Therapeutic agents 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 neuropathy.

Background Art

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

[0003] Patent Documents 1 and 2 describe that the combined use of administration of mesenchymal stem cells and rehabilitation can improve the therapeutic effect for diseases such as neurological diseases.

[0004] Patent Document 3 describes the treatment of damaged parts using a stem cell culture supernatant obtained by culturing dental pulp stem cells in serum-free medium.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, there is a further need to improve the therapeutic effect for neuropathy.

[0007] The present invention has been made in view of the above circumstances, and an object thereof is to provide a therapeutic agent containing a culture supernatant of mesenchymal stem cells, which is more effective for the treatment of neuropathy.

Means for Solving the Problems

[0008] The inventors of the present invention have found that the above problems can be solved by combining the administration of the culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells with the application of stimulation to the nerves of patients, and by adjusting the timing of the application of the stimulation and the site to which the stimulation is applied, and have thus completed the present invention. More specifically, the present invention provides the following.

[0009] (1) A therapeutic agent for nerve disorders, wherein the therapeutic agent is a therapeutic agent containing the 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 stimulation to the nerves of a patient, and the stimulation 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. Therapeutic agent.

[0010] (2) A therapeutic agent for intranasal administration of nerve disorders, wherein the therapeutic agent is a therapeutic agent containing the 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 stimulation to the nerves of a patient, and the application of the stimulation is performed from before the administration of the therapeutic agent to 16 hours after the administration. Therapeutic agent.

[0011] (3) A therapeutic agent for intravenous administration of nerve disorders, wherein the therapeutic agent is a therapeutic agent containing the 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 stimulation to the nerves of a patient, and the application of the stimulation is performed from before the administration of the therapeutic agent to 3 hours after the administration. Therapeutic agent.

[0012] (4) A therapeutic agent for subcutaneous administration of nerve disorders, wherein the therapeutic agent is a therapeutic agent containing the 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 stimulation to the nerves of a patient, and the application of the stimulation is performed from before the administration of the therapeutic agent to 16 hours after the administration. Therapeutic agent.

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

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

[0015] (7) The culture supernatant is used by dissolving its lyophilized product at the time of administration, and the therapeutic agent according to any one of (1) to (5). [Advantages of the Invention]

[0016] According to the present invention, there is provided a therapeutic agent containing a culture supernatant of mesenchymal stem cells, which is effective for the treatment of nerve disorders. [Modes for Carrying Out the Invention]

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

[0018] [Therapeutic Agent] The therapeutic agent for treating nerve disorders of the present invention (hereinafter, also referred to as "the therapeutic agent of the present invention") is a preparation containing 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 in its usage mode, includes 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 one of modes 2 to 4. (Mode 1) A mode in which 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. (Mode 2) When the preparation is a therapeutic agent for nasal administration, a mode in which the application of the stimulus is performed from before the administration of the therapeutic agent to 16 hours after the administration. (Aspect 3) When the preparation is a therapeutic agent for intravenous administration, the aspect in which the stimulation is applied from before the administration of the therapeutic agent to 3 hours after the administration. (Aspect 4) When the preparation is a therapeutic agent for subcutaneous administration, the aspect in which the stimulation is applied from before the administration of the therapeutic agent to 16 hours after the administration.

[0019] It has been conventionally known that the stem cell culture supernatant may be useful for the treatment of neurological diseases and the like (for example, Patent Document 3). However, as a result of the studies by the present inventors, it has been found that the combined use of the administration of the culture supernatant of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells and the stimulation of the patient's nerves particularly enhances the therapeutic effect.

[0020] Furthermore, the present inventors have also found an unexpected finding that the therapeutic effect is particularly high when the stimulation applied to the patient's nerves 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.

[0021] Furthermore, the present inventors have also found an unexpected finding that the therapeutic effect can be further enhanced by adjusting the timing of the stimulation according to the administration route of the culture supernatant.

[0022] The reason why the therapeutic effect of neuropathy is enhanced by the present invention is presumably that by adjusting the site of stimulation and / or the timing of stimulation as described above, the active ingredients and the like contained in the culture supernatant easily migrate to the affected area (nerve injury site, etc.), and the effect of the culture supernatant is promoted.

[0023] In the present invention, "neuropathy" means any disorder that impairs the nerve itself or its function, and the causative disease, the nerve injury site, etc. are not particularly limited.

[0024] The causative disease of neuropathy is not particularly limited, and any disease that causes neuropathy can be mentioned. Examples of these diseases include cerebrovascular diseases, brain tumors, encephalitis, dementia, neurodegenerative diseases, spinal cord injuries, myelitis, intervertebral disc hernias, and other central nervous system diseases and peripheral nerve disorders.

[0025] In the present invention, "treatment of neuropathy" means alleviation or complete cure of various symptoms associated with neuropathy (such as motor disorders, dysarthria, dysphagia, higher brain function disorders, dementia, aphasia, Parkinson's syndrome, ataxia, sensory disorders, pain, coldness, numbness, flushing, etc.). Whether the therapeutic effect of neuropathy is achieved is evaluated based on known criteria and methods such as NIHSS (National Institutes of Health Stroke Scale), mRS (modified Rankin scale), AIS (ASIA Impairment Scale), Frankel classification, SIAS (Stroke Impairment Assessment), BRS (Brunnstrom stage), FMA (Fugl Meyer Assessment), MMT (manual muscle testing), FMA (Fugl Meyer Assessment), standard aphasia examination (SLTA), WAB aphasia examination, token test, MMSE (Mini-Mental State Examinaton), etc.

[0026] In the present invention, "patient" means any organism suffering from neuropathy. For example, mammals such as humans, monkeys, cows, horses, pigs, dogs, cats, etc., birds, reptiles, and other any pet animals, etc. can be mentioned.

[0027] Hereinafter, the composition of the therapeutic agent of the present invention will be described in detail.

[0028] (Mesenchymal stem cells, culture supernatant of cells capable of differentiating into mesenchymal stem cells) The therapeutic agent of the present invention contains mesenchymal stem cells and / or the culture supernatant of 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 the culture supernatant of mesenchymal stem cells and the 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 the culture supernatant of mesenchymal stem cells and the culture supernatant of cells capable of differentiating into mesenchymal stem cells, their mixing ratio is not particularly limited and is appropriately adjusted according to the therapeutic effect to be obtained and the like.

