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WO2022080473A1 - Activateur de cellules nerveuses - Google Patents

Activateur de cellules nerveuses Download PDF

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WO2022080473A1
WO2022080473A1 PCT/JP2021/038149 JP2021038149W WO2022080473A1 WO 2022080473 A1 WO2022080473 A1 WO 2022080473A1 JP 2021038149 W JP2021038149 W JP 2021038149W WO 2022080473 A1 WO2022080473 A1 WO 2022080473A1
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vegf
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三沢幸子
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
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    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
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    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a neuronal cell activating agent used for the treatment of neuropsychiatric disorders and the like, which comprises an indolinone compound as an active ingredient.
  • the present invention has also been discovered as a neurotrophic factor group containing an indolinone compound as an active ingredient, that is, a brain-derived neurotrophic factor (BDNF), a growth factor involved in angiogenesis.
  • BDNF brain-derived neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • VEGF Vascular Endotherial Growth Factor
  • VEGF Vascular Endotherial Growth Factor
  • the present invention is a method for inducing differentiation of pluripotent stem cells into nerve cells, and is an organism excluding the involvement of unidentified components not containing feeder cells and animal-derived serum or matrigel.
  • the present invention relates to a method for inducing differentiation by the environment (zeno-free culture system).
  • the present invention also relates to a differentiation-inducing method in which the process is simpler than that of the conventional method, the number of undifferentiated cells remaining is small, and the mechanism analysis is easy.
  • the first aspect of the present invention In recent years, the number of patients with depression, PTSD, panic disorder and the like has increased with the sophistication and complexity of society, and this has been regarded as a problem. Against this background, mental illnesses have been added to the four major illnesses (cancer, stroke, myocardial infarction, and diabetes) for which countermeasures should be focused on, and they have come to be called the five major illnesses. Deterioration of brain function due to diseases of the brain and nervous system not only causes a serious deterioration of the patient's own quality of life, but also has a great impact on the lives of surrounding people such as family members involved in nursing or long-term care. Therefore, improvement and prevention of deterioration of brain function due to these diseases has become an important issue.
  • BDNF nutritional factor
  • VEGF vascular endothelial growth factor
  • Non-Patent Document 1 There are roughly two methods for regenerative medicine. One is a method of utilizing stem cells such as iPS cells. The other is a method of eliciting self-renewal ability, which is a function responsible for activating cell growth factors including BDNF as a functional substance used in a method of eliciting the self-repairing ability of cells that have been damaged and have stopped functioning. A search for a target substance has been proposed (Non-Patent Document 1).
  • Non-Patent Document 1 Non-Patent Document 1
  • VEGF signaling protein is said to be effective in promoting the proliferation and differentiation of neural stem cells, and is a VEGF receptor that is a receptor for recombinant VEGF-C, recombinant VEGF-D, VEGF-C and VEGF-D.
  • Treatment of cognitive dysfunction has been investigated using a few activating compounds (Patent Documents 3-6).
  • VEGF protein administration and increased VEGF expression using a virus vector brought about a significant improvement effect on ALS disease model animals (Non-Patent Documents 16, 17, 18), VEGF protein was investigated as an ALS therapeutic agent. rice field.
  • Non-Patent Document 7 a powerful fast-acting antidepressant approved by the US FDA in 2019, re-stretches the dendrites of stress-induced nerve cells via VEGF and BDNF, which improves symptoms. It was clarified that it leads to (Non-Patent Document 7). As described above, it is essential that VEGF and BDNF function jointly in order to develop the efficacy of the antidepressant ketamine.
  • Non-Patent Document 19 it is necessary to remove undifferentiated cells remaining after differentiation induction by cell sorting.
  • feeder cells derived from heterologous organisms must be used (Patent Documents 8, Non-Patent Documents 19, 20 and 24), and mouse sarcoma cell-derived matrigels containing unidentified components must be used (Patent Documents 8 and). 9. Non-Patent Documents 19-26).
  • stepwise differentiation such as using serum containing unidentified components derived from heterologous organisms and co-culture of mouse glial cells (Non-Patent Documents 19, 21 and 25), and undergoing germ layer cell (cell aggregate) formation.
  • Patent Documents 8 and 9 Non-Patent Documents 22, 24-26. Further, there is a problem that a reagent having high cytotoxicity must be used (Patent Document 9, Non-Patent Documents 19, 20 and 24).
  • the first aspect of the present invention It is an object of the present invention to provide a nerve cell activating agent used for the treatment of neuropsychiatric disorders and the like.
  • a nerve cell activating agent that activates nerve cells by promoting the expression of proteins related to BDNF and / or VEGF signaling system.
  • the second aspect of the present invention In the present invention, in a biological environment (zeno-free culture system) in which the involvement of unidentified components is excluded, pluripotent stem cells to nerve cells do not go through the process of forming germ layer (cell aggregate). It is an object of the present invention to provide a method for inducing differentiation by suppressing the residual of undifferentiated cells.
  • iPS cells Due to the nature of iPS cells as pluripotent stem cells, research and development and clinical trials have been promoted as one of the pillars of regenerative medicine.
  • Clinical trials such as these have been started, and the development of platelet preparations made from iPS cells is also in progress.
  • the acquisition of iPS cells was a major technological innovation.
  • the chronic myeloid leukemia anticancer drug bosutinib is ALS
  • the immunosuppressant rapamycin is advanced ossifying fibrosis and Pendred syndrome
  • the ergot alkaloid derivative Parkinson's disease drug bromocriptin is It was confirmed that lopiniol , a dopamine D2 receptor system agonist for Alzheimer's disease, which improves Parkinson's disease, can be a therapeutic drug for ALS, respectively.
  • the present inventor Since the present inventor is known to be useful as a human disease model of iPS cells, the quantity and quality of components such as feeder cells, matrigel, and serum can be easily determined from iPS cell culture to the end of nerve cell differentiation.
  • VEGF-A and its families and their receptors which are known to be involved in the avoidance of motor neuronal death in ALS, are shown in the process of differentiation from iPS cells to motor neurons and long-term nerves after the completion of differentiation.
  • VEGF receptor-1 which is a negative regulator of VEGF-A signal
  • VEGF receptor-2 which is said to be responsible for the nerve cell survival activity by VEGF-A signal, also tends to decrease.
  • the indolinone compound of the present invention was differentiated from iPS cells derived from sporadic ALS patients, resulting in motor neuronal cell death. I found that it has the effect of preventing.
  • the indolinone compound of the present invention can maintain the survival of nerve cells without supplementing the screening evaluation system with a neurotrophic factor, and the indolinone compound of the present invention has a neurotrophic factor (BDNF, VEGF family, VEGF). It was confirmed that the autocline of the family receptor) has a nerve cell activating effect and a survival maintenance effect.
  • BDNF neurotrophic factor
  • VEGF vascular endothelial growth factor
  • the present inventor used semaxanib as the indolinone compound of the present invention and diligently investigated this mechanism of action.
  • the present invention maintained the survival of nerve cells by promoting the simultaneous expression of genes responsible for BDNF and VEGF signal transduction system.
  • BDNF vascular endothelial growth factor
  • it is responsible for nerve cell survival maintenance, promotion of dendritic process elongation / branching, spine formation, synaptic neoplasia, and other neuroplasticity, and VEGF signal transduction involved in nerve stem cell proliferation and differentiation promotion.
  • VEGF-A, VEGF-C, VEGF-D, VEGF receptor-2, VEGF receptor-3, VEGF-A and VEGF-C and VEGF-D co-receptors (neuropyrin 2) was found to be promoted and enhanced.
  • the present inventor presents the present invention to muscular atrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, Let's syndrome, and age-related neuropathy.
  • ALS muscular atrophic lateral sclerosis
  • Neurodegenerative diseases such as (dementia, etc.) and neuropsychiatric disorders such as depression, schizophrenia, bipolar disorder, autism spectrum, developmental disorders, post-traumatic stress disorders (PTSD), anxiety disorders, and panic disorders. It has been found that it can function as a nerve cell activator for systemic diseases, recovery of symptoms after stroke, and recovery of function after spinal cord injury, and can prevent or treat these neuropsychiatric diseases. It is also thought to contribute to extending healthy life expectancy.
  • the present inventor has been diligently studying the improvement of the method for inducing differentiation of induced pluripotent stem cells (iPS cells) into nerve cells.
  • iPS cells induced pluripotent stem cells
  • RA retinoic acid
  • the outline of the findings is shown in FIG.
  • the differentiation induction method of the present invention can be roughly divided into the following two stages.
  • step 2 In order to initiate the differentiation of induced pluripotent stem cells (iPS cells) into nerve cells, a germ layer (embyoid body) is formed in the conventional method, but it takes a long time to form nerve cells and the process is complicated. Is. The present inventor has found that this germ layer formation and control is difficult in the first place in a xenofree culture system that does not use feeder cells. Therefore, we considered using the induction of expression of the neurogenin gene family (1, 2, and 3), which is a transcription factor that plays a decisive role in the initiation of neuronal differentiation.
  • the neurogenin gene family (1, 2, and 3
  • a transient expression induction experiment was performed by gene transfer with all combinations of the neurogenin gene family (1, 2, and 3) alone, and neurogenin 2 was found from iPS cells to neurons. It was found that it was necessary and sufficient for inducing differentiation into.
  • neurogenin 2 there is a method of introducing the neurogenin 2 gene into iPS cells by the RNA lipofection method.
  • the neurogenin 2 gene is loaded on a vector and introduced into iPS cells, and for example, doxycycline is used to transiently induce the expression of neurogenin 2 for a short period of time. The method can be appropriately selected depending on the purpose of use of the cells.
  • the method of introducing the neurogenin 2 gene by the RNA lipofection method is desirable, but the RNA lipofection method has low gene transfer efficiency into cells, and the transfer efficiency depends on many factors such as culture conditions and is stable. It is difficult to obtain the results.
  • stable induction of iPS cell differentiation is required. Therefore, the neurogenin 2 gene is loaded into a vector and introduced into iPS cells, and expressed by doxycycline or the like. It is desirable to use induction. Since neurogenin 2 is a differentiation initiation switch, induction of neurogenin 2 expression is required for a certain period of time at the initial stage of differentiation initiation, but it is preferable to discontinue the expression of neurogenin 2 thereafter, and doxycycline.
  • the treatment time of doxycycline is preferably 16 to 20 hours for inducing the expression of neurogenin 2 by the above method.
  • iPS cells introduced with the neurogenin 2 gene whose expression can be induced by the addition of doxycycline are treated with doxycycline for 16 to 20 hours to express neurogenin 2, they are expressed from the cells as shown in FIGS. 8b) and 9b).
  • the processes begin to grow, the cells become smaller, and change to show the morphology of neural progenitor cells (Fig. 8a), compared to the control cells not treated with doxycycline in FIG. 9a). This state was defined as the initiation stage of differentiation in FIG.
  • an iPS maintenance medium containing a Rock inhibitor is preferable as an environment for initiating differentiation of iPS cells.
  • step 3 In order to further promote the differentiation induction of iPS cells whose differentiation was initiated by the transient expression of neurogenin 2 by doxicycline treatment, a culture method and a culture reagent were examined. Differentiation is promoted by stepwise use of primary culture, secondary culture, and 5 kinds of test agents (Rock inhibitor, TGF ⁇ family inhibitor, GSK3 inhibitor, adenylate cyclase activator, Smoothed agonist) as appropriate, and tertiary. By adding a Rock inhibitor and a neurotrophic factor (BDNF, GDNF, IGF-1) to the culture, it became possible to induce nerve cell differentiation in a Xenofree culture system (Fig. 7 Development stage of differentiation).
  • BDNF neurotrophic factor
  • iPS cells derived from unhealthy patients for example, iPS cells derived from ALS patients
  • the cells die during the process of inducing differentiation, and the long-term culture required to complete the differentiation into nerve cells is impossible. Things happen.
  • the addition of the indolinone compound enables long-term culture and can produce patient-derived differentiated neurons.
  • desired nerve cells can be produced from iPS cells of healthy subjects and patients.
