Therapeutic agent for nerve disorders

Abstract

The present invention addresses the problem of providing a therapeutic agent that is more efficacious for treating nerve disorders, said therapeutic agent comprising a culture supernatant of mesenchymal stem cells. The present invention provides a therapeutic agent for nerve disorders, said therapeutic agent comprising a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells and being to be used together with stimulation of patient's nerves, wherein the stimulation is preferentially provided to one or more sites selected from the group consisting of a site of nerve damage, an area surrounding a site of nerve damage, and a site compensating for the function of a site of nerve damage. No Figure

Description

Treatment for neurological disorders

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

[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 a treatment of an injured area using a stem cell culture supernatant obtained by serum-free culture of dental pulp stem cells.

[0005] International Publication No. 2017 / 188457, JP 2013-508013 A, Japanese Patent No. 6296622

[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.

[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 nerve damage, the therapeutic agent comprising a culture supernatant of mesenchymal stem cells and / or cells that can be differentiated into mesenchymal stem cells, and used in combination with stimulating the nerves of a patient, the stimulation being preferentially given to one or more areas selected from the group consisting of the damaged nerve area, the area surrounding the damaged nerve area, and an area compensating for the function of the damaged nerve area.

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

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

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

[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.

[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.

[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 differentiate into mesenchymal stem cells, and is used in combination with stimulating the nerves of a patient. Its usage modes include the following four modes. The therapeutic agent of the present invention may have any one of the following four modes, or may have a combination of Mode 1 and any of Modes 2 to 4. (Mode 1) A mode in which stimulation is preferentially applied to one or more selected from the group consisting of the nerve damaged site, the area surrounding the nerve damaged site, and a site compensating for the function of the nerve damaged site. (Mode 2) A mode in which stimulation is applied from before administration of the therapeutic agent up to 16 hours after administration when the formulation is a therapeutic agent for nasal administration. (Mode 3) A mode in which stimulation is applied from before administration of the therapeutic agent up to 3 hours after administration when the formulation is a therapeutic agent for intravenous administration. (Mode 4) A mode in which stimulation is applied from before administration of the therapeutic agent up to 16 hours after administration when the formulation is a therapeutic agent for subcutaneous administration.

[0019] It has been known that stem cell culture supernatants can be useful in the treatment of neurological disorders and the like (e.g., Patent Document 3). However, as a result of investigations by the present inventors, it has been found that the therapeutic effect is particularly enhanced by combining the administration of culture supernatants of mesenchymal stem cells and / or cells that can differentiate 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 to which stimulation is applied and / or the timing of application of stimulation as described above, the active ingredients and the like 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.). The effectiveness of treatment for neuropathy was assessed using 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), and Fugl-Meyer Assessment (FMA). The assessment is based on known standards and methods such as the Standard Language Assessment (SLTA), the WAB Aphasia Test, the Token Test, and the 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] (Culture supernatant of mesenchymal stem cells and cells capable of differentiating 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 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 capable of differentiating into mesenchymal stem cells, or it 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 between them is not particularly limited and is 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 capable of differentiating into mesenchymal stem cells through normal division and proliferation. Such cells have both self-renewal ability and pluripotency, which allows them to differentiate into a variety of cells. Examples of cells capable of differentiating into mesenchymal stem cells include induced pluripotent stem cells (IPS cells) and embryonic stem cells (ES 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 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] 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), or the like.

[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 whose expression of various markers satisfies any of the following criteria: - positive for at least one selected from CD73, CD90, CD105, and CD200; - negative for at least one selected from 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 JP-A-2013-508013.

[0038] The mesenchymal stem cells are not particularly limited, and any known method 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 the dish and proliferate, and then allowing a portion of the obtained cells to proliferate 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 differentiate into mesenchymal stem cells include DMEM medium, RPMI1640 medium, HamF12 medium, and combinations thereof. The media 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 differentiate 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 cell removal (by filtration, etc.), concentration, freezing, drying, and dilution.