[0029] In the present invention, the mesenchymal stem cells (Mesenchymal Stem Cell, MSC) used are somatic stem cells derived from mesenchyme and having self-renewal ability and differentiation ability, and their preparation method, the tissue from which they are derived, etc. are not particularly limited.

[0030] In the present invention, the "cells capable of differentiating into mesenchymal stem cells" means cells that can be differentiated into mesenchymal stem cells by normal cell division and proliferation. Such cells have both self-renewal ability and pluripotency to differentiate into various cells. Examples of cells capable of differentiating into mesenchymal stem cells include IPS cells (Induced Pluriopotent Stem Cells), ES cells (Embryonic Stem Cells), and the like.

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

[0032] The mesenchymal stem cells and the cells capable of differentiating into mesenchymal stem cells may be derived from the cells (autologous cells) of the patient to be administered, or may be derived from the cells (allogeneic cells) other than the patient.

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

[0034] As mesenchymal stem cells, cells maintaining an undifferentiated state with negative differentiation markers (such as CD24, etc.) are usually used.

[0035] Mesenchymal stem cells may satisfy any of the following in terms of various marker expressions. · At least one selected from CD73, CD90, CD105, and CD200 is positive. · At least one selected from CD19, CD34, CD45, CD74, CD79α, and HLA-DR is negative.

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

[0037] Examples of mesenchymal stem cells include those reported to be applicable to the treatment of various diseases, such as mesenchymal stem cells described in International Publication No. 2017 / 188457, International Publication No. 2009 / 002503, Japanese Patent Publication No. 2013-508013, etc.

[0038] Although not particularly limited as mesenchymal stem cells, any method known as a method for preparing mesenchymal stem cells can be adopted. Preferred preparation methods include the method described in Japanese Patent No. 4061487. This method includes steps of adding fresh bone marrow cells to a culture dish and allowing them to adhere and proliferate on the culture dish, and further proliferating a part of the obtained cells 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 capable of differentiating into mesenchymal stem cells.

[0040] When obtaining the culture supernatant in the present invention, the medium and culture conditions used are not particularly limited and can be appropriately selected according to the type of cells such as mesenchymal stem cells and cells capable of differentiating into mesenchymal stem cells.

[0041] Examples of the medium used for culturing mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells include DMEM medium, RPMI1640 medium, HamF12 medium, and combinations thereof. The medium may contain components used in the culture of stem cells (various sera, bovine serum albumin, antibiotics, vitamins, minerals, etc.).

[0042] After culturing mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells, the cells are appropriately processed from the obtained culture as needed, and the culture supernatant in the present invention is obtained. Such treatments include commonly known steps such as cell removal (by filtration, etc.), concentration, freezing, drying, dilution, etc.

[0043] The culture supernatant in the present invention is preferably obtained by removing cells from the culture of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells.

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

[0045] The isolation and culture of mesenchymal stem cells and / or cells capable of differentiating into mesenchymal stem cells may also be carried out using a commercially available kit.

[0046] In the therapeutic agent of the present invention, the dosage and the number of administrations of the culture supernatant in the present invention can be appropriately adjusted according to the effect to be obtained and the condition of the patient to be administered (age, weight, degree of symptoms, etc.).

[0047] The therapeutic agent of the present invention can adjust the time required for each administration according to the administration method of the culture supernatant in the present invention, the number of administrations, and the like.

[0048] The frequency of implementation of the combination of administration and stimulation of the culture supernatant according to the present invention is not particularly limited, but each may be performed once, or may be repeated two or more times. When repeated two or more times, it may be carried out over several weeks (for example, one week) or more to several months (for example, 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) Conventionally known components can be incorporated into the therapeutic agent of the present invention according to the administration method and the like.

[0050] The therapeutic agent of the present invention can 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, together with the culture supernatant in the present invention, a medium (physiological buffer, sterilized water, physiological saline, glucose solution, medium) and components known to be incorporated into a therapeutic agent for nasal administration (emulsifier, surfactant, stabilizer, etc.) may be incorporated as required.

[0052] When the therapeutic agent of the present invention is a therapeutic agent for intravenous administration, together with the culture supernatant in the present invention, a medium (physiological buffer, sterilized water, physiological saline, glucose solution, medium) and components known to be incorporated into a therapeutic agent for intravenous administration (emulsifier, surfactant, stabilizer, etc.) may be incorporated as required. When the therapeutic agent of the present invention is a therapeutic agent for intravenous administration, it is usually administered via injection or drip.

[0053] When the therapeutic agent of the present invention is a therapeutic agent for subcutaneous administration, together with the culture supernatant in the present invention, a medium (physiological buffer solution, sterilized water, physiological saline, glucose solution, culture medium), and components known to be formulated in a therapeutic agent for intravenous administration (emulsifying agent, surfactant, stabilizer, etc.) may be formulated as necessary.

[0054] The method for storing the therapeutic agent of the present invention is not particularly limited, and examples include frozen storage, freeze-drying, and refrigerated storage. The therapeutically agent stored frozen is thawed and used for treatment. The therapeutically agent that has been freeze-dried is dissolved in a medium (physiological buffer solution, sterilized water, physiological saline, glucose solution, culture medium) and used for treatment.

[0055] The therapeutic agent of the present invention preferably contains a solution obtained by dissolving a freeze-dried product of the culture supernatant in the present invention. Examples of the solvent of the solution include physiological buffer solution, sterilized water, physiological saline, glucose solution, culture medium, etc.

[0056] The therapeutic agent of the present invention may be divided into small portions in a container (such as a vial) for each single-dose or multiple-dose.

[0057] From the viewpoint of convenience, etc., the culture supernatant in the present invention is preferably one that has been frozen and stored in a state of being divided into small portions in a container for each dose.

[0058] (Stimulation) The therapeutic agent of the present invention is used in combination with the application of a stimulus to the patient's nerve, and the application of the stimulus satisfies one of the following four requirements, or a combination of requirement 1 and any one of requirements 2 to 4. (Requirement 1) 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. (Requirement 2) When the preparation is a therapeutic agent for nasal administration, the application of the stimulus is performed from before the administration of the therapeutic agent to 16 hours after the administration. (Requirement 3) When the preparation is a therapeutic agent for intravenous administration, the application of the stimulus is performed from before the administration of the therapeutic agent to 3 hours after the administration. (Requirement 4) When the preparation is a therapeutic agent for subcutaneous administration, the stimulation is performed from before the administration of the therapeutic agent to 16 hours after the administration.

