Method for screening active pharmaceutical ingredient using function of cell or tissue as index

Abstract

An object of the present invention is to provide a novel screening technique for selecting a useful pharmaceutical compound.SOLUTION: The present invention for solving the above-mentioned problems is a method for screening an active ingredient of a medicament, comprising a step of topically administering a candidate substance to a region where a specific tissue develops in an embryo of an animal (excluding human) or the vicinity thereof, a step of observing the specific tissue or a differentiated cell of the specific tissue in the animal after the topical administration, and a step of selecting, as the active ingredient, a candidate substance observed to improve a function of the specific tissue or the differentiated cell as compared with the case where the candidate substance is not administered.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening for a pharmaceutical active ingredient, a method for producing a pharmaceutical, a method for designing a pharmaceutical, and a pharmaceutical composition. [Background technology]

[0002] Adult stem cells are rare, undifferentiated cells found in all tissues of the body. Normally maintained in a quiescent, non-dividing state, these cells can proliferate and differentiate to replace naturally dying cells in tissues and to repair wounds in response to injury. Due to their proliferation and tissue regenerative capacity, adult stem cells have the potential to treat not only aging but also various degenerative diseases (Non-Patent Document 1).

[0003] For example, cardiac stem cells / cardiac progenitor cells (CSCs) that have the ability to differentiate into all cardiac lineage cells are known to exist in the heart (Non-Patent Document 2).

[0004] Furthermore, investigations into the localization of the transcription factor SOX9, which functions to maintain cells in an undifferentiated state, have reported that SOX9 is expressed in crypts containing Lgr5-positive cells, which are stem cells, in the intestine, in the pancreas, and exclusively in the pancreatic duct and its terminal duct cells, the centroacinar cells, and in the liver, in bile duct cells (Non-Patent Document 3).

[0005] In mature mammals, nerve cells generally do not have the ability to divide. Therefore, once damaged, the damage continues for a long time. It was previously believed that the central nervous system, particularly the brain and spinal cord, had no regenerative capacity. It was also said that the lack of regenerative capacity in the central nervous system was one of the reasons why there was no treatment for paralysis caused by external injuries such as spinal cord injury. On the other hand, peripheral nerves have the ability to regenerate, and even after they are severed, axons regrow and function is restored. However, even in this case, regeneration takes a long time, from several months to over a year. This places a heavy burden on patients in improving their quality of life. Furthermore, because nerve regeneration takes such a long time, nerve cells often die during that time, preventing functional recovery.

[0006] Recent studies have revealed the presence of adult neural stem cells in the adult brain, which are responsible for neurogenesis. Adult neural stem cells are a unique cell subpopulation characterized by structural plasticity. In mammals, neurogenesis occurs in two sprouting regions: the SVZ and the SGZ throughout life. To date, various markers have been discovered that are expressed in a multi-step strategy during the progression of adult hippocampal neurogenesis. Radial glia-like neural stem / progenitor cells present in the SGZ are normally relatively quiescent but can be activated by internal and external stimuli. These cells divide symmetrically and asymmetrically to form a pool of neuroblasts. A subset of cells within this pool differentiates into immature neurons (Non-Patent Document 4).

[0007] Due to its medical importance, the induction of neural cell regeneration has been studied extensively and in depth. The development of in vitro technology for reversing differentiation of somatic cells into pluripotent cells (induced pluripotent stem cells, iPS cells) (Patent Document 1) has prompted worldwide efforts to search for and identify genes and compounds that proliferate neural stem cells (e.g., Patent Documents 2 and 3). In parallel with this, efforts are also underway to search for and identify genes and compounds that induce differentiation of neural stem cells or undifferentiated neural precursor cells into mature neurons (Patent Document 4).

[0008] Currently, a method is in the clinical stage in which stem cells are obtained by initializing somatic cells collected from a patient, which are then amplified through self-replication, induced to differentiate into the desired cells, and then transplanted back into the patient to treat the disease (e.g., Non-Patent Documents 5, 6, and 7).

[0009] It is also speculated that the difficulty of regenerating the central nervous system is due to the presence of substances in the central nervous system that inhibit nerve growth. If these nerve regeneration inhibitors present in the central nervous system can be suppressed using antibodies or other methods, it is expected that some nerves will regenerate in the central nervous system and function will be restored. Recently, Nogo has been discovered as an inhibitor of central nervous system regeneration (Non-patent Documents 8 and 9). However, inhibition of Nogo only regenerates a portion of nerve fibers, and it is thought that other regeneration inhibitors may exist. Semaphorins are also thought to be one of the factors that inhibit nerve regeneration in vivo (Non-patent Documents 10 and 11). Furthermore, the present inventors have reported that BRD7 and Ikaros are downstream factors of CRBN, that they are inhibitors of central nervous system development, and that antagonists of BRD7 and Ikaros and activators of CRBN can promote regeneration of the central nervous system (Patent Document 5).