  • RA retinoic acid
  • BMP signal inhibitor which were considered to be necessary in the conventional method for inducing differentiation
  • the indolinone compound By adding the indolinone compound to induce differentiation, even if the iPS cells have low cell activity as in the case of iPS cells derived from neurological disease patients, they are finally differentiated without dying on the way. It became possible to produce nerve cells. 7) It was found that the indolinone compound promotes the gene expression of the apoptosis-inhibiting protein survivin in iPS cells. As a result, in iPS cells derived from ALS patients, the expression of the survivin gene was significantly reduced, but in the induction of differentiation of such iPS cells, the addition of the indolinone compound improved the expression of survivin and differentiated it. Even at the time of induction, differentiated nerve cells can be produced without the cells dying at an early stage. The present inventor has completed the present invention based on the above findings.
  • R 1 , R 2 and R 3 are independently hydrogen atom, halogen atom, substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, formyl group, acetyl group, carboxyl group, substituted or absent. It is a substituted lower alkoxycarbonyl group having 1 to 4 carbon atoms or a substituted or unsubstituted lower alkylamide group having 1 to 4 carbon atoms, and the substituents include a halogen atom, a carboxyl group and a lower alkoxy group having 1 to 4 carbon atoms.
  • R 4 , R 5 , R 6 and R 7 are a hydrogen atom, a halogen atom, a nitro group, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a lower alkoxy group having 1 to 4 carbon atoms, and a carbon number of carbon atoms, respectively.
  • an indolinone compound or a pharmaceutically acceptable salt thereof which is a nerve cell activator.
  • R 1 , R 2 and R 3 independently have a hydrogen atom, a methyl group, an ethyl group, a halogen atom, a formyl group, an acetyl group, a carboxyl group, an ethyloxycarbonyl group, a methoxycarbonylmethyl group and an ethoxycarbonyl.
  • Methyl group, 2-carboshikiethyl group, 2-methoxycarbonylethyl group, 2-ethoxycarbonylethyl group, 2 - diethylaminoethylamide group, and R4 , R5, R6 and R7 are independent of each other.
  • the nerve cell activator according to (1) above which is a hydrogen atom, a halogen atom, or a nitro group.
  • the halogen atoms in R 1 , R 2 and R 3 are chlorine atoms, bromine atoms and iodine atoms, and the halogen atoms in R 4 , R 5 , R 6 and R 7 are fluorine atoms and chlorine atoms.
  • the nerve cell is a central or peripheral nervous system nerve cell.
  • the nerve cell is a transplanted neural stem cell in regenerative medicine.
  • a therapeutic agent for neuropsychiatric disorders which comprises the nerve cell activating agent according to any one of (1) to (8) above as an active ingredient.
  • Psychiatric and neurological disorders include muscular atrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neuropathy (dementia, etc.), depression, schizophrenia, and bipolar. Disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorder, Let syndrome, Down's disease, cerebral ischemic disorder, or spinal cord injury, as described in (9) above.
  • Psychiatric and neurological disease therapeutic agent include muscular atrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neuropathy (dementia, etc.), depression, schizophrenia, and bipolar. Disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorder, Let syndrome, Down's disease, cerebral ischemic disorder, or spinal cord injury, as described in (
  • R 1 , R 2 and R 3 are independently hydrogen atom, halogen atom, substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, formyl group, acetyl group, carboxyl group, substituted or absent. It is a substituted lower alkoxycarbonyl group having 1 to 4 carbon atoms or a substituted or unsubstituted lower alkylamide group having 1 to 4 carbon atoms, and the substituents include a halogen atom, a carboxyl group and a lower alkoxy group having 1 to 4 carbon atoms. It is a carbonyl group and a dilower alkylamino group having 1 to 4 carbon atoms.
  • R 4 , R 5 , R 6 and R 7 are a hydrogen atom, a halogen atom, a nitro group, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a lower alkoxy group having 1 to 4 carbon atoms, and a carbon number of carbon atoms, respectively. It is independently selected from the group consisting of a lower alkylcarbonyl group of 1 to 4, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, a carboxyl group and a hydroxyl group, and the substituents are a halogen atom, a carboxyl group and 1 to 4 carbon atoms. It is a lower alkoxycarbonyl group of.
  • R 1 , R 2 and R 3 independently have a hydrogen atom, a methyl group, an ethyl group, a halogen atom, a formyl group, an acetyl group, a carboxyl group, an ethyloxycarbonyl group, a methoxycarbonylmethyl group and an ethoxycarbonyl.
  • Methyl group, 2-carboshikiethyl group, 2-methoxycarbonylethyl group, 2-ethoxycarbonylethyl group, 2 - diethylaminoethylamide group, and R4 , R5, R6 and R7 are independent of each other.
  • the expression promoter according to (11) above which is a hydrogen atom, a halogen atom, or a nitro group.
  • the halogen atoms in R 1 , R 2 and R 3 are chlorine atoms, bromine atoms and iodine atoms
  • the halogen atoms in R 4 , R 5 , R 6 and R 7 are fluorine atoms and chlorine atoms.
  • (14) The expression promotion according to (11) or (12) above, wherein R 1 and R 3 are methyl groups, R 2 is a hydrogen atom, and R 4 , R 5 , R 6 and R 7 are hydrogen atoms. Agent.
  • (15) The expression-promoting agent according to any one of (11) to (14) above, wherein the promotion of expression is promotion of expression of BDNF or VEGF signaling protein.
  • the VEGF signaling protein is VEGF-A, VEGF-C, VEGF-D, VEGF receptor-2, VEGF receptor-3, and VEGF-A, VEGF-C, and VEGF-D co-receptors.
  • the expression-promoting agent according to any one of (11) to (17) above, wherein the VEGF signal transduction protein is VEGF-A, VEGF-C, and VEGF-D.
  • the VEGF signaling protein is VEGF-A, VEGF-C, VEGF-D, VEGF receptor-2, VEGF receptor-3, and VEGF-A, VEGF-C, and VEGF-D co-receptors.
  • the expression-promoting agent according to any one of (11) to (17) above, which is neuropyrin 2.
  • An iPS cell survivin expression promoter containing an indolinone compound as an active ingredient (21) The survivin expression promoter according to (20) above, wherein the iPS cells are iPS cells derived from an ALS patient. (22) The survivin expression-promoting agent according to (20) or (21) above, wherein the iPS cells are iPS cells that are inducing differentiation. (23) The survivin expression promoter according to any one of (20) to (22) above, wherein the indolinone compound is the compound according to (1) to (4) above. (24) The survivin expression-promoting agent according to (23) above, wherein the indolinone compound is Semaxanib. (25) The survivin expression-promoting agent according to any one of (20) to (24) above, wherein the iPS cells are iPS cells capable of transiently inducing the expression of neurogenin 2.
  • a survivin expression-promoting agent for nerve cells induced to differentiate from iPS cells which comprises an indolinone compound as an active ingredient.
  • the survivin expression promoter according to any one of (26) to (28) above, wherein the indolinone compound is the compound according to (1) to (4) above.
  • (30) The survivin expression-promoting agent according to (29) above, wherein the indolinone compound is Semaxanib.
  • the survivin expression-promoting agent according to any one of (26) to (30) above, wherein the iPS cells are iPS cells capable of transiently inducing the expression of neurogenin 2.
  • the indolinone compound may be brought into contact with iPS cells or nerve cells induced to differentiate from iPS cells at a concentration of 1 M to 1 pM, particularly 10 mM to 10 pM, particularly 100 ⁇ M to 100 pM.
  • the “transient” of the transient expression induction of neurogenin 2 can be 16 to 20 hours.
  • 1) A step of culturing iPS cells into which the human neurogenin 2 gene has been introduced in a laminin-coated container, and 2) A step of transiently inducing the expression of the neurogenin 2 gene for the initiation of differentiation, and 3) iPS cells that have started differentiation are cultured in a laminin-coated container, and as a test agent for promoting the development of differentiation, a small molecule TGF ⁇ family inhibitor, adenylate cyclase activator, GSK3 inhibitor, Smoothened agonist and Rock inhibitor,
  • a method for inducing differentiation of iPS cells into nerve cells which comprises a step of adding at least one of six types of indolinone compounds.
  • step 2) is a step of inducing differentiation of iPS cells into neural progenitor cells
  • step 3) is a step of inducing differentiation of neural progenitor cells into neural cells.
  • the small molecule TGF ⁇ family inhibitor may be contacted with iPS cells or iPS cells that have initiated differentiation at a concentration of 1 mM to 1 nM, particularly 100 ⁇ M to 10 nM.
  • the adenylate cyclase activator may be contacted with iPS cells or iPS cells in which differentiation has started at a concentration of 1 mM to 1 nM, particularly 100 ⁇ M to 10 nM.
  • the GSK3 inhibitor may be contacted with iPS cells or iPS cells in which differentiation has started at a concentration of 1 mM to 1 nM, particularly 100 ⁇ M to 10 nM.
  • the Smoothened agonist may be contacted with the iPS cell or the iPS cell at which differentiation has started at a concentration of 1 mM to 1 nM, particularly 100 ⁇ M to 10 nM.
  • the Rock inhibitor may be brought into contact with the iPS cell or the iPS cell at which differentiation has started at a concentration of 1 mM to 1 nM, particularly 100 ⁇ M to 10 nM.
  • the indolinone compound may be brought into contact with the iPS cells or the iPS cells in which differentiation has started at a concentration of 1M to 1pM, particularly 10 mM to 10pM, particularly 100 ⁇ M to 100 pM.
  • the laminin coating is a coating with a human recombinant laminin fragment.
  • the introduction of the human neurogenin 2 gene is carried out by using an RNA lipofection method or a vector in which the neurogenin 2 gene is mounted on a plasmid vector or a viral vector and whose expression can be transiently induced by doxycycline treatment or the like.
  • the concentration of the Rock inhibitor in the medium can be 1 mM to 1 nM, particularly 100 ⁇ M to 100 nM, and the indolinone compound can be 1 M to 1 pM, particularly 10 mM to 10 pM, and particularly 100 ⁇ M to 100 pM.
  • step 2) when transient induction of neurogenin 2 expression is performed using iPS cells into which the neurogenin 2 gene has been introduced using a vector, it is transient for 16 to 20 hours.
  • the method for inducing differentiation according to any one of (32) to (36) above, wherein an indolinone compound is added as needed.
  • step 3 as a differentiation development promoting agent, corresponding to the stage of differentiation induction, a) In the first step (primary culture), a Rock inhibitor, an adenylate cyclase activator, a GSK3 inhibitor, a small molecule TGF ⁇ family inhibitor are added, and an indolinone compound is added as necessary. b) In the second step (secondary culture), a Rock inhibitor, an adenylate cyclase activator, a Smoothened agonist are added, and an indolinone compound is added as necessary.
  • the nerve cell according to any one of (32) to (37) above, wherein in the third stage (tertiary culture), a Rock inhibitor is added, and a neurotrophic factor and / or an indolinone compound is added as needed.
  • Method of inducing differentiation into in (38), the first stage (primary culture) refers to the stage in which the cells further differentiate from the neural progenitor cells, and the second stage (secondary culture) refers to the state in which the cells are in the state of progressing differentiation into nerve cells.
  • a certain stage, and the third stage (tertiary culture) is a stage in which cells are in a state toward completion of differentiation of nerve cells.
  • the first stage is on days 01-14, and the second stage (secondary culture) is 14-.
  • the third stage can be on the 6th to 40th days, especially on the 6th to 22nd days.
  • the method for inducing differentiation into nerve cells according to any one of (32) to (38) above, wherein the iPS cells are cultured after the initiation of differentiation without using retinoic acid (RA) and LDN193189.
  • the initiation of differentiation refers to the initiation of step 2).
  • the indolinone compound is added to the differentiation-inducing medium in steps 2) and / or 3), according to (38) above.
  • the method for inducing differentiation into nerve cells according to the above. (41) The method for inducing differentiation into nerve cells according to (40) above, wherein the iPS cells are iPS cells derived from a neurological disease patient. (42) The method for inducing differentiation into nerve cells according to (41) above, wherein the nerve patient is a patient with amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • (45) An iPS cell-derived nerve cell produced by the method for inducing differentiation according to any one of (32) to (44) above.