[0059] While the culture supernatant in the present invention that has been administered remains in the affected area (nerve injury site such as the brain, etc.) or its vicinity, if the blood flow volume to the affected area or the like increases or the metabolic amount in the same part increases, the culture supernatant in the present invention migrates to the affected area, and the therapeutic effect can be efficiently enhanced. The specific methods are the above Requirements 1 to 4.

[0060] When using the therapeutic agent of the present invention in a manner that satisfies the above Requirement 1, stimulation (exercise stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, and higher brain function stimulation, etc. described later) is applied at an arbitrary 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 volume of the affected area (nerve injury site, etc.) and the metabolic amount in the same part increase preferentially over other nerve regions, so the amount of the culture supernatant in the present invention that migrates to the affected area can be increased.

[0061] When using the therapeutic agent of the present invention in a manner that satisfies any of the above Requirements 2 to 4, stimulation (whole body movement, local movement, and exercise stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, and higher brain function stimulation, etc. described later) is applied within the administration period defined by these requirements of the therapeutic agent. By applying stimulation so as to satisfy any of the above Requirements 2 to 4, at a timing when the remaining amount of the culture supernatant in the present invention in the blood or cerebrospinal fluid is large, the blood flow volume to the affected area (brain, etc.) increases or the metabolic amount in the same part increases, so the amount of the culture supernatant in the present invention that migrates to the affected area can be increased.

[0062] In the above Requirements 2 to 4, "the stimulation is performed before the administration of the therapeutic agent" means that the start time of the stimulation is before the start time of the administration of the therapeutic agent. In the above Requirements 2 to 4, "the stimulation is performed within n hours after the administration of the therapeutic agent" means that the start time of the stimulation is before the time point when n hours have elapsed from the start time of the administration of the therapeutic agent.

[0063] In the above-mentioned requirement 2, from the viewpoint that the effects of the present invention are likely to be achieved, the stimulation is preferably performed from before the administration of the therapeutic agent to 10 hours after the administration. In such a case, particularly from the viewpoint that the effects of the present invention are likely to be achieved, after the culture supernatant in the present invention is administered intranasally, the stimulation is preferably performed from the administration of the therapeutic agent to 3 hours after the administration.

[0064] In the above-mentioned requirement 2, from the viewpoint that the effects of the present invention are likely to be achieved, preferably at the time point immediately after to 1 hour after intranasal administration, more preferably at the time point 5 minutes to 1 hour after intranasal administration, and most preferably at the time point 30 minutes to 1 hour after intranasal administration, it is preferable to perform the stimulation.

[0065] In the above-mentioned requirement 2, the reason why it is preferable to perform the stimulation at the time point immediately after to 1 hour after intranasal administration is as follows. The culture supernatant in the present invention that has been administered usually has an increased blood concentration and cerebrospinal fluid concentration immediately after administration. Subsequently, the culture supernatant concentration rapidly decreases in the blood for about 24 hours and in the cerebrospinal fluid for about 12 hours, and decreases to a level that cannot be detected in the affected part (such as the brain) 24 hours after administration. In the case of intranasal administration, as a route for the culture supernatant to migrate into the cerebrospinal fluid, in addition to passing through the blood, there is also a more direct migration route at an earlier stage, that is, after the administered culture supernatant penetrates the nasal mucosal epithelial cells, it reaches the cerebrospinal fluid around the olfactory nerve bundle and further migrates to the cerebrospinal fluid in the subarachnoid space. Through such a route, the culture supernatant, for example, has a cerebrospinal fluid concentration that is several tens of times or more higher than the blood concentration about 15 to 30 minutes after administration. Since there is no migration barrier for substances between the cerebrospinal fluid and the brain tissue, the amount of the drug (culture supernatant) in the cerebrospinal fluid can correspond to the amount of the drug (culture supernatant) in the extracellular fluid of the brain tissue. Therefore, the time point immediately after to 1 hour after intranasal administration is the timing when the amount of the drug in the extracellular fluid of the brain tissue is the highest. Therefore, by performing the stimulation at this time point, the effects of the present invention can be achieved more efficiently.

[0066] In another aspect of the above requirement 2, from the viewpoint that the effects of the present invention are likely to be achieved, the culture supernatant in the present invention may be administered intranasally before sleep (for example, 0 to 3 hours before sleep), and stimulation may be applied after sleep (for example, immediately after waking up to 5 hours after waking up). It is considered that the blood concentration and cerebrospinal fluid concentration of the culture supernatant (supernatant) increase by taking sleep. In addition, resynthesis of neural circuits is likely to occur by sleep stimulation. From the above, by combining sleep stimulation after intranasal administration, the effects of the present invention can be achieved more efficiently.

[0067] In the above requirement 2, in order to make it as easy as possible to retain the culture supernatant in the nasal cavity after intranasal administration regardless of the start of stimulation, the lying position may be maintained for at least about 15 minutes to 30 minutes after administration. Also, a short sleep state (for example, within 1 hour) may be shifted to immediately after administration.

[0068] In the above requirement 2, a particularly preferred embodiment is as follows. Any stimulation (preferably magnetic stimulation in a lying position) is applied over, for example, 1 to 20 minutes immediately after intranasal administration of the culture supernatant in the present invention. During the stimulation, a transition to a sleep state may occur. After the stimulation, another arbitrary stimulation (preferably exercise stimulation) may be further applied. When a transition to a sleep state occurs during the stimulation, it is preferable to perform intranasal administration before falling asleep and fall asleep as soon as possible (for example, within 1 hour after administration). In the above embodiment, the intranasal administration may be performed once or two or more times. For example, it may be performed before the transition to the sleep state or immediately before the stimulation.

[0069] In the above requirement 3, from the viewpoint that the effects of the present invention are likely to be achieved, the stimulation is preferably performed from before the administration of the therapeutic agent to 1 hour after the administration.