[0043] The culture supernatant of the present invention is preferably one obtained by removing cells 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 a 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 the effects of the present invention are to be achieved. Therefore, the culture supernatant of 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 differentiate into mesenchymal stem cells, or one that has been concentrated (by ultrafiltration, etc.) in terms of soluble solids 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 performed 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 the Therapeutic Agent) The therapeutic agent of the present invention can 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, 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, the culture supernatant of the present invention may be optionally blended with a vehicle (physiological buffer solution, sterile water, physiological saline, glucose solution, medium) or components known to be blended in therapeutic agents for intravenous administration (emulsifiers, surfactants, stabilizers, etc.) When the therapeutic agent of the present invention is a therapeutic agent for intravenous administration, it is usually administered via injection or infusion.

[0053] When the therapeutic agent of the present invention is a therapeutic agent for subcutaneous 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.

[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 (such as vials) 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 selected from the group consisting of the nerve damaged area, the area surrounding the nerve damaged area, and an area compensating for the function of the nerve damaged area. (Requirement 2) When the formulation is a therapeutic agent for nasal administration, the stimulation is applied from before administration of the therapeutic agent up to 16 hours after administration. (Requirement 3) When the formulation is a therapeutic agent for intravenous administration, the stimulation is applied from before administration of the therapeutic agent up to 3 hours after administration. (Requirement 4) When the formulation is a therapeutic agent for subcutaneous administration, the stimulation is applied from before administration of the therapeutic agent up to 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 at the affected area increases, the culture supernatant of the present invention will migrate to the affected area, thereby efficiently enhancing 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 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 applied 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 nerve damage site) increase preferentially 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 an embodiment that satisfies any one of the above requirements 2 to 4, stimulation (such as whole-body exercise, local exercise, or the motor, sensory, electrical, magnetic, verbal, and higher brain function stimulation described below) is administered within the administration period of the therapeutic agent specified by the requirement. By providing stimulation so as to satisfy any one of the above requirements 2 to 4, the amount of culture supernatant of the present invention that migrates to the affected area (such as the brain) can be increased by increasing the blood flow rate to the affected area or the metabolic rate in the affected area at a time when the remaining amount of the culture supernatant of the present invention in the blood or cerebrospinal fluid is high, thereby increasing the amount of the culture supernatant of the present invention that migrates 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 stimulation starts before the administration of the therapeutic agent starts. In the above requirements 2 to 4, "the stimulation is applied up to n hours after the administration of the therapeutic agent" means that the stimulation starts before n hours have elapsed since the administration of the therapeutic agent started.

[0063] In the above requirement 2, from the viewpoint of easily achieving the effects of the present invention, the stimulation is preferably performed from before administration of the therapeutic agent until 10 hours after administration. In such a case, from the viewpoint of easily achieving the effects of the present invention, the stimulation is preferably performed from the administration of the therapeutic agent until 3 hours after administration after nasal administration of the culture supernatant of the present invention.

[0064] In the above requirement 2, from the viewpoint of easily achieving the effects of the present invention, it is preferable to apply stimulation preferably 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, it is preferable to apply stimulation immediately to one hour after nasal administration for the following reasons. The administered culture supernatant of the present invention typically increases in blood and cerebrospinal fluid concentrations immediately after administration. The culture supernatant concentration then rapidly decreases over approximately 24 hours in the blood and approximately 12 hours in the cerebrospinal fluid, reaching undetectable levels in the affected area (e.g., brain) 24 hours after administration. In the case of nasal administration, the culture supernatant can be transferred to the cerebrospinal fluid via an earlier, more direct transfer route, in addition to the blood route. That is, the administered culture supernatant permeates 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 the blood concentration, for example, within approximately 15 to 30 minutes after administration. Since there is no 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 brain tissue. Therefore, the amount of drug in the extracellular fluid of brain tissue is highest immediately after intranasal administration to one hour after administration, and therefore, the effects of the present invention can be more efficiently achieved by applying stimulation at this time point.

[0066] In another embodiment of the above requirement 2, from the viewpoint of facilitating the achievement 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 stimulation may be performed after sleep (e.g., immediately after awakening to 5 hours after awakening). Sleeping is thought to increase the blood and cerebrospinal fluid concentrations of the culture supernatant (supernatant liquid). Furthermore, sleep stimulation facilitates resynthesis of neural circuits. For these reasons, the effects of the present invention can be achieved more efficiently by combining sleep stimulation after nasal administration.