  • (46) The iPS cell-derived nerve cell according to (45) above, wherein the nerve cell is a motor nerve cell.
  • (47) The iPS cell-derived nerve cell according to (45) or (46) above, which can be maintained in a Xenofree culture system for a long period of time.
  • 48 The iPS cell-derived nerve cell according to (46) above, wherein the long-term culture maintenance is the culture maintenance for at least 4 months.
  • (49) The iPS cell-derived nerve cell according to any one of (45) to (48) above, wherein the iPS cell is an iPS cell derived from a neurological disease patient.
  • 50) The iPS cell-derived nerve cell according to (49) above, wherein the neurological disease patient is an ALS patient.
  • iPS cells derived from ALS patients into which the human neurogenin 2 gene has been introduced are subjected to transient induction of neurogenin 2 expression in a xenofree culture system to initiate differentiation induction, and are cultured in the presence of an indolinone compound.
  • the nerve cells obtained in the above are subjected to transient induction of neurogenin 2 expression in a xenofree culture system to initiate differentiation induction, and are cultured in the presence of an indolinone compound.
  • (52) A method for screening a therapeutic agent for a neuropsychiatric disorder using the iPS cell-derived nerve cell according to any one of (45) to (51) above.
  • (53) The method for screening a neurological disease therapeutic agent according to (52), wherein the iPS cell-derived nerve cell is an iPS cell-derived nerve cell of an ALS patient, and the neurological disease therapeutic agent is an ALS therapeutic agent.
  • the screening methods of (52) and (53) include, for example, the step of bringing the iPS cell-derived nerve cell according to any one of (45) to (51) into contact with the test substance, and the biomarker for neuropsychiatric disorders.
  • test substance to be increased can be selected, and for biomarkers that increase in neuropsychiatric disorders, the test substance to be decreased can be selected.
  • biomarker for a psychiatric nervous system disease changes in gene expression and changes in the type and / or amount of produced protein, which are specific to the disease, can be used.
  • neurodegenerative diseases such as ALS and Alzheimer's disease
  • increased neuronal degradation products such as neurofilament light chains, can also be a biomarker for the degree of disease state.
  • a cell activator containing an indolinone compound as an active ingredient which is a cell group committed to the differentiation of iPS cells of ALS patients into nerve cell differentiation by inducing differentiation. That is, a cell activator of a cell group directed to differentiate into a nerve cell by inducing differentiation from iPS cells of an ALS patient containing an indolinone compound as an active ingredient.
  • the cell activating agent according to (54) above wherein the differentiation-induced nerve cells are activated so that they can be cultured and maintained for at least one month.
  • the indolinone compound is Semaxanib.
  • the indolinone compound of the present invention Since the indolinone compound of the present invention has an excellent nerve cell activating effect, it is useful for activating damaged or dysfunctional central / peripheral nerve cells or transplanted neural stem cells. Is. As a result, the nerve cell activator of the present invention is useful as a preventive and / or therapeutic agent for neuropsychiatric disorders. Furthermore, since the compound of the present invention is a small molecule compound, it is inexpensive and excellent in stability as compared with BDNF and VEGF signal transduction protein agents. In addition, since the VEGF protein drug does not cross the blood-brain barrier, it needs to be administered intraventricularly, which causes a great problem in clinical use. Can be used for. It is also effective for peripheral nerve cells such as lower motor neurons.
  • the indolinone compound of the present invention simultaneously promotes the expression of genes responsible for BDNF and / or VEGF signaling system, promotes the proliferation and differentiation of neural stem cells, and re-elongates and branches dendrites atrophic due to stress or disease. Promotes and promotes spine formation to improve neuroplasticity. As a result, activation of nerve cells occurs. Furthermore, the expression of autocline in BDNF and VEGF signaling neurotrophic factor neurons improves nerve cell maintenance, neuroplasticity, neural stem cell proliferation and promotion of differentiation, and promotes memory reconstruction in the hippocampus.
  • the indolinone compound of the present invention can be used for muscle atrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neuropathy (dementia, etc.), depression, and schizophrenia.
  • ALS muscle atrophic lateral sclerosis
  • PTSD post-traumatic stress disorder
  • anxiety disorder anxiety disorder
  • panic disorder autism spectrum disorder
  • developmental disorder developmental disorder
  • Let syndrome, Down syndrome cerebral ischemic disorder
  • spinal cord injury It is useful as a therapeutic or prophylactic agent for system diseases, a therapeutic or prophylactic agent in regenerative medicine, and a nerve cell activator.
  • it is thought that it contributes to the extension of healthy life expectancy by suppressing cerebral atrophy due to aging.
  • the neural activator of the present invention is the basis for maintaining the normal function of the nervous system, and in order to maintain the normal function of the nervous system, it is necessary to act in combination under physiological conditions.
  • the scientific basis for the simultaneous activation of the factors to be a futuristic therapeutic agent for not only the ALS whose effects have been demonstrated in the examples but also the various mental degenerative diseases and mental diseases exemplified above will be described below. ..
  • the nerve activator of the present invention avoids cell death of nerve cells derived from ALS patients, exhibits an effect of maintaining survival of nerve cells derived from ALS patients, and stops the addition of BDNF which is considered to be essential for maintaining the culture of nerve cells. Since the nerve cells could be maintained, it was inferred that they have the activity of promoting the autocline of the nerve cell nutrition factor essential for maintaining the survival of various nerve cells. As a result of the measurement, it was proved that the expression of VEGF family, VEGF receptor family, and VEGF co-receptor neuropilin-2 (Neuropilin-2) was activated in addition to BDNF.
  • VEGF and its receptor family were discovered as growth factors involved in angiogenesis, but subsequently have important activity as neurotrophic factors and play important roles in neuronal survival, neurogenesis, and nerve axon elongation. (Jin K., et al., Proc. Natl. Acad. Sci.
  • VEGF Vascular Endotherial Growth Factor
  • ALS is a motor neurodegenerative disease with a relatively rapid progression of disease, and often dies 3 to 5 years after diagnosis due to bulbar palsy, muscular atrophy of limbs, and respiratory muscle palsy.
  • riluzole which has a life-prolonging effect
  • edaravone which further improves QOL
  • Familial ALS with known gene mutations accounts for 5-10%, and sporadic ALS accounts for 90-95%.
  • VEGF protein cannot cross the blood-brain barrier, it is administered intraventricularly when used as a drug, but it has been shown that VEGF administration delays the onset and has a life-prolonging effect in ALS model animals (Storkebaum E. et al.).
  • VEGF from ADNI (Alzheimer's Disease neuroimaging initiative), a clinical research group aimed at predicting the onset of Alzheimer's disease and establishing a method for determining the effect of therapeutic agents, is used for Alzheimer's disease and dementia due to aging. It became a biomarker and showed that the higher the concentration of VEGF in the cerebrospinal fluid, the higher the cognitive ability and the larger the volume of the hippocampus (Hohman TJ., Et al., JAMA (2015) 72 (5); 520-529. "The roll of Vascalar endothelial gross factor in Neurodegeneration and cognitive declination").
  • VEGF also has a neuroprotective effect in Huntington's disease (Ellison SM, et al., Mol Ther. (2013) 21: 1862-75 “Dose-dependent neuroprotraction of VEGF165 in Huntington's disease”. stripeum ").
  • ALS neurological degenerative diseases
  • the VEGF family, VEGF receptor family, and VEGF co-receptors have a neuroprotective effect and are therapeutic agents for peripheral neuropathy and epilepsy.
  • VEGF Vascular Endothelial Growth Factor
  • angiogenesis-promoting factor in the hippocampus. This factor indicates that the research group was previously enhanced by electroconvulsive (ECS) therapy.
  • VEGF mRNA The content of VEGF mRNA was increased in the hippocampal granule cell layer of rats treated with fluoxetine (a serotonin reuptake inhibitor) or desipramine (a norepinephrine reuptake inhibitor) for 14 days, similar to the content of VEGF in hippocampal homogenates.
  • Pharmacoinhibition of VEGF receptor Flk-1 (also known as VEGF receptor-2) inhibits increased cell proliferation in the subcapsular granule cell zone (SGZ) produced by ECS or chronic exposure to fluoxetine or desipramine.
  • SGZ subcapsular granule cell zone
  • intracerebroventricular administration of VEGF isoform promoted SGZ cell proliferation.
  • VEGF is responsible for the onset of the effect of the fast-acting and powerful antidepressant ketamine approved by the US FDA in 2019 (Deyama S., et al., Am J Psychiatry. (2019) 176 (5). ); 388-400 "Role of Natural VEGF Signaling in the Frontal Cortex in the Rapid Antidepressant Effects of Ketamine"), and BDNF and VEG (.
  • VEGF-C and VEGF-D and their receptors, VEGF Receptor-2 and VEGF Receptor-3 (note that VEGF Receptor-2 is VEGF-A, C).
  • D receptor, VEGF receptor-3 is a VEGF-C, D receptor) promotes the proliferation and differentiation of neural precursor cells in the hippocampus and is essential for neurogenesis in the adult hippocampus. It shows that there is (Han J., et al., Cell Reports (2015) 10; 1158-1172 "Vascular Endothelial Growth Factor Receptor 3 Controls Natural Station" Stem Cell.
  • Patent Documents 2 and 3 VEGF-C and VEGF-D proteins and their receptors, VEGF receptor-2 / 3, activators are useful as therapeutic agents for aging and neurodegenerative diseases. It states that there is.
  • the neurostimulator of the present invention has great potential as a futuristic therapeutic agent for many neuropsychiatric disorders.
  • BDNF is a neuronutrient factor that controls the neoplasia of nerve cells, the differentiation and growth of neurite progenitor cells, and the promotion of neurite outgrowth.
  • Elongation (Deyama S, et al., Biological Psychity (2019) 86; 143-152 Neurotrophic and antidepressant actions of Brain-derivated neuron neuriter Supports the construction of complex networks. In mature neurons, it controls the formation and disappearance of synapses and the plasticity of nerves related to learning and memory, and maintains normal brain function (Mattson and Wan, Neuromolecular Med (2008) 10; 157-158) (Miranda M). Et al., Front Cell Neurosci.
  • BDNF Brain-Derived Neurotropic Factor: A Key Molecular for Memory in the Health.
  • BDNF has been shown to be a biomarker for many neurodegenerative and psychiatric disorders and has a therapeutic effect on neuropsychiatric disorders and spinal cord injuries.
  • a group at Tokyo Medical & Dental University revealed that early motor function recovery can be obtained by administering BDNF mRNA to the injured site of spinal cord injury model mice to enhance BDNF expression at the injured site.
  • BDNF Alzheimer's disease and age-related cognitive impairment
  • Brain-Derived Neurotrophic Factor A Key Molecule for Memory in the Healthy and the Pathological Brain
  • Tala-Arancibia et al Brain Res Rev (2008) 59: 201-220 New insights into brain BDNF function in Normal aging and Alzheimer's disease "), considered to be an effective therapeutic agent for Alzheimer's disease and Parkinson's disease (Sampario, T. et al., Natural Regen. Res.
  • BDNF is a bio-marker (frontiers in Molecular Neuroscience (2020) 12: article 335 "Evaluation of Biochemical and epigenetic measures of peripheral Brain-Derived Neurotrophic factor (BDNF) as Biomarker in Huntington's disease patients") ..
  • depression The lifetime prevalence of depression is very high at about 10 to 20%, and it is said that 60 to 70% of suicides are depressed, so it is an urgent society to analyze the pathophysiology of depression and develop treatment methods. This is a subject (Kenji Hashimoto, Brain and Psychiatric Medicine (2009) 20 (1); 55-60 "Brain-derived neuronutrient factor and depression”). Depression and anxiety disorders are associated with a decrease in BDNF (Martinowich K., et al., Nature Neurosci. (2007) 10; 1089-1093 "New insights into BDNF faction in depression” antidepressant and antidepressant.