[0010] Other types of adult stem cells are known to exist in the adult body, including hematopoietic stem cells, which originate from the bone marrow and have the ability to differentiate into all blood cells; satellite cells, which are found in mature muscles, located between the basement membrane and the sarcolemma, and supply new cells to muscle fibers; hair follicle stem cells, which give rise to new hair follicles and maintain all cell lineages of hair follicles over a long period of time; mammary stem cells, which are the source of cells for growing mammary glands during puberty and pregnancy; mesenchymal stem cells, which originate from the bone marrow stroma and can differentiate into various tissues; endothelial stem cells, which differentiate into vascular endothelium and are responsible for remodeling blood vessels; olfactory mucosa stem cells, which can be harvested from the olfactory mucosa and have the ability to differentiate into many different cells; and neural crest stem cells, which can differentiate into neurons, Schwann cells, myofibroblasts, chondrocytes, and melanocytes. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2007 / 069666 publication [License 2] WO2016 / 002854 [License 3] Special Announcement No. 2015-071565 [License 4] Special Announcement No. 2017-145215 [Patent Document 5] WO2015 / 127351 [Non-licensed literature]

[0012] [Non-licensed Document 1] Ann NY Acad Sci. 2020 Feb; 1462(1):27-36. [Non-licensed Document 2] Oxid Med Cell Longev. 2019; 2019: 5813147. [Non-licensed Document 3] Nature Genetics, 43, 34-41 (2011) [Non-licensed Document 4] Biomed Res Int.2015;2015:727542. [Non-licensed Document 5] New England Journal of Medicine 2017;376:1038-1046 [Non-licensed Document 6] Journal of the Japanese Cataract Society, Volume 27, No. 1, 2015, p.32-34 [Non-licensed Document 7] Regenerative Medicine: Journal of the Japanese Society for Regenerative Medicine 14(4)=62:2015.11 p.319-329 [Non-licensed Document 8] Nature 403, 434, 2000. [Non-licensed Document 9] Nature 403, 439, 2000. [Non-licensed Document 10] Cell 75, 217, 1993. [Non-licensed Document 11] Cell 75, 1389, 1993.

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[0013] Until now, it has been thought that stem cell self-renewal and differentiation induction are induced by different factors. Because of this common technical knowledge, factors that induce stem cell self-renewal and factors that induce differentiation induction have been screened separately in in vitro test systems. Specifically, cultured stem cells are exposed to a candidate substance, and factors that induce self-renewal are screened using the proliferation-promoting effect as an indicator. Conversely, cultured stem cells are exposed to a candidate substance, and factors that induce differentiation induction are screened using the differentiation-inducing effect into the desired mature cells as an indicator. However, the factors selected in this two-stage in vitro screening test system have only been confirmed to be effective in vitro, and therefore their use is primarily focused on regenerative medicine using ES cells and iPS cells, i.e., in vitro stem cell expansion and differentiation induction processes. In the prior art, there exist substances that induce self-replication and differentiation of undifferentiated cells in vivo as a single agent, but there was no idea of ​​screening for these substances.

[0014] In view of the above circumstances, an object of the present invention is to provide a novel screening technique for selecting useful pharmaceutical compounds. [Means for solving the problem]

[0015] As a result of the inventor's diligent research efforts, it was discovered that by administering a candidate substance to undifferentiated or differentiated cells in a living body, it is possible to screen for compounds that can realize "regenerative medicine" in the living body as a single agent.The active ingredient screened using this living body method was then demonstrated to have tissue regeneration effects in adults.Furthermore, it was also demonstrated that the active ingredient has the ability to forcibly induce the differentiation of cancer cells into normal cells.This invention was completed based on these discoveries.

[0016] That is, the present invention, which solves the above-mentioned problems, is a method for screening for an active ingredient for treating or preventing a disease, disorder, or illness, or a symptom thereof, comprising the following steps A, B and / or C, and D: [Step A] A step of administering a candidate substance to an animal (excluding humans). [Step B] A step of observing undifferentiated cells in the animal that has undergone Step A. [Step C] A step of observing differentiated cells in the animal that has undergone Step A. [Step D] A step of selecting as the active ingredient a candidate substance that increases the amount of undifferentiated cells, or a candidate substance that increases the amount of differentiated cells or the function of tissue composed of differentiated cells, compared to when the candidate substance is not administered.

[0017] The present invention uses living animals as a screening tool. Then, undifferentiated and / or differentiated cells in the animals to which a candidate substance has been administered are observed. This technical feature of the present invention makes it possible to efficiently screen for active ingredients that can realize regenerative medicine in vivo as a single agent. Furthermore, the present invention makes it possible to screen for active ingredients that have the effect of forcibly differentiating cancer cells into normal cells.