[0067] In the above 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 as much as possible after administration. Also, the subject may be put into a sleep state for a short period (for example, 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, an arbitrary stimulus (preferably, a magnetic stimulus in a supine position) is applied, for example, over 1 to 20 minutes. The subject may transition to a sleep state during the application of the stimulus. After the application of the stimulus, another arbitrary stimulus (preferably, a motor stimulus) may be further applied. If the subject transitions to a sleep state during the application of the stimulus, it is preferable to perform nasal administration before falling asleep, and to fall asleep as quickly as possible (for example, within 1 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 the stimulus.

[0069] In the above requirement 3, 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 facilitating 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 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 patient's nerve" means that stimulation is applied to the whole or part of the patient's body to cause 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 applied 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 stimulation application means can simultaneously apply one or more of these stimuli. For example, a voluntary movement-assisted electrical stimulation device can simultaneously apply motor stimulation, sensory stimulation (somatosensory stimulation), and electrical stimulation to the 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 the patient.

[0075] [Motor Stimulation] In the present invention, "motor stimulation" refers to motor stimulation targeted at the affected area. However, motor stimulation targeted at the affected area does not exclude motor stimulation accompanied by whole-body exercise (such as exercise using a treadmill, etc.). Examples of motor stimulation include a combination of neuromuscular facilitation (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 constrained movement therapy on the non-paralyzed side (CI therapy: Constrained-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 (e.g., "Robot Suit HAL" (trademark)), repeated motor stimulation and sensory stimulation can be simultaneously applied to the patient.

[0076] For example, a preferred stimulation for a patient with dysphagia is a combination of any exercise stimulation and swallowing training. For a patient with dysarthria, a preferred stimulation is a combination of any exercise stimulation and articulation training.

[0077] Motor stimulation is stimulation of motor pathways. Motor pathways 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. Furthermore, motor pathways synapse with lower motor neurons, and their axons extend as peripheral nerves and synapse with extrapyramidal muscle fibers, contracting the target muscle and causing 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 motor and sensory pathways constantly interact with each other.

[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" collectively refers 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, the term "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] The means of providing visual stimulation to patients are not particularly limited, but include 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 stimuli are usually stimuli to the somatosensory system (sensations obtained from the skin, mucous membranes, joints, muscles, tendons, etc.). Somatosensory sensations are broadly divided into four modalities (pain, temperature, touch, and deep (proprioception) sensations), and specialized sensory receptors, nerve fibers, conduction pathways, etc. are used for each. The sensory neural pathways through which somatosensory sensations reach the cerebral cortex have been clarified, and the following pathways are known, for example: Deep sensation / fine touch sensation: Passes through the dorsal column-medial ciliary tract system (receptor → primary neuron (enters the spinal cord, ascends the ipsilateral dorsal column, and terminates in the ipsilateral dorsal column nucleus of the medulla oblongata) → secondary 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 → primary neuron (enters the spinal cord and terminates in the dorsal horn of the spinal cord) → secondary 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, "electrical stimulation" refers to stimulation applied electrically using an electric current. For example, this includes stimulation in which an electrode is attached to the affected area and a neural circuit is excited by electricity (low frequency, medium frequency, high frequency, interference wave, etc.).

[0092] The means for applying electrical stimulation to a patient is not particularly limited, and may include a method using a voluntary movement-assisted electrical stimulation device (such as IVES) or a method conventionally known as 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 with stimulation using low, medium, or high frequency waves, interference waves, etc. Exciting neural circuits with electrical stimulation can increase the pain threshold, as explained by gate control theory, and lower the motor threshold, making it easier to move the paralyzed limb.