  • Reduced BDNF is a biomarker of bipolar disorder (Fernandes et al. BMC schizophrenia (2015) 13; 289 “Peripheral brain-developed neurotrophic factor” (BDNF) as a biomarker ), A biomarker of severe psychosis with delusional hallucinations such as schizophrenia (Nieo et al. Frontiers in Psychosis (2021) 12; ), A biomarker of autism spectrum disorder (Saghazadeh A. and Rezaei N. , J Autism Dev Disord. (2017) 47 (4): 1018-1029 “Brain-Derived Neurotrophic Factor Levels in Analysis: A Systematic Review and Meta-Analysis”).
  • BDNF neurotrophic factor
  • the nerve cell activator of the present invention is a small molecule compound, it has advantages that it is inexpensive, easy to store, and is considered to pass through the blood-brain barrier.
  • the neurostimulator of the present invention has not been considered in drug discovery because of its functionality of activating multiple neurotrophic factors physiologically required for normal nervous system function. Providing futuristic drugs with physiological functionality and contributing to the treatment of Alzheimer's disease, Parkinson's disease, dementia, and depression-these have many social impacts-not limited to ALS. there is a possibility. Therefore, it makes a huge social and economic contribution.
  • iPS cells artificial pluripotent stem cells
  • up to 6 types of differentiation development promoters can be added, and up to 9 types of reagents can be used for culturing with the addition of neurotrophic factors or indolinone compounds in the final differentiation, facilitating process control.
  • reagents can be used for culturing with the addition of neurotrophic factors or indolinone compounds in the final differentiation, facilitating process control.
  • impurities In particular, in the case of iPS cells derived from patients with neurological disorders, even if the cell activity is low, the addition of the indolinone compound makes it possible to promote the expression of the surviving gene, and the cells die on the way. It has become possible to produce finally differentiated nerve cells without any problems.
  • iPS cells derived from human disease patients can be induced to differentiate into nerve cells without using retinoic acid (RA) or LDN193189, which are differentiation-inducing agents used in conventional differentiation culture of nervous system cells. It became possible to maintain cells for a long period of time.
  • RA retinoic acid
  • LDN193189 differentiation-inducing agents used in conventional differentiation culture of nervous system cells. It became possible to maintain cells for a long period of time.
  • the culture of motor neurons derived from ALS patients obtained in the Xenofree culture system is unprecedented, and the congestion of a large number of unidentifiable compositions and the amount of congestion of each composition cannot be controlled. It does not contain impurities derived from biological culture aids (serum, feeder cells, matrigel), which has a big problem, and it is simplified to use only materials whose origin is clear and the amount of addition can be consistently controlled.
  • the differentiation culture method of the present invention mature nerve cells can be easily induced to differentiate from iPS cells derived from healthy subjects or disease patients, so that it is easy to screen for therapeutic agents for neurological diseases using these mature nerve cells, and it is possible to create. It can provide useful means for the drug.
  • FIG. 3 is a microscopic image showing that acclimation of iPS cell lines to a feeder-free and xeno-free culture system has been established.
  • A. It is a figure which showed the phase-contrast microscope image of the iPS cell derived from a healthy person.
  • B It is a figure which showed the phase-contrast microscope image of the iPS cell derived from a healthy person.
  • FIG. 1 It is a figure which showed the phase-contrast microscope image of the motor neuron (the 33rd day of the differentiation induction) differentiated from the iPS cell derived from a healthy person.
  • C It is a schematic diagram which showed the expression dynamics of a marker gene at the time of differentiation from iPS cells into motor neurons.
  • the neural progenitor cell marker (PAX6) is first expressed, but then the expression decreases as the differentiation progresses.
  • a motor neuron marker (HB9) is expressed, and its expression increases with differentiation into motor neurons.
  • mature motor nerve markers are expressed.
  • the neurotransmitter receptor GluA1 was used as a marker for mature motor neurons.
  • FIG. 2C It is a figure which shows the expression change of the differentiation marker mRNA described in FIG. 2C accompanying the differentiation from iPS cells to motor neurons.
  • the expression of the neural progenitor cell marker (PAX6) increased about 3200 times on the 15th day of differentiation induction compared with the control, and then decreased about 1500 times on the 33rd day of differentiation with differentiation (upper figure).
  • the expression of the motor nerve marker (HB9) increased about 2200 times on the 15th day of differentiation induction and about 6600 times on the 33rd day with differentiation (middle figure).
  • the expression of the mature neuron marker (neurotransmitter receptor GluA1) remained almost unchanged until the 15th day of differentiation induction, and increased about 600-fold on the 33rd day (see the figure below).
  • FIG. 6 is a microscopic image showing the effect of addition of an indolinone compound (semaxanib) on the maintenance of survival of neurons derived from sporadic ALS patients.
  • Semaxanib an indolinone compound
  • FIG. 3 is a microscopic image showing that when iPS cells derived from sporadic ALS patients were differentiated into motor neurons, the addition of semaxanib prevented the motor neurons from cell death. There is no difference due to the addition of semaxanib in the early stage of differentiation (1st day of differentiation induction) (left column). However, on the 15th day of differentiation induction, cells died in the control, but cells with extended neurites were observed in the semaxanib-added group (middle row), and on the 27th day of differentiation induction, they were equivalent to motor neurons derived from healthy subjects. A phase-contrast microscope image was observed (right figure). It is a figure which showed the effect of promoting BDNF expression by the addition of semaxanib.
  • BDNF expression in healthy subject-derived neurons increased approximately 4.6-fold compared to controls, and sporadic ALS patient-derived neurons increased approximately 3.4-fold compared to iPS cells. bottom. It is shown that the addition of semaxanib results in autoclination by the BDNF neurons themselves. Left figure: from healthy subjects, right figure: from sporadic ALS patients.
  • the effects of the addition of semaxanib on the maintenance of nerve cell survival, neuroplasticity (promotion of dendrite elongation and branching), and the expression of genes related to the proliferation and differentiation promotion of neural stem cells were investigated using cells derived from healthy subjects.
  • VEGF-A related to neuroplasticity (promotion of dendrite elongation and branching) and survival maintenance was increased about 3 times that of the control (upper left figure).
  • the expression of VEGF-C, which is involved in the proliferation and differentiation promotion of neural stem cells, was increased about 9.3 times that of the control (upper middle figure).
  • the expression of VEGF-D, which is involved in the proliferation and differentiation promotion of neural stem cells, was increased about 18 times that of the control (upper right figure).
  • VEGF receptor-2 which is a receptor for VEGF-A, VEGF-C, and VEGF-D, decreased to 50% of iPS cells with differentiation into nerve cells, but the addition of semaxanib reduced the expression to about 2 of the control.
  • VEGF receptor-3 which is a receptor for VEGF-C and VEGF-D involved in the proliferation and differentiation promotion of neural stem cells
  • neuropyrin 2 which is a co-receptor of VEGF receptor-2 and VEGF receptor-3 involved in the proliferation and differentiation promotion of neural stem cells
  • Semaxanib has been shown to increase the expression of genes involved in the VEGF signaling system as described above.
  • VEGF-D which is involved in the proliferation and differentiation promotion of neural stem cells
  • VEGF receptor-2 which is a receptor for VEGF-A, VEGF-C, and VEGF-D
  • VEGF receptor-3 which is a receptor for VEGF-C and VEGF-D involved in the proliferation and differentiation promotion of neural stem cells, was reduced in about 73% of iPS cells (lower middle figure).
  • neuropyrin 2 which is a co-receptor of VEGF receptor-2 and VEGF receptor-3 involved in the proliferation and differentiation promotion of neural stem cells, was increased about 5-fold as that of iPS cells (lower right figure).
  • Semaxanib has been shown to increase the expression of genes involved in the VEGF signaling system, except for VEGF receptor-2 and VEGF receptor-3 as described above. It is a figure which showed the outline of the nerve cell differentiation induction method of this invention.
  • IPS cells derived from healthy individuals into which the human neurogenin 2 gene, which can be transiently induced to be expressed with Doxycycline were cultured for 16 hours in the presence of Doxycycline (DOX) (right figure) and 16 hours in the absence of DOX.
  • DOX Doxycycline
  • FIG. 8 is a diagram showing changes in cells when the neural stem cell-like cells of FIG. 8 are further cultured.
  • the expression of the motor neuron marker HB9 increases sharply on the 15th day of induction of differentiation (about 2200 times when the expression in iPS cells is 1), and increases as the cells mature into nerve cells (middle figure b).
  • the expression of GluA1, one of the subunits of the neurotransmitter glutamate receptor increased sharply on the 18th day of differentiation induction (about 660 times when the expression in iPS cells is 1), and the high expression is maintained thereafter (see the figure below). c). From these facts, differentiation progresses from iPS cells by the method to mature motor neurons via neural stem cells, and mature motor neurons are established by the 33rd day of differentiation induction.
  • the expression level of survivin was reduced in about 18% of iPS cells derived from healthy subjects in iPS cells derived from ALS patients.
  • the expression level of survivin mRNA was increased three-fold when the indolinone compound was added, as compared with the neurons without the addition.
  • iPS cells derived from ALS patients die when differentiated into neurons, the expression level of survivin in neurons derived from ALS patients differentiated by adding an indolin compound increases about 3 times as compared with iPS cells derived from ALS patients. It reached 80% of the neurons derived from healthy subjects (without the addition of indolinone).
  • the first aspect of the present invention The "indolinone compound” of the present invention is the general formula (1).
  • R 1 , R 2 and R 3 are independently hydrogen atom, halogen atom, substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, formyl group, acetyl group, carboxyl group, substituted or absent. It is a substituted lower alkoxycarbonyl group having 1 to 4 carbon atoms or a substituted or unsubstituted lower alkylamide group having 1 to 4 carbon atoms, and the substituents include a halogen atom, a carboxyl group and a lower alkoxy group having 1 to 4 carbon atoms. It is a carbonyl group and a dilower alkylamino group having 1 to 4 carbon atoms.
  • R 4 , R 5 , R 6 and R 7 are a hydrogen atom, a halogen atom, a nitro group, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a lower alkoxy group having 1 to 4 carbon atoms, and a carbon number of carbon atoms, respectively. It is independently selected from the group consisting of a lower alkylcarbonyl group of 1 to 4, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, a carboxyl group and a hydroxyl group, and the substituents are a halogen atom, a carboxyl group and 1 to 4 carbon atoms. It is a lower alkoxycarbonyl group of.
  • the lower alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an i-propyl group and an n-butyl group.
  • the methyl group and the ethyl group can be increased.
  • the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • Preferred examples include a fluorine atom and a chlorine atom.
  • the indolinone compound the compound described in US57929783 (or Japanese Patent No.
  • R 1 , R 2 and R 3 are independently hydrogen atoms and methyl groups, respectively.
  • R 1 , R 2 and R 3 are independently hydrogen atoms and methyl groups, respectively.
  • the indolinone compound of the present invention can be synthesized according to the method described in US57929783.
  • the pharmaceutically acceptable salt of the indolinone compound include acid addition salts.
  • the acid addition salt include inorganic acid salts such as hydrochloride, hydrobromide and sulfate, and organic acid salts such as citrate, oxalate, tartrate, fumarate and maleate. Can be done.
  • the "brain-derived neurotrophic factor" of the present invention refers to BDNF (Brain-deved neurotrophic factor). BDNF is widely distributed in the brain, triggering the differentiation of nerve cells and further promoting maturation.
  • VEGF Vascular Endothelial Growth Factor
  • VEGF receptor tyrosine kinase VEGF receptor
  • VEGF Vascular Endothelial Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • VEGF-A VEGF-C
  • VEGF-D VEGF Receptor-2
  • VEGF-C VEGF-C
  • VEGF-D Receptor VEGF Receptor-3
  • Neuropyrin 2 VEGF-A, VEGF-C, and VEGF-D Co-Receptor
  • VEGF-A regulates angiogenesis and vascular permeability
  • VEGF-C and VEGF-D primarily regulate lymphangiogenesis.
  • VEGF-A plays an important role in maintaining the survival of nerve cells.
  • VEGF-A plays an important role in neuroplasticity, such as spine formation and synaptogenesis reduced by depression and PTSD, and dendrite re-elongation and bifurcation.
  • VEGF-C and VEGF-D play important roles in promoting neural stem cell proliferation and differentiation.