[0070] In the above requirement 4, from the viewpoint that the effects of the present invention are likely to be achieved, the stimulation is preferably performed from before the administration of the therapeutic agent to 10 hours after the administration, more preferably 4 hours after the administration, and even more preferably up to 3 hours after the administration.

[0071] In the present invention, "stimulation" means something that brings about a physiological change (at least one, preferably two or more of electrical changes, blood flow changes, metabolic changes, etc.) to the site where the stimulation is applied. In the present invention, the intensity of the stimulation to be applied can be appropriately adjusted according to the effect to be obtained and the condition of the patient (age, weight, degree of symptoms, etc.) who is the administration target.

[0072] In the present invention, "applying stimulation to the nerves of a patient" means that applying the stimulation causes an arbitrary reaction in the nerves by applying the stimulation to the whole or a part of the patient's body. However, a mode in which the stimulation is also applied to any tissue adjacent to the nerves is not excluded. The stimulation to the patient may be performed at only one location in the nerves or at a plurality of locations.

[0073] The stimulation of the present invention may be performed at any one or more time points before, during, or after the 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 realize the stimulation to the nerves. From the viewpoint of being likely to enhance the therapeutic effect according to 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 a plurality of these stimulations. For example, according to a voluntary movement assistance type electrical stimulation device, motor stimulation, sensory stimulation (somatosensory stimulation), and electrical stimulation can be simultaneously applied to the patient. If a robot-assisted training device (for example, "Robot Suit HAL" (trademark)) is used in combination, motor stimulation and sensory stimulation can be simultaneously applied to the patient.

[0075] [Motor Stimulation] In the present invention, "motor stimulation" means motor stimulation targeted at the affected part. However, as the motor stimulation targeted at the affected part, motor stimulation accompanied by whole body movement (such as movement using a treadmill, etc.) is not excluded. As movement stimuli, in order to increase the amount of stimulation to the target nerve pathway, there are modes of combined use of neuro-muscular facilitation methods (PNF method, Brunnstrom method, Bobath method, etc.), Kawahira method (facilitation repetitive therapy), modes of concentrated repetitive movement stimuli such as Arm Basisis training, modes of forcibly giving movement stimuli such as non-paralyzed side constraint movement therapy (CI therapy: Constrain-Induced Movement Therapy), and modes of combining the stimuli to be given with not only movement stimuli but also sensory stimuli, electrical stimuli, and magnetic stimuli. In addition, by using a robot-assisted training device (for example, "Robot Suit HAL" (trademark)), repetitive movement stimuli and sensory stimuli can be simultaneously given to the patient.

[0076] For example, as stimuli given to patients with dysphagia, the combined use of arbitrary movement stimuli and swallowing training is mentioned as a preferable stimulus. For patients with dysarthria, the combined use of arbitrary movement stimuli and articulation training is mentioned as a preferable stimulus.

[0077] Movement stimuli are stimuli to the motor nerve pathway. The motor nerve pathway is a pathway that transmits movement information from upper motor neurons (starting from the primary motor cortex of the cerebral cortex or the brainstem) to lower motor neurons. This pathway includes the lateral corticospinal tract, rubrospinal tract, reticulospinal tract, vestibulospinal tract, tectospinal tract, corticobulbar tract, etc. In addition, the motor nerve pathway synapses with lower motor neurons, and its axon extends as a peripheral nerve and synapses with extrafusal muscle fibers to contract the target muscle and cause movement. Note that the primary motor cortex receives regulation from the premotor area, supplementary motor area, cingulate motor cortex, thalamus, primary somatosensory cortex, superior parietal lobule, etc., and functions while the motor nerve pathway and sensory nerve pathway continuously regulate each other.

[0078] [Sensory Stimuli] In the present invention, "sensory stimulation" means a stimulation to any of the senses (vision, hearing, touch, etc.) related to neuropathy. From the viewpoint of being likely to exhibit the effects of the present invention, as the sensory stimulation in the present invention, somatosensory stimulation, auditory stimulation, and visual stimulation are preferable.

[0079] In the present invention, "somatosensory stimulation" means the general term for cutaneous sensation, deep sensation, and visceral sensation. Specifically, sensations obtained from the skin, mucous membranes, joints, muscles, tendons, etc. (pain sensation, temperature (low temperature to high temperature) sensation, tactile pressure sensation, etc.) can be mentioned.

[0080] The means for applying somatosensory stimulation to a patient is not particularly limited, and examples include tactile pressure, acupuncture and moxibustion, heat, weights, vibration, etc.

[0081] Examples of tactile pressure include rehabilitation for a part having neuropathy (such as hands, feet, etc.) (for example, massage performed while visually confirming a part having a disorder).

[0082] Examples of acupuncture and moxibustion include methods using needles and moxibustion (burning of mugwort).

[0083] Examples of heat include methods using moxibustion (burning of mugwort), hot packs, hydrotherapy, etc.

[0084] Examples of weights include methods using a weight band for performing a mild resistance exercise used in rehabilitation.

[0085] Examples of vibration include methods using vibration.

[0086] In the present invention, "auditory stimulation" means a stimulation imparted by sound. The sound is not particularly limited, and examples include the voices of humans, etc., any music (a certain rhythmic rhythm, etc.).

[0087] The means for applying an auditory stimulus to a patient is not particularly limited, and it may be, for example, Rhythmic Auditory Stimulation (RAS).

[0088] In the present invention, "visual stimulus" means a stimulus imparted by visual information. The visual information is not particularly limited, and examples thereof include any information existing in space (characters, pictures, videos, etc.).

[0089] The means for applying a visual stimulus to a patient is not particularly limited, and examples thereof include functional training (visual exploration 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 somatosensation (sensations obtained from the skin, mucous membranes, joints, muscles, tendons, etc.). Somatosensation is roughly classified into four modalities (pain sensation, temperature sensation, touch-pressure sensation, deep (proprioceptive) sensation), and sensory receptors, nerve fibers, conduction paths, etc. specialized for each reception are used. The sensory nerve pathways by which somatosensation reaches the cerebral cortex have been clarified, and for example, the following pathways are known. Deep sensation and fine touch-pressure sensation: Pass through the posterior column-medial lemniscus pathway system (receptor → primary neuron (enters the spinal cord, ascends the ipsilateral posterior column, and terminates in the ipsilateral posterior column nucleus of the medulla oblongata) → secondary neuron (crosses and ascends the contralateral medial lemniscus, and terminates in the contralateral thalamic VPL) → tertiary neuron (reaches the contralateral cerebral cortex somatosensory area).). Warmth-pain sensation and crude touch-pressure sensation: Pass through the spinothalamic tract (receptor → primary neuron (enters the spinal cord and terminates in the posterior horn of the spinal cord) → secondary neuron (crosses and ascends the contralateral anterior funiculus, ascends the contralateral spinothalamic tract, and terminates in the contralateral thalamic VPL) → tertiary neuron (reaches the contralateral cerebral cortex somatosensory area).).