[0094] [Magnetic Stimulation] In the present invention, the term "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 (for example, transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS) which stimulates with a weak current, and deep brain stimulation (DBS) which inserts electrodes deep into the brain and continuously applies electrical stimulation to the nervous system to control its functions) may be used. While typical TMS techniques include 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, "theta burst stimulation (TBS)," which involves a burst of three 50 Hz stimuli administered at a frequency of 5 Hz, may also be used. Intermittent TBS (iTBS) involves theta burst stimulation (5 Hz, three 50 Hz stimuli) administered for 2 seconds followed by an 8-second pause, which increases the excitability of the motor cortex. Continuous TBS (cTBS) involves continuous theta burst stimulation (5 Hz, three 50 Hz stimuli) which suppresses the excitability of the motor cortex. In other words, continuous TBS exerts an inhibitory effect, while intermittent TBS exerts an excitatory effect. TBS can be performed at a lower stimulation intensity than low-frequency or high-frequency rTMS and has the characteristic of having a longer duration of action.

[0096] [Linguistic Stimulus] In the present invention, the term "linguistic stimulus" refers to a stimulus given through 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 function" is a general term for mental (psychological) functions including cognitive processes (perception, memory, learning, thinking, judgment, etc.) and emotions (emotions) of actions. In the present invention, "higher brain function stimulation" refers to 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 target of stimulation is the frontal lobe function, higher brain function stimulation usually refers to stimulation at one of the stages in a pyramidal hierarchy (from the lowest level): arousal → inhibition / activation → attention and concentration → information processing → memory → executive function / logical thinking ("General Rehabilitation, May 2006 issue (Igaku-Shoin)").

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

[0103] [Confirmation and Evaluation of Stimulation] 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 (MRI) and single photon emission CT (SPECT).

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

[0108] [Timing of Stimulation] Stimulation may be performed once or multiple times at any time within the period specified in the present invention, although this does not exclude the possibility of stimulation being performed 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 to an increase in blood flow and metabolic rate to the affected area (e.g., nerve damage site) 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 a time when blood flow and metabolic rate in the affected area are higher. Specifically, when the stimulation is sensory stimulation or verbal stimulation, the time from application of stimulation to an increase in blood flow and metabolic rate in the affected area tends to be longer than when the stimulation is electrical stimulation or magnetic stimulation. Therefore, when the stimulation is sensory stimulation or verbal stimulation, it is preferable to set the time between application of stimulation and administration of the therapeutic agent longer, or to set the time between administration of the therapeutic agent and application of the stimulation shorter. On the other hand, when the stimulation is electrical stimulation or magnetic stimulation, it is preferable to set the time between application of stimulation and administration of the therapeutic agent shorter, and it is also possible to set the time between administration of the therapeutic agent and application of the stimulation longer.

[0111] [Area to which stimulation is applied] When stimulation is preferentially applied to one or more areas selected from the group consisting of the area of ​​nerve damage, the area around the area of ​​nerve damage, and areas that compensate for the function of the area of ​​nerve damage, it does not exclude the possibility of stimulation being applied to areas other than these areas.

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

[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 motor stimulation] When nerve damage occurs in the right primary motor cortex (part that controls the fingers), the "damaged nerve area" is the right primary motor cortex (part that controls the fingers). When nerve damage occurs in the right primary motor cortex (part that controls the fingers), the "surroundings of the damaged nerve area" are the undamaged right primary motor cortex (other than the part that controls the fingers) to the premotor cortex, supplementary motor area, right primary sensory area, etc. When nerve damage occurs in the right primary motor cortex (part 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 to the premotor cortex, supplementary motor area, etc.

[0119] If nerve damage occurs in the right primary motor cortex (the area that controls the fingers), it is possible to preferentially stimulate the nerve-damaged area by attempting to move the paralyzed fingers of the left hand.If nerve damage occurs in the right primary motor cortex (the area that controls the fingers), it is possible to preferentially stimulate the area around the nerve-damaged area by attempting to move areas close to the paralyzed fingers of the left hand (uninjured fingers of the left hand, left wrist, etc.).If nerve damage occurs in the right primary motor cortex (the area that controls the fingers), it is possible to preferentially stimulate areas that compensate for the function of the nerve-damaged area by encouraging various tasks and gross movements that use the paralyzed fingers of the left hand.

