers of the affected primary motor field), the area surrounding the damaged nerve (other than the area controlled by the fingers of the affected primary motor field), and the area compensating for the function of the damaged nerve 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 were given placebo affected area magnetic stimulation (2000 pulses per session, approximately 11 minutes) that only emitted sound, and the culture supernatant was administered in the same manner as above. Note that the placebo affected area magnetic stimulation does not actually apply magnetic stimulation to the patient.

[0149] [Language stimulation] We administered language stimulation to patients with aphasia among cerebral hemorrhage patients in their 70s and 80s. The language stimulation consisted of communication tasks (reading, writing, listening, speaking, repetition, and calculation). The stimulation time was set to 60 min.

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

[0151] [Reference test] 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 was administered to the patients, the therapeutic effect for each symptom was evaluated using the following indexes, and the evaluation results were classified according to the following criteria. The results are shown in Table 1.

[0153] In addition, "after the longest test time has elapsed since administration of the culture supernatant" in each evaluation criterion 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 / 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 (the longest test time after administration of the 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 (the longest test time after administration of the culture supernatant). ○: A therapeutic effect was observed compared with the results of stimulation of the healthy side (the 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 with the results of placebo 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 affected side magnetic stimulation (the longest test time after administration of culture supernatant). △: A slight therapeutic effect was observed compared to the results of placebo magnetic stimulation on the affected side (the longest test time after administration of the 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, a 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 the soluble solid content was adjusted before administration. As described above, improvement of the symptoms of neuropathy was observed. However, the effect was strongest when the culture supernatant was not diluted, and when it was diluted, the effect was stronger at lower dilution rates.

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

[0177] In addition, when intranasal administration was performed, 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, regardless of the type of culture supernatant, and then motor stimulation was applied immediately thereafter (although this is not shown in the data).

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

Claims

1. A therapeutic agent for nasal administration for nerve disorders, comprising: The therapeutic agent is a therapeutic agent containing a culture supernatant, and is used in combination with stimulation of the nerves of a patient; The culture supernatant is a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into 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 performed from before administration of the therapeutic agent to 16 hours after administration. Therapeutic agent.

2. A therapeutic agent for intravenous administration for a neurological disorder, comprising: The therapeutic agent is a therapeutic agent containing a culture supernatant, and is used in combination with stimulation of the nerves of a patient; The culture supernatant is a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into 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 performed from before administration of the therapeutic agent to up to 3 hours after administration. Therapeutic agent.

3. A therapeutic agent for subcutaneous administration for nerve disorders, comprising: The therapeutic agent is a therapeutic agent containing a culture supernatant, and is used in combination with stimulation of the nerves of a patient; The culture supernatant is a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into 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 performed from before administration of the therapeutic agent to up to 4 hours after administration. Therapeutic agent.

4. The therapeutic agent according to any one of claims 1 to 3, wherein the culture supernatant is undiluted or concentrated in terms of soluble solids content.

5. The therapeutic agent according to claim 1 , wherein the culture supernatant is a lyophilized product which is dissolved at the time of administration.

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