[0091] [Electrical Stimulation] In the present invention, "electrical stimulation" means a stimulus electrically applied using an electric current. For example, it includes a stimulus that attaches electrodes to the affected part and excites the nerve 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, and methods using a voluntary movement assistance type electrical stimulation device (such as IVES) or methods conventionally known as current stimulation therapies (transcutaneous electrical nerve stimulation (TENS) method, functional electrical stimulation (FES) method, therapeutic electrical stimulation (TES) method, Transcranial Direct Current Stimulatiuon (tDCS), Deep Brain Stimulation (DBS) method, etc.) may be used.

[0093] Electrical stimulation generally involves attaching electrodes to paralyzed limbs and repeatedly exciting nerve circuits by stimulation with low frequency, medium frequency, high frequency, interference waves, etc. By exciting nerve circuits through electrical stimulation, it is possible to increase the pain threshold as explained by the gate control theory or lower the movement threshold to make it easier to move the paralyzed limb.

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

[0095] The means for applying magnetic stimulation to a patient is not particularly limited, and methods conventionally known as magnetic stimulation (for example, Transcranial Magnetic Stimulation (TMS), Transcranial Direct Current Stimulatiuon (tDCS) that stimulates with a weak current, Deep Brain Stimulation (DBS) that inserts electrodes deep into the brain and continuously applies electrical stimulation to the nervous system for control treatment of its function, etc.) may be used. In TMS, classical rTMS methods such as low-frequency rTMS (1 Hz or less) that acts suppressively or high-frequency rTMS (5 Hz or more) that acts excitatorily are representative. However, "theta burst stimulation (TBS)" in which burst stimulation consisting of triple pulses of 50 Hz is performed at a frequency of 5 Hz may also be used. In intermittent TBS (iTBS: intermittent TBS), pulse stimulation is performed by performing theta burst stimulation (triple pulses of 50 Hz at 5 Hz) for 2 seconds and then pausing for 8 seconds, which enhances the excitability of the motor cortex. In continuous TBS (cTBS: continuous TBS), the excitability of the motor cortex is suppressed by continuously performing theta burst stimulation (triple pulses of 50 Hz at 5 Hz). That is, TBS has an inhibitory effect when performed continuously and an excitatory effect when performed intermittently. TBS can be performed with a lower stimulus intensity than low-frequency or high-frequency rTMS and has the characteristic of a more sustained action time.

[0096] [Language stimulation] In the present invention, "language stimulation" means a stimulation given by communication through language.

[0097] The means for applying language stimulation to a patient is not particularly limited, but examples include prompting the patient to perform actions such as reading, writing, drawing, listening, speaking, repeating, and calculating.

[0098] Language stimulation is usually a stimulation to brain regions related to language (Broca's area, Wernicke's area, left angular gyrus (Brodmann area 39), left supramarginal gyrus (Brodmann 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 (affects) of behavior. In the present invention, "higher brain function stimulation" means memory training, attention training, executive function training, and social behavior training.

[0100] The means for imparting higher brain function stimulation to a patient is not particularly limited, and examples include prompting the patient to perform memory training, attention training, executive function training, social behavior training, and the like.

[0101] Higher brain function stimulation, when the object of stimulation is the frontal lobe function or the like, is usually stimulation at any stage of a pyramidal hierarchy (from the lowest layer: arousal → inhibition / activation → attention and concentration → information processing → memory → executive function / logical thinking, as described in the May 2006 issue of "Comprehensive Rehabilitation" (Medical Review)).

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

[0103] [Confirmation and Evaluation of Stimulation] In the present invention, whether or not a stimulation has been applied to a patient and its intensity are specified by the presence or degree of a physiological change in the patient.

[0104] Examples of physiological changes serving as indices related to the application of stimulation include electrical changes, changes in blood flow volume, changes in metabolic amount (metabolic amount of oxygen, etc.). Usually, the greater the intensity of the applied stimulation, the greater the amount of change in these physiological changes. For example, the greater the intensity of the applied stimulation, the greater the blood flow volume and metabolic amount in the brain.

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

[0106] Changes in blood flow volume are specified, for example, for changes in the blood flow volume of the brain, 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) examination, etc.

[0108] [Timing of Stimulation Application] Stimulation application may be performed once or multiple times at any point within the period defined in the present invention. However, performing stimulation application after the period defined in the present invention is not excluded.

[0109] When stimulation application is performed before the administration of a therapeutic agent, the timing of stimulation application can be appropriately set according to the type of stimulation, the patient's condition, etc.

[0110] Depending on the type of stimulation, the time from stimulation application until the blood flow and metabolic rate to the affected part (nerve injury site, etc.) increase, and the duration of the increase in blood flow and metabolic rate may vary. Therefore, it is preferable to adjust the timing of the administration of the therapeutic agent so that it overlaps with the timing when the blood flow and metabolic rate of the affected part are higher. Specifically, when the stimulation is a sensory stimulation or a language stimulation, the time from stimulation application until the blood flow to the affected part increases or the metabolic rate increases tends to be longer than when the stimulation is an electrical stimulation or a magnetic stimulation. Therefore, when the stimulation is a sensory stimulation or a language stimulation, it is preferable to set a longer time between stimulation application and therapeutic agent administration, or to set a shorter time between therapeutic agent administration and stimulation application. On the other hand, when the stimulation is an electrical stimulation or a magnetic stimulation, it is preferable to set a shorter time between stimulation application and therapeutic agent administration, and it is also possible to set a longer time between therapeutic agent administration and stimulation application.

[0111] [Site to Which Stimulation is Applied] When the stimulation is preferentially applied to one or more selected from the group consisting of the nerve injury part, the periphery of the nerve injury part, and the part that compensates for the function of the nerve injury part, it is not excluded that the stimulation application is applied to other than these parts.