  • VEGF also binds to a receptor complex consisting of both neuropilin and VEGF receptors. This receptor complex is known to have VEGF signaling activity. Therefore, it has been studied to use recombinant VEGF-C and VEGF-D protein preparations as therapeutic agents for various neurodegenerative diseases such as Down's syndrome and Alzheimer's disease (Patent Documents 4 and 5). .. However, since these are protein preparations, it is difficult to control delivery to target tissues in the brain.
  • the indolinone compound of the present invention has a feature of enhancing or promoting the expression of the above-mentioned BDNF and / or VEGF signaling protein group.
  • some indolinone compounds have the characteristic of simultaneously enhancing the expression of BDNF and the VEGF signaling protein group, as shown in, for example, semaxanib. It has been recognized that BDNF and the VEGF signaling protein group need to work in conjugation in order to exhibit a therapeutic effect physiologically (Non-Patent Document 7). As a result, it was newly found that it promotes neuroplasticity (re-elongation and branching of dendrites, spine formation and synaptic formation), and promotes proliferation and differentiation of neural stem cells.
  • nerve plasticity is promoted in addition to maintenance of nerve cell survival, proliferation and differentiation of nerve stem cells are promoted, and muscle atrophic lateral sclerosis (ALS), Alzheimer's disease. , Parkinson's disease, Huntington's disease, age-related neuropathy (such as dementia), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum It is thought that it can improve neuropsychiatric disorders such as disorders, developmental disorders, Let's syndrome, and Down's syndrome.
  • the lost neural cells are supplemented, so that in symptom recovery after cerebral ischemia, functional recovery after spinal cord injury, extension of healthy life span, and regenerative medicine. It is also considered to be useful for maintenance and colonization of transplanted neural stem cells.
  • Non-Patent Document 7 As a powerful and fast-acting antidepressant, the dissociative anesthetic ketamine was approved by the US FDA in 2019. In order for ketamine to exert antidepressant action, administration of BDNF and amplification of expression alone are not sufficient, and conjugation by VEGF is required (Non-Patent Document 7). That is, in order to ensure the antidepressant action, it is considered essential to promote the expression of both BDNF and the VEGF signal transduction protein at the same time for the expression of the physiological function as an antidepressant.
  • BDNF and VEGF signaling proteins by simultaneously promoting the expression of BDNF and VEGF signaling proteins and physiologically conjugating them, for example, with muscle atrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, and aging.
  • Neurological disorders such as dementia
  • depression schizophrenia, bipolar disorders, post-traumatic stress disorders (PTSD), anxiety disorders, panic disorders, autism spectrum disorders, developmental disorders, Let syndrome, Down syndrome
  • PTSD post-traumatic stress disorders
  • anxiety disorders anxiety disorders
  • panic disorders panic disorders
  • autism spectrum disorders developmental disorders
  • Let syndrome, Down syndrome It is considered to be more effective for the treatment and symptom improvement of various neuropsychiatric diseases including, cerebral ischemia, and nerve damage.
  • the compound of the present invention is a small molecule compound, it can be easily transferred into the brain, and it is stable and easy to store.
  • the compound of the present invention is stable and easy to store, it is possible to develop simple pharmaceuticals such as nasal drops and patches, which could not be realized by protein preparation
  • the “neuronal activator” of the present invention refers to neuronal plasticity (re-elongation and branching of dendritic processes, formation of spines) in damaged nerve cells, dysfunctional nerve cells or transplanted nerve stem cells. And synapse formation), avoiding apoptosis (cell death of damaged nerve cells) to help maintain nerve cell survival, or promote growth and differentiation of nerve stem cells to maintain nerve cells.
  • Examples of the damaged nerve cell include a damaged cell in a nerve cell related to cerebral ischemia disorder and spinal cord injury.
  • the damaged nerve cells include, for example, muscle atrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, and other diseases in which damaged nerve cells are shed. be able to.
  • Neurological cells that have caused dysfunction include, for example, depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), panic / anxiety disorder, Rett syndrome, Down syndrome, autism spectrum disorder, and developmental disorder.
  • Nerve cells that cause age-related neuropathy including dementia include transplanted neural stem cells used for the treatment of spinal cord injury, Alzheimer's disease or Parkinson's disease, and nerve cells derived from iPS cells used for the treatment of macular degeneration. ..
  • the "psychiatric and neurological disorders" of the present invention include, for example, muscular atrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neuropathy (dementia, etc.), depression, and schizophrenia.
  • Diseases, bipolar disorders, post-traumatic stress disorders (PTSD), anxiety disorders, panic disorders, autism spectrum disorders, developmental disorders, Let's syndrome, Down's syndrome and the like can be mentioned.
  • the "expression promoter" of the present invention refers to promoting or enhancing the expression of BDNF and / or VEGF signaling protein.
  • BDNF and VEGF signaling protein In the case of co-expression of BDNF and VEGF signaling protein, neuroplasticity and neural stem cell proliferation and differentiation can be promoted more effectively.
  • the expression-promoting agent of the present invention has an excellent BDNF expression-promoting effect, and increased expression of BDNF causes re-elongation and branching of atrophied dendrites, promotes spine formation, and improves neuroplasticity. Furthermore, memory reconstruction in the hippocampus is promoted.
  • the expression-promoting agent of the present invention has excellent expression promotion of VEGF and VEGF receptors and their co-receptors, and motor nerve cell death in ALS due to increased expression of VEGF and VEGF receptors and their co-receptors. Is suppressed, and in addition, the proliferation of nerve stem cells and their differentiation are promoted.
  • the expression-promoting agent of the present invention when BDNF and VEGF signaling protein are simultaneously expressed, synergistic effects can be expected, and the frame of therapeutic effect that has been tried using each single substance so far.
  • BDNF and VEGF signaling protein Due to the synergistic effect of promoting the simultaneous expression of BDNF and VEGF signaling protein, muscle atrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neuropathy (dementia, etc.), depression , Sticking dysfunction, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorder, let syndrome, down syndrome, post-stroke symptom recovery, post-spinal injury It has become possible to treat neuropsychiatric disorders such as functional recovery more effectively.
  • the conjugate of BDNF and VEGF signaling protein is essential for amelioration of depression.
  • the "therapeutic agent” of the present invention refers to a pharmaceutical composition containing the compound of the present invention as an active ingredient and used for the prevention or treatment of neuropsychiatric disorders.
  • These pharmaceutical compositions are prepared as general pharmaceutical preparations and administered orally or parenterally. When administered orally, it can be administered in the dosage form usually used in the art. When administered parenterally, it can be administered in the form of a topical administration (such as a transdermal agent), an injection, or a nasal agent.
  • a topical administration such as a transdermal agent
  • an injection or a nasal agent.
  • examples of the oral preparation include tablets, powders, granules, capsules, liquids and the like.
  • Examples of the injection solution include sterile solutions or suspensions.
  • Examples of the topically administered agent include ordinary patches and nasal drops. These formulations are prepared using commonly used pharmaceutical carriers.
  • the pharmaceutical carrier a substance that is commonly used in the pharmaceutical field and does not react with the compound of the present invention is used.
  • Specific examples of pharmaceutical carriers used in the production of tablets, capsules, granules and powders include excipients such as lactose, corn starch, sucrose, mannitol, calcium sulfate, crystalline cellulose, carmellose sodium and modified starch. , Disintegrants such as carmellose calcium, binders such as methylcellulose, gelatin, gum arabic, ethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, lubricants such as light anhydrous silicic acid, magnesium stearate, starch, hardened oil Can be mentioned.
  • the tablets may be coated by a well-known method using a usual coating agent.
  • Specific examples of carriers used in the production of syrups include sweeteners such as sucrose, glucose and fructose, suspending agents such as gum arabic, tragant, sodium carmellose, methyl cellulose, sodium alginate, crystalline cellulose and bee gum.
  • Dispersants such as sorbitan fatty acid ester, sodium lauryl sulfate, polysorbate 80 can be mentioned.
  • Injectable preparations are usually prepared by dissolving the above active ingredients in distilled water for injection, but if necessary, lysis aids, buffers, pH regulators, tonicity agents, soothing agents, preservatives, etc. Can be added.
  • the compound may be in the form of a suspended injection in which the compound is suspended in distilled water for injection or vegetable oil, and a base, a suspending agent, a viscous agent and the like can be added as needed.
  • the "indolinone compound” is a low molecular weight compound having an indolinone skeleton, which has a survivin gene expression-promoting action and neurotrophic activity such as BDNF. Those that have the effect of promoting the expression of factors. For example, Semaxanib can be mentioned.
  • the "iPS cell” of the present invention is an induced pluripotent stem cell produced by incorporating Yamanaka factor or the like into a human somatic cell. As the human somatic cells, somatic cells such as healthy subjects and patients with neurological disorders can be used.
  • ALS muscular atrophic lateral sclerosis
  • Alzheimer's disease Parkinson's disease
  • Huntington's disease age-related neurological disorders (such as dementia), depression, schizophrenia, and bipolar disorder.
  • Post-psychiatric stress disorder PTSD
  • anxiety disorder panic disorder
  • autism spectrum disorder developmental disorder
  • let syndrome down syndrome
  • cerebral ischemic disorder or spinal cord injury.
  • the "survivin” of the present invention is a protein that inhibits the activation of caspase and suppresses apoptosis.
  • Survivin is often highly expressed in cancer cells, whereas it is rarely expressed in short-term turnover cells such as many fully differentiated skin tissues. Therefore, survivin has been regarded as a good target for cancer treatment, but it has also become clear that it is essential for normal proliferation and differentiation of blood stem cells, T lymph cells, erythroblasts, etc., and survivin was targeted. There is a concern about cancer treatment (Bird. Nature Reviews Immunology (2004) 4:166.) (Lung et al., J Exp Med (2007) 204: 1603-11).
  • survivin is maintained in the cerebral cortex where mature nerve cells are maintained for a long period of time, and plays a major role in the long-term maintenance of nerve cells. It is known that the expression of survivin mRNA increases with the initiation of differentiation induction of neural progenitor cells, and survival is essential for the proliferation and differentiation of neural progenitor cells in the adult hippocampus, especially in the adult hippocampus after traumatic brain injury. It has been shown to play a major role in neurogenesis and recovery after traumatic brain injury in (Zhang et al., Neuroscience (2015) 300: 219-228).
  • the "expression-promoting agent” of the present invention refers to a substance that promotes the expression of the cybabin gene and enables the expressed survivin protein to suppress apoptosis and activate cells.
  • "Xenofree” in the present invention means "xenogeneic component free” and is used in abbreviated form as the word "xeno-free”. That is, the word xenofree means "does not contain heterologous components", “does not contain animal origin", or “does not contain non-human origin components”.
  • the "Xeno-free culture system” of the present invention means that the culture environment of cells is Xeno-free, and for example, animal-derived components (bovine-derived serum, feeder cells, mouse sarcoma-derived matrigel, etc.) are used in the culture medium. If not, it is said to be a Xenofree culture system.
  • animal-derived components bovine-derived serum, feeder cells, mouse sarcoma-derived matrigel, etc.
  • EB embryoid body
  • the "human neurogenin 2 gene” of the present invention is one of the factors promoting the differentiation of neural stem cells into neurons, and is a bLHL type transcription factor.
  • the "iPS cell into which the human neurogenin 2 gene has been introduced” in the present invention is an iPS cell into which the human neurogenin 2 gene has been introduced by the RNA lipofection method, or a plasmid vector or a virus vector in which the neurogenin 2 gene is loaded. , Refers to iPS cells into which a gene has been introduced with a vector capable of transiently inducing expression.
  • RNA lipofection method is a method in which positively charged cationic liposomes bind around negatively charged DNA to form a complex, and the DNA is taken up from the cell surface into the cell by endocytosis phenomenon.
  • a vector in which the neurogenin 2 gene is mounted on a plasmid vector or a viral vector and whose expression can be transiently induced is prepared according to the method of Kim et al. (Method in Molecular Biology (2016) 1357: 111-131). can.
  • Kim et al. A vector in which doxycycline transiently expresses neurogenin 2 can be prepared and a gene can be introduced into iPS cells.