[0120] [Example of providing sensory stimulation] When the right primary sensory cortex (tertiary neurons) is damaged, the "damaged area" refers to the right primary sensory cortex. When the right primary sensory cortex (tertiary neurons) is damaged, the "surroundings of the damaged area" refers to the undamaged right primary sensory cortex to right primary motor cortex, etc. When the right primary sensory cortex (tertiary neurons) is damaged, the "areas compensating for the function of the damaged area" refers to the left primary sensory cortex, the secondary somatosensory cortex connected to the primary sensory cortex, the parietal association cortex, the motor cortex, the visual cortex, etc.

[0121] In the case of nerve damage to the right primary sensory cortex (tertiary neurons), providing sensory stimulation to the affected area with sensory impairment allows for preferential stimulation of the nerve-damaged area.In the case of nerve damage to the right primary sensory cortex (tertiary neurons), providing stimulation to an area close to the area with sensory impairment allows for preferential stimulation of the area surrounding the nerve-damaged area.In the case of nerve damage to the right primary sensory cortex (tertiary neurons), providing sensory stimulation to the area with sensory impairment or its surroundings by having the patient visually confirm it, or providing sensory stimulation to the same degree simultaneously with the healthy side, allows for preferential stimulation of the area that compensates for the function of the nerve-damaged area.

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

[0123] If the second finger on the right side has nerve damage, applying electrical stimulation to the damaged area of ​​the second finger on the right side can prioritize stimulation of the damaged area.If the second finger on the right side has nerve damage, applying electrical stimulation to the first, third, fourth, or fifth finger on the right side, which are adjacent to the damaged second finger, can prioritize stimulation of the area surrounding the damaged area.If the second finger on the right side has nerve damage, applying electrical stimulation to the right wrist, forearm, upper arm, or shoulder can prioritize stimulation of the area that compensates for the function of the damaged nerve.

[0124] [Example of application of magnetic stimulation] If the right primary motor cortex (part controlling the fingers) is damaged, the "damaged nerve area" is the right primary motor cortex (part controlling the fingers). If the right primary motor cortex (part controlling the fingers) is damaged, the "surroundings of the damaged nerve area" are the undamaged right primary motor cortex (other than the part controlling the fingers) to the premotor cortex, supplementary motor area, right primary sensory area, etc. If the right primary motor cortex (part controlling the fingers) is damaged, the "areas compensating for the function of the damaged nerve area" are the right parietal lobe, left primary motor cortex to the premotor cortex, supplementary motor area, etc.

[0125] In the event of nerve damage to the right primary motor cortex (the area controlling the fingers), it is possible to preferentially stimulate the damaged nerve 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).In the event of nerve damage to the right primary motor cortex (the area controlling the fingers), it is possible to preferentially stimulate the area surrounding the damaged nerve area by applying excitatory magnetic stimulation to the right primary motor cortex (other than the area controlling the fingers) - the premotor cortex, supplementary motor area, and right primary sensory cortex, which are areas close to the damaged nerve area.In the event of nerve damage to the right primary motor cortex (the area controlling the fingers), it is possible to preferentially stimulate the area that compensates for the function of the damaged nerve by applying excitatory magnetic stimulation to the right parietal lobe. Furthermore, 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 language stimulation] When Wernicke's area is damaged, the "area of ​​nerve damage" is Wernicke's area. When Wernicke's area is damaged, the "surroundings of the area of ​​nerve damage" are 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 area of ​​nerve damage" 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, providing sensory language stimulation can prioritize stimulation of the damaged area. When Wernicke's area is damaged, providing repetition or motor language stimulation can prioritize stimulation of the area surrounding the damaged area. When Wernicke's area is damaged, providing tasks such as phonology, vocabulary, grammar, reading comprehension, and arithmetic can prioritize stimulation of areas that compensate for the damaged area.

[0128] [Example of providing higher brain function stimulation] When executive dysfunction occurs due to nerve damage, the "area of ​​nerve damage" refers to the part of the brain that controls executive function. When executive dysfunction occurs due to nerve damage, the "area surrounding the nerve damage" refers to the part of the brain that controls memory and information processing, which are at a lower level than executive function. When executive dysfunction occurs due to nerve damage, the "area that compensates for the function of the nerve damage" refers to the part of the brain that controls attention, concentration, inhibition, motivation, alertness, etc., which are at an even lower level than memory and information processing.