[0112] In the present invention, "preferably applied to (a predetermined site)" means that when starting to apply a stimulus, the stimulus is first applied to at least one of the nerve injury site, the area around the nerve injury site, and the site that compensates for the function of the nerve injury site.

[0113] In the present invention, the "nerve injury site" means the site itself where nerve injury (atrophy, nerve block, severance, fracture, defect, brain injury, spinal cord injury, etc.) has occurred. Usually, the nerve injury site is the cause of neuropathy.

[0114] In the present invention, the "area around the nerve injury site" means a site around the nerve injury site but not the nerve injury site itself (for example, a site surrounding the nerve injury site or a site close to the nerve injury site).

[0115] In the present invention, the "site that compensates for the function of the nerve injury site" means a site that attempts to compensate for the function of the nerve injury site (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 cortex, etc. on the healthy side).

[0116] Examples of the site where the stimulus is applied to the patient include, for example, the head (brain, etc.), face, eyes, ears, mouth, upper limbs, lower limbs, trunk, speech organs, swallowing organs, etc.

[0117] Examples of the site where the stimulus can be applied and the method of applying the stimulus are illustrated below for each type of stimulus.

[0118] [Examples of applying motor stimulation] When the right primary motor cortex (finger control area) is damaged by nerve injury, the "nerve injury site" is the right primary motor cortex (finger control area). When the right primary motor cortex (finger control area) is damaged by nerve injury, the "area around the nerve injury site" is the undamaged right primary motor cortex (other than the finger control area) - premotor cortex, supplementary motor cortex, right primary sensory cortex, etc. When the right primary motor area (finger control part) is damaged by nerve injury, the "sites that compensate for the function of the nerve injury part" are the right parietal lobe, the left primary motor area - premotor area, supplementary motor area, etc.

[0119] When the right primary motor area (finger control part) is damaged by nerve injury, by trying to move the paralyzed left fingers, it is possible to preferentially stimulate the nerve injury part. When the right primary motor area (finger control part) is damaged by nerve injury, by trying to move the sites adjacent to the paralyzed left fingers (the left fingers that have escaped injury, the left wrist, etc.), it is possible to preferentially stimulate the area around the nerve injury part. When the right primary motor area (finger control part) is damaged by nerve injury, by promoting various tasks and gross movements that use the paralyzed left fingers, it is possible to preferentially stimulate the sites that compensate for the function of the nerve injury part.

[0120] [Examples of applying sensory stimulation] When the right primary somatosensory area (third neuron) is damaged by nerve injury, the "nerve injury part" is the right primary somatosensory area. When the right primary somatosensory area (third neuron) is damaged by nerve injury, the "area around the nerve injury part" is the undamaged right primary somatosensory area - right primary motor area, etc. When the right primary somatosensory area (third neuron) is damaged by nerve injury, the "sites that compensate for the function of the nerve injury part" are the left primary somatosensory area, the secondary somatosensory area that communicates with the primary somatosensory area, the parietal association area, the motor area, the visual area, etc.

[0121] When the right primary somatosensory area (third neuron) is damaged by nerve injury, by applying sensory stimulation to the affected part with sensory impairment, it is possible to preferentially stimulate the nerve injury part. When the right primary somatosensory area (third neuron) is damaged by nerve injury, by applying stimulation to the site adjacent to the site with sensory impairment, it is possible to preferentially stimulate the area around the nerve injury part. When the right primary sensory cortex (tertiary neuron) is damaged, when giving sensory stimulation to the area with sensory impairment or its vicinity, the patient can be asked to visually confirm, or the same degree of sensory stimulation can be given to the healthy side simultaneously, so that the area that compensates for the function of the nerve injury part can be preferentially stimulated.

[0122] [Example of applying electrical stimulation] When the right second finger is damaged by nerve injury, the "nerve injury part" is the right second finger. When the right second finger is damaged by nerve injury, the "vicinity of the nerve injury part" refers to the right first, third, fourth, and fifth fingers that have escaped nerve injury. When the right second finger is damaged by nerve injury, the "area that compensates for the function of the nerve injury part" refers to the right wrist, forearm, upper arm, and shoulder.

[0123] When the right second finger is damaged by nerve injury, by applying electrical stimulation to the right second finger which is the nerve injury part, the stimulation to the nerve injury part can be preferentially given. When the right second finger is damaged by nerve injury, by applying electrical stimulation to the right first, third, fourth, or fifth finger adjacent to the right second finger with nerve disorder, the stimulation to the peripheral part of the nerve injury part can be preferentially given. When the right second finger is damaged by nerve injury, by applying electrical stimulation to the right wrist, forearm, upper arm, or shoulder, the stimulation to the area that compensates for the function of the nerve injury part can be preferentially given.

[0124] [Example of applying magnetic stimulation] When the right primary motor cortex (finger control area) is damaged by nerve injury, the "nerve injury part" is the right primary motor cortex (finger control area). When the right primary motor cortex (finger control area) is damaged by nerve injury, the "vicinity of the nerve injury part" refers to the right primary motor cortex (other than the finger control area) that has escaped injury - the premotor cortex, supplementary motor area, right primary sensory cortex, etc. When the right primary motor cortex (finger control area) is damaged by nerve injury, the "area that compensates for the function of the nerve injury part" refers to the right parietal lobe, left primary motor cortex - premotor cortex, supplementary motor area, etc.

[0125] When the right primary motor area (finger control area) is damaged by nerve injury, excitatory magnetic stimulation (high-frequency rTMS <5 Hz or higher> or intermittent TBS, etc.) can be applied to the right primary motor area (finger control area) to preferentially stimulate the damaged nerve area. When the right primary motor area (finger control area) is damaged by nerve injury, excitatory magnetic stimulation can be applied to the right primary motor area (other than the finger control area) - premotor area, supplementary motor area, right primary sensory area, which are sites close to the damaged nerve area, to preferentially stimulate the area around the damaged nerve area. When the right primary motor area (finger control area) is damaged by nerve injury, excitatory magnetic stimulation can be applied to the right parietal lobe 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 <1 Hz or lower> or continuous TBS) to the left primary motor area, the suppression of activity from the left cerebral hemisphere to the right cerebral hemisphere (interhemispheric suppression) can be reduced. As a result, the damaged nerve area (right primary motor area <finger control area>), the surrounding area (right primary motor area <other than the finger control area> - premotor area, supplementary motor area, right primary sensory area), and the compensatory area (right parietal lobe) can be released from suppression, and their blood flow and excitability can be increased.