  • the "laminin coating” of the present invention means coating a culture vessel (culture dish, etc.) with a human recombinant laminin fragment. It is preferable to coat with iMatrix-511 (Nippi). For the differentiation of iPS cells and iPS cells into neurons and the maintenance of the culture of the differentiated neurons, it is desirable to perform two-dimensional culture on a laminin-coated culture dish. For differentiation of iPS cells into nerve cells and maintenance of nerve cells, a 1: 1 mixed medium of Neurobasal Medium or Neurobasal Medium and DMEM / F12 can be used.
  • the "transient expression induction" of the present invention means to introduce the human neurogenin 2 gene into iPS cells and temporarily express the transcription factor neurogenin 2 protein.
  • Expression induction is performed by lipofection in the RNA lipofection method, but when a vector is used, for example, since a vector in which neurogenin 2 is transiently expressed by doxycycline is used, only in the presence of doxycycline is used. Neurogenin 2 can be expressed transiently.
  • the "small molecule TGF ⁇ family inhibitor" of the present invention refers to a small molecule compound that has a structure similar to that of TGF- ⁇ and inhibits the signal transduction of the TGF ⁇ family that transmits signals by a similar pathway.
  • the TGF ⁇ family is involved in a wide variety of cell functions such as cell proliferation, differentiation, development, and regulation of apoptosis. By suppressing or inhibiting this action, for example, proliferation and function of blood cells and lymphocytes are also performed. Can be promoted.
  • TGF ⁇ family examples include SB431542, LY364947 (4- [3- (2-pyridinyl) -1H-pyrazole-4-yl) -quinoline), LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, SB505124, GW788388 and the like can be mentioned.
  • the "adenylate cyclase activator” of the present invention is a compound that activates adenylate cyclase, and adenylate cyclase converts cAMP, which is a second messenger of many neurotransmitters and hormones, from ATP. It is an enzyme that is synthesized by Examples of the activator of adenylate cyclase include F0855 (forskolin) and DG02877 (corhorsin daropert hydrochloride).
  • the "GSK3 inhibitor” of the present invention refers to an inhibitor of glycogen synthase 3 (Glycogen kinase kinase 3) involved in cell division, cell proliferation, cell motility and cell survival.
  • CHIR-99021 (6-[[2-[[4- (2,4-Dicholopenyl) -5- (4-methyl-1H-imidazole-2-yl) -2-pyrimidinyl] amino] ethyl] amino] nicotinonirile), A1070722 (1- (7-Methyloxyquinolin-4-yl) -3- [6- (trifluoromethyl) pyrridin-2-yl] urea), BIO ((2'Z, 3'E) -6-Bromodirubin- 3'-oxyme), SB216763 (3- (2,4-Dicholopenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione), SB415286 (3-[ (3-Chloro-4-hydroxyphenyl) amineo] -4- (2-nitrophenyl) -1h-pyrrole-2,5-dione), TC-G24 (N- (3- (3-
  • the “Smoothened agonist” of the present invention is an agonist (promoter) of the 7-transmembrane membrane protein Smoothened that increases the proliferation and survival of nerve cells.
  • agonist promoter
  • palmorfamine (2- (1-Naphthoxy) -6- (4-morpholinonaylino) -9-cyclohexylpurine
  • SAG 3-Chloro-N- [trans-4- (methylamino) cyclohexyl] -N- [3. -(4-Pyridinyl) benzyl] -1-benzothiophene-2-carboxamide
  • the “Rock inhibitor” of the present invention refers to an inhibitor of ROCK (p160-Rho-associated coiled-coil kinase), which is a phosphorylating enzyme involved in the basics of cell functions such as cell proliferation and gene expression.
  • ROCK p160-Rho-associated coiled-coil kinase
  • the ROCK inhibitor is a substance that exhibits a very strong cell death inhibitory action, and examples thereof include Y-27632, Thiazovivin, RKI-1447, GSK-4229286A, and Fasudil Hydrochloride.
  • the "nerve growth factor” of the present invention is a cell growth / growth factor or cytokine that has an action on nerves, and is a nerve growth factor (NGF) or a brain-derived neurotrophic factor (brain-derived).
  • nerve growth factor / BDNF
  • neurotropin-3 NT-3
  • neurotropin-4 / 5
  • Glyal Cell Line-derivated Neurotropic Factor GDNF
  • IGF-1 Insurin-like Growth Factor-1 Vascular Endothelial Growth Factor (VEGF)
  • VEGF Vascular Endothelial Growth Factor
  • the term "cell activity is weak" in the present invention means that the cell is inactive as if it stagnates or dies during the development of differentiation.
  • iPS cells when inducing differentiation of iPS cells derived from patients with organically problematic diseases caused by genetic mutations or epigenetic abnormalities, the iPS cells are overloaded and the cell activity is weakened. In the middle of differentiation, cell differentiation will be stagnant or cells will die.
  • the "patient with a neuropsychiatric disorder” of the present invention refers to a disease in which a lesion of the nerve itself such as the brain, spinal cord, or peripheral nerve or a lesion of the muscle itself causes movement disorder or cognitive disorder.
  • a lesion of the nerve itself such as the brain, spinal cord, or peripheral nerve or a lesion of the muscle itself causes movement disorder or cognitive disorder.
  • Examples include Parkinson's disease, spinocerebellar degeneration, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, neuritis and myelitis caused by viruses and fungi, myasthenia gravis, muscular dystrophy, and polymyelitis.
  • cerebrovascular disorders having a wide range of pathological conditions from dementia, acute stroke to gradually progressing vascular dementia, Huntington's disease, aging and the like.
  • regression of nerve protrusions decreased spine density, depression with diminished neurogenesis in the hippocampus, schizophrenia, bipolar disorder, panic disorder, PTSD, panic disorder, anxiety disorder and the like can be mentioned.
  • developmental disorders such as autism spectrum disorder due to abnormal synaptic pruning during brain development and attention deficit hyperactivity disorder can be mentioned.
  • the "screening method for a therapeutic agent for neurological disease” of the present invention is a disease-specific method that induces differentiation of iPS cells derived from a neurological disease patient into nerve cells, particularly motor nerve cells, and reflects the patient's nerve cells (particularly motor nerve cells). It is a method of producing a typical nerve cell and using the cell to screen for an optimal therapeutic drug for the disease.
  • the disease-specific neurons induced to differentiate in the present invention can be used to screen compounds effective for treating neurological disorders (eg, pharmaceutical compounds, solvents, small molecules, peptides, or polynucleotides). For example, screening compounds can be evaluated by screening the drug alone or in combination with other compounds and analyzing differentiation-induced disease-specific morphological or functional changes in neuronal cells.
  • morphological changes it can be evaluated by extension of dendrites, increase in the number of dendrite branches, increase in spines on dendrites, extension of axons, and the like.
  • evaluation of functional changes changes in gene expression, or changes in the type or amount of produced protein can be used for evaluation.
  • the differentiation-induced disease-specific neurons of the present invention can be used for analysis of neurological disease-specific neuronal cell-specific kinetics.
  • the "cell activator" of the present invention can simultaneously promote the activity of a plurality of factors that are required to work together in a physiological environment, and is a drug discovery targeting a single molecule so far. It is the basis of a completely new type of therapeutic drug with physiological functionality.
  • ALS muscle atrophic lateral sclerosis
  • PTSD post-traumatic stress disorder
  • anxiety disorder panic disorder
  • autism spectrum disorder developmental disorder
  • let syndrome down syndrome
  • spinal cord injury etc.
  • it is a small molecule compound, inexpensive and easy to store, and has a high possibility of crossing the blood-brain barrier, which sets it apart from the neurotrophic factor replacement therapy that has been sought for use. ..
  • “Culture can be maintained” in the present invention means that stable and finally differentiated neurons can be cultured and maintained for at least 4 months under Xeno-free culture conditions containing no unidentified impurities using a small number of steps and reagents. To say. In the case of iPS cells derived from neurological disease patients, even if the cell activity is low, for example, by adding an indolinone compound, finally differentiated nerve cells without dying on the way are prepared and cultured. It means something that can be maintained.
  • the first aspect of the present invention Establishment of feeder-free iPS cell culture system (1) Materials / reagents 1) Healthy person-derived iPS cells (HSP0003, 0009, 0029) and ALS patient-derived iPS cells (HSP0290 (familial), 0292 (Family), 0134 (Sporadic), 0140 (Family)) (National Development Agency RIKEN BioResource Center (hereinafter RIKEN BRC); 3) iPS cell culture medium StemFit AK02N Ajinomoto (Takara Bio); 4) iMatrix-511 Nippi (Takara Bio); 5) Y-27632 10 mM solution (Fuji Film Wako Pure Chemical Industries, Ltd.); 6) TrypLE Select (Thermo Fisher); 7) STEM-CELLBANKER (Takara Bio); 8) D-PBS (-) (Nacalai Tesque); 10) Bisel freezing container (Fun
  • the StemFit medium was added and pipeted to disperse the cells well and transferred to a centrifuge tube, and iPS cells were collected by centrifugation.
  • the collected iPS cells were dispersed in 1 ml of StemFit + Y medium and counted, and about 4x10 4 iPS cells per culture dish were dispersed in 4 ml of StemFit + Y medium and seeded in a laminin-coated culture dish. After several passages, it was possible to acclimatize to the feeder-free culture system.
  • FIG. 1A A microscopic image of a culture of healthy iPS cells is shown in FIG. 1A.
  • Vector-incorporated iPS cells were selected by G418 (gradual increase from 50 ⁇ g / mL to 100 ⁇ g / mL) and transiently neurogenin 2 expression by 5 or more doxycyclines for each iPS cell line by limiting dilution. A cell line that can be induced in Japan was established. (Hereinafter, iPS cells in which neurogenin 2 is transiently expressed by doxycycline are collectively referred to as iPS-PBN2).
  • Neurogenin is a transcription factor that is a switch when pluripotent stem cells differentiate into nerve cells.
  • the classical method is to differentiate stem cells into nerve cells via cell cells (embryonic cells (cell aggregates) formation), but in culture without feeder cells, germ layer cell (cell aggregates) formation occurs. Difficult, this method requires complicated processes and the addition of many drugs-many of which show cytotoxicity-and the time required for differentiation is long, one month or longer, so it is a simple method. Was developed.
  • NDM + 10 ⁇ M Y-27632 + 3 ⁇ M SB431542 + 3 ⁇ M CHIA99021 + 3 ⁇ M horskolin medium was removed from the culture dish, washed once with 4 mL of D-PBS (-), added with 1 mL of TrypLE Select, and placed in a 5% CO 2 incubator at 37 ° C. After reacting for 9 minutes, 4 ml of NDM medium was added and pipetting was performed to disperse the cells well and transferred to a centrifuge tube, and the cells collected by centrifugation were dispersed in 1 ml of NDM medium to count the number of cells.
  • NDM + 10 ⁇ M Y-27632 + 3 ⁇ M horscholine + 3 ⁇ M palmolfamine medium seeded in a laminin-coated 60 mm culture dish (D4).
  • the medium was replaced with NDM + Y-27632 + neurotrophic factor (10 ng / mL recombinant BDNF + 10 ng / mL recombinant GDNF + 10 ng / mL recombinant IGF-1) medium (D6). Every 3 days, the medium was replaced with fresh NDM + Y-27632 + neurotrophic factor medium.
  • RNA was extracted over time by a conventional method using isogen, and a reverse transcription reaction was performed using PrimeScript RT reagent Kit. Using the cDNA product obtained by the reverse transcription reaction and the TB Green Premix EX taq II reagent, the change in mRNA expression of ⁇ -actin, PAX6, HB9, and GluA1 over time was measured using SmartCyclor.
  • HB9 is widely used as the most reliable motor nerve marker. Since it is essential for mature neurons to functionally transmit signals by neurotransmitters, the neurotransmitter receptor GluA1 was used as one of the markers for mature neurons. PAX6 was used because the primer design could be performed smoothly.
  • FIG. 2A shows the morphological changes of the iPS cell line HSP0003-PBN2 # 2 derived from healthy subjects due to the differentiation into motor neurons by induction of neurogenin 2.