[0129] When nerve damage causes executive dysfunction, executive function training can prioritize stimulation of the nerve damage area.When nerve damage causes executive dysfunction, memory and information processing training, which are at the lower level of executive function, can prioritize stimulation of the area surrounding the nerve damage.When nerve damage causes executive dysfunction, attention, concentration, inhibition, motivation, and alertness, which are at the lower level of memory and information processing, can prioritize stimulation of the area that compensates for the nerve damage.

[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] The following cultures were all carried out at a temperature of 37°C and CO ２ The cells were cultured in an incubator at a concentration of 5% for 3 weeks.

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

[0135] (3) Culturing of dental pulp-derived mesenchymal stem cells Using the same method as in "(1) Culturing 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 0.1-0.22 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) Culturing of Umbilical Cord-Derived Mesenchymal Stem Cells Using the same method as in "(1) Culturing 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 0.1-0.22 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 of Patients> The culture supernatant prepared above was administered to patients with the following symptoms of neurological disorders. Each patient was administered either intranasally, intravenously, or subcutaneously. In addition, stimulation (one of motor stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, and higher brain function stimulation) was also administered.

[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. "n hours before administration" means that the stimulation started n hours before the administration of the culture supernatant started. "Immediately after administration" means that the stimulation started immediately after the administration of the culture supernatant (any time within 15 minutes after the administration of the culture supernatant). "n hours after administration" means that the stimulation started n hours after the administration of the culture supernatant started.

[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 into both nasal cavities using a standard nasal dropper. Administration was performed daily for 14 weeks. Nasal administration was performed immediately after administration, 30 minutes after administration, 3 hours, 10 hours, and 20 hours after administration. Two patients were administered the same nasal dose, with and without a 1-hour sleep interval.

[0142] (2) Intravenous Administration: Each patient received 5 ml of culture supernatant by 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 surface of the upper arm every 7 days for 4 weeks.

[0144] (Application of 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 as motor stimulation. In this example, stimulation was administered to the nerve damage site (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 site (left elbow to shoulder, left hip). The stimulation duration per 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 per session) and were administered the culture supernatant in the same manner as above.

[0146] Sensory Stimulation: A group of patients in their 40s and 50s with cerebral hemorrhage who had sensory impairments received sensory stimulation of the upper and lower limbs. The patients were given cooling stimuli using ice packs on the paralyzed limbs, auditory stimuli (verbal prompts), and instructions to visually confirm the position of the upper and lower limbs. In this example, stimulation was administered to the nerve damage site (left fingers, left foot to lower leg), areas surrounding the nerve damage (left wrist to forearm, left knee to thigh), and areas compensating for the nerve damage site (left elbow to shoulder, left hip). Each stimulation session lasted 30 minutes. The above sensory stimulation was administered to the affected upper and lower limbs. In addition, a group of patients in their 40s and 50s with similar symptoms received 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] Low-frequency stimulation was applied to the paralyzed upper and lower limbs of a group of cerebral hemorrhage patients in their 40s and 50s via electrodes attached to the limbs. In this example, stimulation was applied to the nerve damage site (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 nerve damage (left elbow to shoulder, left hip). The stimulation duration per session was set to 20 minutes for each site. The above electrical stimulation was applied to the affected upper and lower limbs. Furthermore, electrical stimulation (20 minutes per session) was also applied to the healthy upper and lower limbs of a group of patients in their 40s and 50s with similar symptoms, 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 a group of cerebral hemorrhage patients in their 40s and 50s. Specifically, intermittent TBS (iTBS; 1 burst 50 Hz, 3 stimuli) was administered at 5 Hz (time interval 200 ms) at 80% motor threshold intensity, with stimulation for 2 seconds followed by an 8-second pause, for a total of 2000 pulses. In this example, stimulation was administered to the affected nerve area (the area innervating the primary motor field on the affected side), the area surrounding the nerve injury (other than the area innervating the primary motor field on the affected side), and the area compensating for the function of the nerve injury (the right parietal lobe). The stimulation duration per session was set at 2000 pulses per area, approximately 11 minutes. The above magnetic stimulation was administered to the affected temporal region. In addition, a group of patients in their 40s and 50s with similar symptoms received 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] Language stimulation was administered to patients with aphasia among a group of cerebral hemorrhage patients in their 70s and 80s. Language stimulation consisted of communication tasks (reading, writing, listening, speaking, repetition, and calculation). The stimulation duration was set to 60 minutes.