[0126] [Examples of giving language stimulation] When the Wernicke area is damaged by nerve injury, the "damaged nerve area" refers to the Wernicke area. When the Wernicke area is damaged by nerve injury, the "area around the damaged nerve area" refers to the language circuit, etc., including the Wernicke area and Broca area that have escaped nerve injury, and the conduction pathway (arcuate fasciculus) connecting the two. When the Wernicke area is damaged by nerve injury, the "area that compensates for the function of the damaged nerve area" refers to the left angular gyrus (Brodmann area 39), left supramarginal gyrus (Brodmann area 40), cerebellum, thalamus, basal ganglia of the brain, etc.

[0127] When the Wernicke area is damaged by nerve injury, sensory language stimulation can be given to preferentially stimulate the damaged nerve area. When the Wernicke area is damaged by nerve injury, by having the patient repeat or giving motor language stimulation, the area around the damaged nerve area can be preferentially stimulated. When the Wernicke area is damaged, by giving tasks such as phonology, words, grammar, reading comprehension, calculation, etc., it is possible to preferentially stimulate the parts that compensate for the function of the damaged nerve area.

[0128] [Examples of imparting higher brain function stimulation] When a performance disorder occurs due to nerve damage, the "nerve damage area" is the part of the brain that controls the performance function. When a performance disorder occurs due to nerve damage, the "area around the nerve damage area" is the part of the brain that controls the memory and information processing underlying the performance function. When a performance disorder occurs due to nerve damage, the "part that compensates for the function of the nerve damage area" is the part of the brain that controls attention, concentration, inhibition, activation, arousal, etc., further below the memory and information processing.

[0129] When a performance disorder occurs due to nerve damage, by performing performance function training, it is possible to preferentially stimulate the nerve damage area. When a performance disorder occurs due to nerve damage, by performing memory and information processing training underlying the performance function, it is possible to preferentially stimulate the area around the nerve damage area. When a performance disorder occurs due to nerve damage, by performing training on attention, concentration, inhibition, activation, arousal, which are further below the memory and information processing, it is possible to preferentially stimulate the part that compensates for the function of the nerve damage area.

Example

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

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

[0132] In addition, all of the following cultures were cultured for 3 weeks in an incubator at a temperature of 37°C and a CO2 concentration of 5%.

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

[0134] (2) Culture of adipose-derived mesenchymal stem cells Using the same method as in the above "(1) Culture of bone marrow-derived mesenchymal stem cells", the culture supernatant of adipose-derived mesenchymal stem cells was obtained using mesenchymal stem cells obtained from human adipose tissue. Cells were removed from the obtained culture using a 0.1 - 0.22 μm PVDF filter to obtain the 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) Culture of dental pulp-derived mesenchymal stem cells Using the same method as in the above "(1) Culture of bone marrow-derived mesenchymal stem cells", the culture supernatant of dental pulp-derived mesenchymal stem cells was obtained using mesenchymal stem cells obtained from human dental pulp tissue. Cells were removed from the obtained culture using a 0.1 - 0.22 μm PVDF filter to obtain the culture supernatant of bone marrow-derived mesenchymal stem cells (hereinafter also referred to as "culture supernatant-3"). The obtained culture supernatant had a soluble solid content of 2 mg / ml.

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

[0137] <Treatment of patients> The above-prepared culture supernatant was administered to patients having various symptoms of the following neuropathy. The administration was performed for each patient by any one of the methods of nasal administration, intravenous administration, or subcutaneous administration. In addition, stimulation (any one of motor stimulation, sensory stimulation, electrical stimulation, magnetic stimulation, language stimulation, and higher brain function stimulation) was performed.

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

[0139] The timing of administration of the culture supernatant was changed for each individual patient according to the administration method. The details of the timing of administration of the culture supernatant are as shown in the leftmost column of each of Tables 1 - 15. Note that "n hours before administration" means that the start time of stimulation was n hours before the start time of administration of the culture supernatant. "Immediately after administration" means that the start time of stimulation was immediately after the administration of the culture supernatant (any time within 15 minutes after administration of the culture supernatant). "n hours after administration" means that the start time of stimulation was n hours after the start time of administration of the culture supernatant.

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

[0141] (1) Nasal administration 1 ml of the culture supernatant was administered into both nasal cavities of each patient using a normal nasal drop container. The administration was performed daily for 14 weeks. In addition, nasal administration was performed in two cases each with and without a one-hour sleep interval after administration, in the examples at immediately after the start of administration, 30 minutes after administration, 3 hours after administration, 10 hours after administration, and 20 hours after administration.

[0142] (2) Intravenous administration To each patient, 5 ml of the culture supernatant was administered by intravenous drip over 1 hour. The administration was performed every 7 days for 4 weeks.

[0143] (3) Subcutaneous administration To each patient, 2 ml of the culture supernatant was administered to the extensor side of the upper arm. The administration was performed every 7 days for 4 weeks.

[0144] (Application of stimulation) The details of the stimulation given to each patient are as follows.

[0145] [Motor stimulation] For the group of patients with cerebral hemorrhage in their 40s and 50s, facilitation repetitive therapy was performed as motor stimulation. In this example, the sites of stimulation application were set to the nerve injury site (left fingers, left foot to lower leg), the area around the nerve injury (left hand joint to forearm, left knee joint to thigh), and the site compensating for the function of the nerve injury site (left elbow joint to shoulder joint, left hip joint). The stimulation application time per session was set to 30 minutes for each site. In addition, patients in their 40s and 50s with similar symptoms were made to perform whole-body exercise (exercise on a treadmill for 30 minutes per session), and the culture supernatant was administered in the same manner as above.