  • axons and dendrites were elongated from the cell body, and the characteristics as nerve cells were morphologically confirmed (FIG. 2B).
  • Induction of differentiation into motor neurons was also observed in other iPS-PBN2 strains (iPS0009-PBN2 # 2, iPS0029-PBN2 # 2).
  • FIG. 2C is a schematic representation of the expression patterns of neural precursor cell markers (PAX6), motor nerve markers (HB9), and mature nerve cell markers (neurotransmitter glutamate receptor 1 (GluA1)) associated with the differentiation of iPS cells into motor nerves.
  • PAX6 neural precursor cell markers
  • HB9 motor nerve markers
  • GluA1 mature nerve cell markers
  • FIG. 3 shows an example of changes in the expression of marker gene mRNA associated with the differentiation of HSP0003-PBN2 # 2 derived from a healthy subject into motor neurons.
  • Expression of the neural progenitor cell marker (PAX6) showed a transient and significant increase after induction of differentiation, and then decreased with the maturation of motor neurons.
  • HB9 motor neuron marker
  • GluA1 neurotransmitter receptor GluA1
  • Example 1-3 Effect of addition of semaxanib during differentiation from iPS cells to motor neurons (1) Materials / reagents 1) Healthy subject-derived iPS cell line HSP0003 that can transiently induce neurogenin 2 expression by docycycline -PBN2 # 2 and iPS cell line derived from sporadic ALS patients HSP0134-PBN2 # 1; 3) iPS cell culture medium StemFit AK02N Ajinomoto (Takara Bio); 4) iMatrix-511 Nippi (Takara Bio); 5) Y-27632 10 mM solution (Fuji Film Wako Pure Chemical Industries, Ltd.); 6) Doxycycline 1 mg / mL (LAT Laboratories); 7) 10 mM SU5416 (Semaxanib) (Selleck S2845); 8) TrypLE Select (Thermo Fisher); 9) DMEM / HAM's F12 medium (Fuji Film Wako
  • NDM + 10 ⁇ M Y-27632 + 3 ⁇ M SB431542 + 3 ⁇ M CHIA99021 + 3 ⁇ M forskolin medium D1.
  • Semaxanib was added to the culture medium to a final concentration of 2.5 ⁇ M.
  • differentiated neural progenitor cells proliferate actively and form colonies.
  • the cells were collected by the method shown in (Example 1-1) in order to space the cells so as to be suitable for the subsequent observation of nerve cells, that is, first, NDM + 10 ⁇ M Y-27632 + 3 ⁇ M SB431542 + 3 ⁇ M CHIA99021 + 3 ⁇ M Holscholine medium was removed from the culture dish.
  • the cells were washed once with 4 mL of D-PBS (-), 1 mL of TripLE Select was added, and the cells were reacted at 37 ° C. in a 5% CO 2 incubator for 9 minutes, and then 4 ml of StemFit medium was added for pipetting.
  • the cells were well dispersed, transferred to a centrifuge tube, and collected by centrifugation. After that, except for the addition of semaxanib, (Example 1-2) was followed.
  • the collected cells were dispersed in 1 ml of NDM medium, the number of cells was counted, and 2x10 5 cells per culture dish were suspended in 4 mL of NDM + 10 ⁇ M Y-27632 + 3 ⁇ M horskolin + 3 ⁇ M palmolfamine medium and coated with laminin.
  • the seeds were sown in a 60 mm culture dish (D4).
  • the medium was replaced with NDM + Y-27632 + neurotrophic factor (10 ng / mL recombinant BDNF + 10 ng / mL recombinant GDNF + 10 ng / mL recombinant IGF-1) medium (D6).
  • the medium was replaced with fresh NDM + neurotrophic factor + 2.5 ⁇ M semaxanib medium or NDM + neurotrophic factor + DMSO medium.
  • the medium was divided into a group in which the medium was replaced with a new NDM + Y-27632 + neurotrophic factor + 2.5 ⁇ M semaxanib medium and a group in which the medium was not renewed.
  • RNA was extracted over time by a conventional method using isogen, and a reverse transcription reaction was carried out using PrimeScript RT reagent Kit.
  • FIG. 4A the iPS cell line HSP0003-PBN2 # 2 derived from healthy subjects differentiated into motor neurons with or without the addition of semaxanib. Microscopic images of motor neurons on the first day of induction of differentiation (left column of FIG. 4A) and the 33rd day of induction of differentiation (right column of FIG. 4A) are shown.
  • FIG. 4B shows a microscopic image of the first day of differentiation induction.
  • both the control group and the semaxanib-added group were used until the fourth day.
  • iPS cell lines derived from healthy subjects In HSP0003PBN2 # 2, a 5-fold increase in BDNF expression was detected in the semaxanib-added group as compared with the control group, and a 14-fold increase in BDNF expression was detected as compared with iPS cells.
  • the control group died on the 15th day and could only be compared with iPS cells, but a 3.5-fold increase in BDNF expression was detected compared with iPS cells. rice field.
  • semaxanib has an effect of promoting the expression of BDNF in the motor neurons differentiated from the iPS cells derived from healthy subjects and the iPS cells derived from ALS patients, respectively. Further, from the microscopic image of FIG. 4, it is recognized that the motor neuron has almost 100% neurite outgrowth, indicating that the motor neuron itself is an autocline. In healthy subjects, nerve cells converted to express BDNF necessary for themselves by autocrine, but in ALS patients, this conversion could not be performed, and it is speculated that BDNF expression decreased and died. To. Thus, it was revealed that semaxanib acts on motor neurons and promotes the expression of BDNF by autocline. It is considered that semaxanib can efficiently activate motor neurons by promoting the expression of BDNF autocrine by the motor neurons themselves.
  • Example 1-4 Evaluation of expression of protein genes related to survival maintenance, neuroplasticity, proliferation and differentiation promotion of nerve cells in motor nerve cells differentiated from iPS cells
  • Materials / reagents 1) Neurogenin 2 by doxicycline Normal human-derived iPS cell line HSP0003-PBN2 # 2 and sporadic ALS patient-derived iPS cell line HSP0134-PBN2 # 1; which can induce expression transiently; 3) iPS cell culture medium StemFit AK02N Ajinomoto (Takara Bio); 4) iMatrix-511 Nippi (Takara Bio); 5) Y-27632 10 mM solution (Fuji Film Wako Pure Chemical Industries, Ltd.); 6) Doxycycline 1 mg / mL (LAT Laboratories); 7) 10 mM SU5416 (Semaxanib) (Selleck S2845); 8) TrypLE Select (Thermo Fisher); 9) DMEM / HAM
  • the collected cells were dispersed in 1 ml of NDM medium, the number of cells was counted, and 2x10 5 cells per culture dish were divided into 4 mL of NDM + 10 ⁇ M Y-27632 + 3 ⁇ M horskolin + 3 ⁇ M palmorfamine + 2.5 ⁇ M semaxanib medium or 4 mL.
  • the cells were suspended in NDM + 10 ⁇ M Y-27632 + 3 ⁇ M horskolin + 3 ⁇ M palmolfamine + DMSO medium and seeded in a laminin-coated 60 mm culture dish (D4).
  • ⁇ -actin Using the cDNA product obtained by the reverse transcription reaction and the TB Green Premix EX taq II reagent, ⁇ -actin, VEGF-A, C, D and VEGF receptors-1, 2, 3 and neuropyrin 2 over time. Changes in mRNA expression were measured using a Smart Cycler.
  • the real-time PCR reactions are for ⁇ -actin, Vascular Endothelial Growth Factor (VEGF) -A, VEGF-C, VEGF-D, VEGF Receptor-1, VEGF Receptor-2, VEGF Receptor-3, and Neuropyrin 2, respectively. It was performed using the primer pair of No. 1 and the TB Green Premix EX taq II reagent, and detected by SmartCycler.
  • VEGF Vascular Endothelial Growth Factor
  • Results Fig. 6A shows the evaluation results of protein gene expression promotion related to survival maintenance, neuroplasticity, nerve cell proliferation and differentiation promotion in motor nerve cells differentiated from healthy person-derived iPS cell line HSP0003-PBN2 # 2. rice field.
  • the indolinone compound (semaxanib) -added group obtained the following gene expression-promoting effects.
  • the expression of VEGF-A increased about 3-fold (upper left figure).
  • the expression of VEGF-C and VEGF-D increased about 9.5-fold (upper middle figure) and about 18-fold (upper right figure), respectively.
  • VEGF receptor-2 decreases with differentiation into nerve cells, but the expression was maintained by the addition of semaxanib (lower left figure).
  • FIG. 6B shows the evaluation results of survival maintenance, neuroplasticity, and promotion of protein gene expression related to nerve cell proliferation and differentiation promotion in motor neurons differentiated from the iPS cell line HSP0134-PBN2 # 1 derived from sporadic ALS patients. .. In the control group, the cells died by 15 days after differentiation, but in the semaxanib-added group, the expression promoting effect of each gene was obtained as follows. 1) The expression of VEGF-A increased about 2-fold as compared with iPS cells (upper left figure).
  • VEGF-C and VEGF-D increased about 5.5 times (upper middle figure) and about 4 times (upper right figure), respectively, as compared with iPS cells.
  • VEGF receptor-2 which decreases with differentiation into nerve cells, was maintained at about 30% compared with iPS cells by the addition of semaxanib (lower left figure).
  • Expression of VEGF receptor-3 was maintained at about 80% compared to iPS cells (lower middle figure).
  • Expression of VEGF receptor-2VEGF and receptor-3 co-receptor neuropilin 2 was increased about 5-fold as compared with iPS cells (lower right figure).
  • semaxanib has an effect of promoting the expression of VEGF signal transduction protein on motor neurons differentiated from iPS cells.
  • semaxanib acts on motor neurons and promotes the expression of VEGF signal transduction protein as an autocline.
  • the following materials were used to induce differentiation of iPS cells into motor neurons.
  • (Materials / reagents) 1) Healthy subject-derived iPS cells (HSP0003, 0009, 0029) and ALS patient-derived iPS cells (HSP0290 (familial), 0292 (familial), 0134 (sporadic), 0140 (familial)) (RIKEN BRC) ; 2) iPS cell culture medium StemFit AK02N Ajinomoto (Takara Bio); 3) iMatrix-511 Nippi (Takara Bio); 4) ROCK inhibitor (Y-27632) 10 mM solution (Fujifilm Wako Pure Chemical Industries, Ltd.); 5) TrypLE Select (Thermo Fisher); 6) Transposon vector for human iPS cells piggyBac, All-in-One PB-TAG-ERN (RIKEN BRC) (Kim et al., Method in Molecular
  • Example 2-1 Preparation of healthy person-derived iPS cells and ALS patient-derived iPS cells into which the human neurogenin 2 gene has been introduced
  • Method A stop codon by inserting a base into the human neurogenin 2 gene purchased from Kazusa Technologies according to a conventional method. Was added and cloned into pENTR1A. According to Kim et al. (2016, Method in Molecular Biology. 1357: 111-131), iPS cells in which doxycycline transiently expresses neurogenin 2 were generated. However, the gene transfer was performed by using the reverse transfection method using TransIT-LT1 instead of the electroporation method.
  • the iPS cells into which the vector was incorporated were selected by G418 (gradual increase from 50 ⁇ g / mL to 100 ⁇ g / mL), and neurogenin 2 expression was transiently expressed by doxycycline from each iPS cell line by the limiting dilution method.
  • As cell lines that can be induced to sex iPS0003-PBN2 # 1, 2, 3, 4, 5, iPS0009-PBN2 # 1, 2, 3, 4, 5, iPS0029-PBN2 # 1, 2, from normal human-derived iPS cells, Established 3, 4, and 5.
  • iPS0290-PBN2 # 1, 2, 3, 4, 5, iPS0292-PBN2 # 1, 2, 3, 4, 5, iPS0134-PBN2 # 1, 2, 3, 4, 5, iPS0140-PBN2 # 1, 2, 3, 4, 5 was established (hereinafter, iPS cells in which neurogenin 2 is transiently expressed by doxycycline are collectively referred to as iPS-PBN2).