[0150] [Higher brain function stimulation] Higher brain function stimulation was administered to patients in their 70s and 80s who had suffered from cerebral hemorrhage and were diagnosed with higher brain function disorders (attention disorders). Attention process training was administered as the higher brain function stimulation. The stimulation duration was set to 60 minutes.

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

[0152] <Therapeutic Effect on Neuropathy> After each treatment was administered to the patient, the therapeutic effect for each symptom was evaluated using the index below, and the evaluation results were classified according to the criteria below. 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 / sensory impairment: SIAS (Stroke Impairment Assessment), FMA (Fugl-Meyer Assessment) Cerebral infarction: NIHSS (National Institutes of Health Stroke Scale) Aphasia: Standardized Lung Test for Aphasia (SLTA) Higher brain function: MMSE (Mini-Mental State Examination)

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

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

[0157] (Evaluation criteria - magnetic stimulation) ◎: A significant therapeutic effect was observed compared to the results of placebo patient-side magnetic stimulation (after the longest test time has elapsed since administration of the culture supernatant). ○: A therapeutic effect was observed compared to the results of placebo patient-side magnetic stimulation (after the longest test time has elapsed since administration of the culture supernatant). △: A slight therapeutic effect was observed compared to the results of placebo patient-side magnetic stimulation (after the longest test time has elapsed since 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.

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[0160]

[0161]

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[0163]

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[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. The same improvement in symptoms of neuropathy was observed. However, the effect was greatest when the culture supernatant was not diluted. Furthermore, when the culture supernatant was diluted, the effect was greater at lower dilution rates.

[0176] When nasal administration was performed, the therapeutic effect tended to be higher when stimulation was performed 5 to 30 minutes after nasal 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 nasal administration of the culture supernatant, and then motor stimulation was applied immediately thereafter, 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 administration tended to be more effective than falling asleep over more than one hour.

Claims

1. A therapeutic agent for neuropathy, wherein the therapeutic agent comprises a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells, and is used in combination with the application of a stimulus to the nerves of a patient, and the stimulus is preferentially applied to one or more selected from the group consisting of the nerve injury site, the periphery of the nerve injury site, and the site compensating for the function of the nerve injury site.

2. A therapeutic agent for intranasal administration of neuropathy, wherein the therapeutic agent comprises a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells, and is used in combination with the application of a stimulus to the nerves of a patient, and the application of the stimulus is performed from before the administration of the therapeutic agent to 16 hours after the administration.

3. A therapeutic agent for intravenous administration of neuropathy, wherein the therapeutic agent comprises a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells, and is used in combination with the application of a stimulus to the nerves of a patient, and the application of the stimulus is performed from before the administration of the therapeutic agent to 3 hours after the administration.

4. A therapeutic agent for subcutaneous administration of neuropathy, wherein the therapeutic agent comprises a culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells, and is used in combination with the application of a stimulus to the nerves of a patient, and the application of the stimulus is performed from before the administration of the therapeutic agent to 16 hours after the administration.

5. The therapeutic agent according to any one of claims 1 to 4, wherein the stimulus is one or more selected from the group consisting of motor stimulus, sensory stimulus, electrical stimulus, magnetic stimulus, language stimulus, and higher brain function stimulus.

6. The therapeutic agent according to any one of claims 1 to 5, wherein the culture supernatant is undiluted or concentrated with respect to the soluble solid content.

7. The therapeutic agent according to any one of claims 1 to 5, wherein the culture supernatant is used by dissolving its lyophilized product at the time of administration.