[0146] [Sensory stimulation] For the group of patients with cerebral hemorrhage in their 40s and 50s, as sensory stimulation to the upper and lower limbs of patients with sensory impairment, while applying a cooling stimulus to the paralyzed limb with an ice pack, an auditory stimulus (calling) was given, and furthermore, the patients were instructed to visually confirm the position of the upper and lower limbs. In this example, the sites of stimulation application were set to the nerve injury site (left fingers, left foot to lower leg), the area around the nerve injury (left hand joint to forearm, left knee joint to thigh), and the site compensating for the function of the nerve injury site (left elbow joint to shoulder joint, left hip joint). The stimulation time per session was set to 30 minutes for each site. The above sensory stimulation was performed on the upper and lower limbs of the affected side. In addition, sensory stimulation (30 minutes per session) was performed on the upper and lower limbs of the healthy side of a group of patients aged 40 to 50 with similar symptoms, and the culture supernatant was administered in the same manner as above.

[0147] [Electrical stimulation] For a group of patients with cerebral hemorrhage aged 40 to 50, low-frequency stimulation was applied to the paralyzed upper and lower limbs of the patients through electrodes attached to the hands and feet of the paralyzed patients as electrical stimulation. In this example, the stimulation sites were set to the nerve injury site (left fingers, left foot to lower leg), the area around the nerve injury (left hand joint to forearm, left knee joint to thigh), and the site compensating for the function of the nerve injury site (left elbow joint to shoulder joint, left hip joint). The stimulation time per session was set to 20 minutes for each site. The above electrical stimulation was performed on the upper and lower limbs of the affected side. In addition, electrical stimulation (20 minutes per session) was performed on the upper and lower limbs of the healthy side of a group of patients aged 40 to 50 with similar symptoms, and the culture supernatant was administered in the same manner as above.

[0148] [Magnetic stimulation] Transcranial magnetic stimulation was performed on the heads of a group of patients with cerebral hemorrhage aged 40 to 50 using a TMS device (manufactured by CR Technology). Specifically, intermittent TBS (iTBS: intermittent TBS; 1 burst 50Hz, 3 stimuli) was used to apply stimulation at an intensity of 80% of the motor threshold at 5Hz (time interval 200ms), and the stimulation was performed for 2 seconds and then paused for 8 seconds, resulting in a total of 2000 pulse stimulations. In this example, the stimulation sites were set to the nerve injury site (the finger-dominating part of the primary motor cortex on the affected side), the area around the nerve injury (other than the finger-dominating part of the primary motor cortex on the affected side), and the site compensating for the function of the nerve injury site (the right parietal lobe). The stimulation time per session was set to 2000 pulses, approximately 11 minutes, for each site. The above magnetic stimulation was performed on the head of the affected side. In addition, for a group of patients in their 40s to 50s with similar symptoms, placebo affected area head magnetic stimulation that only emits sound (2000 pulses per session, approximately 11 minutes) was performed, and the culture supernatant was administered in the same manner as above. Note that the placebo affected area head magnetic stimulation does not actually apply magnetic stimulation to the patients.

[0149] [Language stimulation] Among the group of patients with cerebral hemorrhage in their 70s to 80s who showed aphasia, language stimulation was given. As the language stimulation, stimulation using communication tasks (reading, writing, listening, speaking, repetition, calculation) was given. The stimulation time was set to 60 minutes.

[0150] [Higher brain function stimulation] Among the group of patients with cerebral hemorrhage in their 70s to 80s who showed higher brain function impairment (attention impairment), higher brain function stimulation was given. As the higher brain function stimulation, Attention Process Training was performed as attention training. The stimulation time was set to 60 minutes.

[0151] [Reference test] For reference, among the group of patients with cerebral hemorrhage in their 70s to 80s who showed aphasia, they were passively made to listen to the radio or watch TV for 1 hour.

[0152] [Treatment effect of neuropathy] After each treatment was performed on the patients, the treatment effect was evaluated for each symptom using the following indicators, and the evaluation results were classified according to the following criteria. The results are shown in Table 1.

[0153] Note that "after the longest test time has elapsed since the administration of the culture supernatant" in each evaluation criterion means the time point when the longest test time has elapsed since the administration by each method. For example, in the case of nasal administration, "after the longest test time has elapsed since the administration of the culture supernatant" means "20 hours after the administration".

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

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

[0157] (Evaluation criteria - magnetic stimulation) ◎: A significant therapeutic effect was observed compared to the results of placebo affected-side magnetic stimulation (after the longest test time elapsed since the administration of the culture supernatant). ○: A therapeutic effect was observed compared to the results of placebo affected-side magnetic stimulation (after the longest test time elapsed since the administration of the culture supernatant). △: A slight therapeutic effect was observed compared to the results of placebo affected-side magnetic stimulation (after the longest test time elapsed since the 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 the administration of the culture supernatant and the application of the stimulus. ○: A therapeutic effect was observed compared to before the administration of the culture supernatant and the application of the stimulus. △: A slight therapeutic effect was observed compared to before the administration of the culture supernatant and the application of the stimulus. ×: No change was observed compared to before the administration of the culture supernatant and the application of the stimulus.

[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, according to the therapeutic agent used in a usage that satisfies the requirements of the present invention, a significant improvement in the symptoms of neuropathy was observed.

[0175] In addition, in each administration method, when the culture supernatant obtained in the above <Preparation of Mesenchymal Stem Cell Culture Supernatant> was diluted about 3 to 10 times with the medium used for cell culture and administered with the same soluble solid content, improvement in the symptoms of neuropathy was observed as above. However, the effect was highest when the culture supernatant was not diluted. Also, when diluted, the lower the dilution ratio, the higher the effect.

[0176] When nasal administration was performed, when stimulation was applied at the time point from 5 minutes to 30 minutes after nasal administration, there was a tendency for a high therapeutic effect.

[0177] Also, when nasal administration was performed, although not shown in the data, regardless of the type of culture supernatant, when magnetic stimulation was applied over 30 minutes in a lying position 5 minutes after nasal administration of the culture supernatant and then motor stimulation was applied immediately thereafter, the therapeutic effect was particularly high.

[0178] When nasal administration was performed, even when the interval between administration and stimulation was the same, the effect tended to be higher when the patient's sleep was involved than when it was not. In such a case, when falling asleep within 1 hour after nasal administration, the effect tended to be higher than when falling asleep over 1 hour.

Claims

1. A therapeutic agent for a neuropathy, 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 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.

2. The method of claim 1 , wherein the culture supernatant is undiluted or concentrated in terms of soluble solids content.

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

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