  • Example 2-2 Incubation of iPS cells derived from healthy subjects into which the human neurogenin 2 gene has been introduced (1) Method Add 20 ⁇ l of iMatrix-511 to a 60 mm culture dish containing 1 mL of D-PBS (-) and 37 ° C. Laminin coating was performed by reacting in a 5% CO 2 incubator for 60 minutes or more. Next, the solution is removed, replaced with StemFit medium, and placed in an incubator for familiarization. A ROCK inhibitor (Y-27632) was added to the required amount (4 mL culture dish) of StemFit medium to a final concentration of 10 ⁇ M, placed in an incubator, and each iPS-PBN2 obtained in Example 1 was suspended.
  • Y-27632 ROCK inhibitor
  • the seeds were seeded on a laminin-coated culture dish from which StemFit medium had been removed.
  • 5x10 4 iPS-PBN2 cells per 60 mm culture dish coated with laminin as described above were suspended in 4 mL (StemFit + Y) + 1 ⁇ g / mL doxycycline (DOX), seeded and cultured for 16 hours. ..
  • Example 2-2 Induction of differentiation of neural stem cell-like cells from healthy person-derived iPS cells into nerve cells (1) Method The following is a culture dish as it is, which was cultured in the presence of DOX for 16 hours in Example 2. The medium was exchanged as in the above, and differentiation was induced into nerve cells. a) Primary culture NDM medium containing ROCK inhibitor (10 ⁇ M Y-27632) + TGF ⁇ family inhibitor (3 ⁇ M SB431542) + GSK3 inhibitor (3 ⁇ M CHIR99021) + adenylate cyclase activator (3 ⁇ M forskolin) Exchanged for.
  • neural progenitor cells proliferated actively and reached about 70% of the culture area.
  • NDM + 10 ⁇ M Y-27632 + 3 ⁇ M SB431542 + 3 ⁇ M CHIA99021 + 3 ⁇ M horskolin medium wash once with 4 mL of D-PBS (-), add 1 mL of TripLE Select, and react in a 5% CO 2 incubator at 37 ° C. for 9 minutes. After that, 4 ml of NDM medium was added and pipetted to disperse well, transferred to a centrifuge tube, and cells were collected by centrifugation.
  • the collected cells were dispersed in 1 ml of NDM medium and the number of cells was counted.
  • the medium contains ROCK inhibitor (Y-27632) + neurotrophic factor (10 ng / mL recombinant BDNF + 10 ng / mL recombinant GDNF + 10 ng / mL recombinant IGF-1) in the culture dish as it is. It was replaced with NDM medium. Then, every 3 days, the medium was replaced with a new NDM medium containing a neurotrophic factor.
  • Use Isogen over time typically controlling day 1 without doxycycline, 1st, 4th, 15th, 18th, 22nd, 33rd, etc.
  • FIG. 11 shows the expression dynamics of nerve cell differentiation marker mRNA.
  • the expression of the neural stem cell marker PAX6 first increased sharply and decreased with the progress of differentiation (above figure a). Subsequently, the expression of the motor neuron marker HB9 rapidly increased, and the expression level was maintained at a high level while gradually increasing with the progress of differentiation (middle figure b).
  • GluA1 When the expression of GluA1 was examined as a marker for mature neurons, GluA1 rapidly increased on the 18th day when the expression of PAX6 began to decrease, and the high expression was maintained as it was (Fig. C below). Differentiated neurons could be observed for up to 4 months. As described above, it is considered that the majority of differentiated neurons become the majority after the 18th day after the start of the differentiation induction, and the differentiation induction is completed to become mature neurons about one month later.
  • Example 2-3 Incubation of iPS cells derived from ALS patients into which the human neurogenin 2 gene was introduced (1) Method 20 ⁇ l of iMatrix-511 was added to a 60 mm culture dish containing 1 mL of D-PBS (-), and the temperature was 37 ° C. Laminin coating was performed by reacting in a 5% CO 2 incubator for 60 minutes or more. The solution was then removed, replaced with StemFit medium and placed in an incubator for familiarization.
  • a ROCK inhibitor (Y-27632) was added to the required amount (4 mL / petri dish) of StemFit medium to a final concentration of 10 ⁇ M, and the mixture was placed in an incubator to obtain iPS0134 from a sporadic ALS patient obtained in Example 1.
  • PBN2 # 1 was suspended in StemFit + Y-27632 medium in an incubator and seeded on a laminin-coated culture dish.
  • 5x10 4 ALS patient-derived iPS0134-PBN2 # 1 cells per 60 mm cultured dish coated with laminin as described above were suspended in 4 mL (StemFit + Y) + 1 ⁇ g / mL doxycycline (DOX) and seeded.
  • DOX ⁇ g / mL doxycycline
  • Example 2-4 Induction of differentiation of neural progenitor cell-like cells from ALS patient-derived iPS cells into nerve cells (1)
  • the indolinone compound (semaxanib) -added group a final concentration of 2.5 ⁇ M was added at the same time as the initiation of differentiation by the addition of doxycycline (DOX).
  • DOX doxycycline
  • a) Primary culture Medium of neural stem cell-like cells contains ROCK inhibitor (10 ⁇ M Y-27632) + TGF ⁇ family inhibitor (3 ⁇ M SB431542) + GSK3 inhibitor (3 ⁇ M CHIR99021) + adenylate cyclase activator (3 ⁇ M forskolin) The medium was replaced with NDM medium. In the group to which the indolinone compound (semaxanib) was added, a final concentration of 2.5 ⁇ M was added. By the 4th day from the start of differentiation by adding DOX, neural progenitor cells proliferated vigorously and reached about 70% of the culture area.
  • NDM + 10 ⁇ M Y-27632 + 3 ⁇ M SB431542 + 3 ⁇ M CHIA99021 + 3 ⁇ M horskolin medium wash once with 4 mL of D-PBS (-), add 1 mL of TripLE Select, and react in a 5% CO 2 incubator at 37 ° C. for 9 minutes. After that, 4 ml of NDM medium was added and pipetted to disperse well, transferred to a centrifuge tube, and cells were collected by centrifugation. The collected cells were dispersed in 1 ml of NDM medium and the number of cells was counted.
  • Example 2-5 Effect of promoting expression of survivin gene by indolinone compound (semaxanib) (1) Method Similar to Example 5, an indolinone compound (semaxanib) -added group and an indolinone compound (semaxanib) -added group were formed, and iPS cells derived from healthy subjects iPS0003 -PBN2 # 2 and iPS cells iPS0134-PBN2 # 1 derived from sporadic ALS patients were differentiated into neurons.
  • RNA was extracted over time by a conventional method using isogen, and a reverse transcription reaction was performed using PrimeScript RT reagent Kit. Using the cDNA product obtained by the reverse transcription reaction and the TB Green Premix EX Taq II reagent, changes in the mRNA expression of survivin were measured using a Smart Cycler.
  • the nerve cells induced to differentiate from the iPS cells derived from the ALS patient died on the 15th day of the induction of differentiation, but the indolinone compound (semaxanib) was killed.
  • the nerve cells induced to differentiate from the iPS cells derived from ALS patients are still alive and maintained even on the 21st day of the induction of differentiation, and as shown in FIG. 13, the surviving gene is maintained.
  • the expression was increased (up to about 3 times that of iPS cells derived from ALS patients and up to about 80% of healthy person-derived nerve cells to which no indolinone compound was added).
  • the gene expression of survivin was increased 2.7-fold on the 22nd day of the induction of differentiation.
  • the indolinone compound (semaxanib) promotes the expression of the cybabin gene in nerve cells induced in both healthy human-derived iPS cells and ALS patient-derived iPS cells by addition from the start of differentiation. ..
  • the effect of promoting the expression of the cybabin gene at the time of inducing differentiation can suppress the death of cells after induction of neuronal differentiation derived from ALS patients, which are vulnerable to the stress of inducing neuronal differentiation and are prone to death.
  • the indolinone compound of the present invention has an effect of acting on motor neurons and promoting the expression of BDNF and / or VEGF signaling protein as an autocline. Therefore, it has been clarified that the indolinone compound of the present invention is an extremely useful therapeutic agent for a wide range of neuropsychiatric disorders. Furthermore, as shown by semaxanib, in the case of an indolinone compound having the effect of promoting the simultaneous expression of BDNF and VEGF signaling protein, when BDNF and VEGF signaling protein perform a physiological function in a coupled manner (depression).
  • Non-Patent Documents 7 and / or synergistic effects further improve nerve cell maintenance and neuroplasticity, promote proliferation and differentiation of neural stem cells, and activate the biological dynamics of motor nerve cells. Can be transformed into.
  • ALS muscle atrophic lateral sclerosis
  • PTSD post-traumatic stress disorder
  • anxiety disorder panic disorder
  • autism spectrum disorder developmental disorder
  • Let syndrome, Down syndrome, post-stroke It is an extremely useful therapeutic agent for a wide range of neuropsychiatric disorders such as symptom recovery and functional recovery after spinal cord injury, and as an excellent adjunct to regenerative medicine by nerve stem cell transplantation, it is used to treat Parkinson's disease, Alzheimer's disease, and spinal cord injury.
  • Second Embodiment of the present invention With the cybabin expression promoter of the present invention, even cells in which the cell activity of iPS cells is weak, the expression of the cybabin gene is reduced, and the cells are likely to die at the time of differentiation induction can be finally differentiated.
  • the expression of the cybabin gene is significantly reduced, but by adding the indolinone compound at the time of differentiation induction, the expression of survivin is improved and there is no premature cell death. Differentiated nerve cells can now be produced.
  • the method for inducing differentiation of the present invention has enabled efficient differentiation of neurons from induced pluripotent stem cells and long-term culture of differentiated neurons.
  • a xenofree culture system that does not contain unidentified components derived from non-human organisms such as feeder cells, mouse sarcoma-derived matrigel, and bovine serum, which have lot differences.
  • nerve cells peculiar to ALS can be easily produced from iPS cells derived from ALS patients, so that a compound for treating ALS (for example, a pharmaceutical compound, a solvent, a small molecule, a peptide, or a polynucleotide) can be easily produced. It has become possible to perform screening.
  • the screening results are not affected by unidentified components, and more accurate screening has become possible. Furthermore, by inducing the differentiation of nerve cells from iPS cells derived from individual patients, it has become possible to evaluate the therapeutic effect of the drug on the neurological disease of each patient in advance. In addition, the ability to maintain nerve cell culture over a long period of time in a Xenofree culture system has made it possible to evaluate changes over time due to neurological diseases and screen therapeutic agents in more detail.
  • the cyberbin expression promoter of the present invention which promotes the expression of survivin, which plays an important role in maintaining healthy brain activity, activates neurogenesis in the adult hippocampus, especially in the adult hippocampus after traumatic brain injury.

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Abstract

Les présents inventeurs ont découvert que le composé indolinone de la présente invention a un excellent effet d'activation des cellules nerveuses. Les présents inventeurs ont en outre découvert que le mécanisme de fonctionnement de l'effet d'activation des cellules nerveuses est basé sur la promotion de l'expression d'une protéine qui s'apparente au système de signalisation du BDNF et/ou au système de signalisation du VEGF. Ainsi, le composé indolinone de la présente invention peut être utilisé en tant qu'agent thérapeutique pour diverses maladies neuropsychiatriques. De plus, ce composé peut protéger des cellules progénitrices neurales transplantées et, par conséquent, peut être utilisé comme auxiliaire en médecine régénérative.　Les présents inventeurs ont établi un procédé pour induire la différenciation de cellules souches pluripotentes induites (cellules iPS) en cellules nerveuses à un rendement élevé sans passer par la formation d'un corps embryonnaire (agrégat cellulaire) dans un système de culture exempt de xéno-contaminants. Grâce à ce procédé, des cellules nerveuses, qui ne contiennent pas de facteur dérivé d'un organisme différent, n'ont été affectées ni par des composants non identifiés, ni par des différences entre lots et avaient des caractéristiques d'un patient atteint d'une maladie, ont été préparées avec succès. Par conséquent, il devient possible de cribler et d'analyser de manière stable un composé pour le traitement d'une maladie neurologique pour la découverte de médicaments.
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