[0018] A preferred embodiment of the present invention is a method for screening active ingredients of regenerative medicines and / or anticancer agents. According to the present invention, active ingredients of regenerative medicines or anticancer agents can be screened efficiently.

[0019] A preferred embodiment of the present invention is a method for screening for an active ingredient for treating or preventing a disease, disorder, or illness of the nervous system, heart, or pancreas, or a symptom thereof. According to the present invention, an active ingredient for treating or preventing a disease, disorder, or illness of the nervous system, heart, or pancreas, or a symptom thereof, can be efficiently screened.

[0020] A preferred embodiment of the present invention is a method for screening for an active ingredient for treating blood cancer. According to the present invention, active ingredients of therapeutic agents for blood cancer can be efficiently screened.

[0021] In a preferred embodiment of the present invention, the candidate substance is one or more selected from a low molecular weight compound, a protein, a peptide, and a nucleic acid. The types of candidate substances that can be subjected to the screening method of the present invention are not limited.

[0022] A preferred embodiment of the present invention comprises the steps B and C. By observing both undifferentiated cells and differentiated cells after administration of a candidate substance, it is possible to screen for an active ingredient with greater accuracy.

[0023] In a preferred form of the invention, the animal is a vertebrate. In a more preferred embodiment of the invention, the animal is a mammal, fish, bird, reptile or amphibian. In a preferred embodiment of the invention, the animal is a zebrafish or a mouse. By using these animals as screening tools, active ingredients that exert pharmacological effects in humans can be screened more efficiently.

[0024] In a preferred embodiment of the present invention, a candidate substance is administered to an animal embryo in step A. By observing the amount and / or function of undifferentiated cells and / or differentiated cells in the animal embryo to which the candidate substance has been administered, it is possible to screen for an active ingredient that is effective in an adult.

[0025] In a preferred embodiment of the present invention, the animal is a zebrafish embryo. Zebrafish embryos are useful as screening tools because they are transparent and easy to handle. Active ingredients screened in a screening system using zebrafish embryos also exhibit pharmacological effects in mammals.

[0026] In a preferred embodiment of the present invention, the candidate substance is locally administered to the animal in the step A. Local administration is preferred because it determines the location of cell observation in the subsequent steps B and / or C.

[0027] In a preferred embodiment of the present invention, the candidate substance is locally administered to the animal embryo in step A. By observing the state of undifferentiated cells and / or differentiated cells during embryonic development at the site of local administration, highly accurate screening can be achieved.

[0028] In a preferred embodiment of the present invention, the candidate substance is locally administered to a region in the animal embryo where a specific tissue will develop or in the vicinity thereof in step A. By observing the state of undifferentiated cells and / or differentiated cells during the embryonic development of a specific tissue, highly accurate screening can be achieved.

[0029] In a preferred embodiment of the present invention, undifferentiated cells of the specific tissue are observed in step B. By observing undifferentiated cells of the specific tissue, the effect of the candidate substance on the self-renewal ability of undifferentiated cells can be evaluated.

[0030] In a preferred embodiment of the present invention, differentiated cells of the specific tissue are observed in step C. By observing differentiated cells of the specific tissue, the differentiation-inducing effect of the candidate substance can be evaluated.

[0031] In a preferred embodiment of the present invention, step A comprises locally administering the candidate substance to or near a region in a zebrafish embryo where a specific tissue will develop, step B comprises observing undifferentiated cells in the specific tissue, and step C comprises observing differentiated cells in the specific tissue. As mentioned above, zebrafish embryos are transparent and easy to handle, allowing for efficient screening.

[0032] In a preferred embodiment of the present invention, the specific tissue is the nervous system, heart, or pancreas. Thus, the compound may be administered to any of the germ layer tissues, ectoderm, endoderm, and mesoderm.

[0033] In a preferred form of the invention, the nervous system is the central nervous system. By administering the candidate substance in a form that acts directly on the central nervous system, it is possible to effectively screen for active ingredients that have the effect of regenerating a central nervous system that is impossible or extremely difficult to regenerate.

[0034] In a preferred embodiment of the invention, the central nervous system is the brain, spinal cord or optic nerve. By administering the candidate substance in a form that acts directly on the brain, spinal cord, or optic nerve, it is possible to effectively screen for active ingredients that have the effect of regenerating the brain, spinal cord, or optic nerve, which belong to the central nervous system and are impossible or extremely difficult to regenerate.

[0035] In a preferred embodiment of the present invention, the method is a method for screening for an active ingredient for treating or preventing a disease, disorder, or illness of the same tissue as the specific tissue, or symptoms thereof, which enables efficient screening of an active ingredient for a therapeutic drug for a disease of the specific tissue, etc.

[0036] In a preferred embodiment of the present invention, the present invention is a method for screening for an active ingredient for treating or preventing a disease, disorder, or illness or symptoms thereof in a tissue different from the specific tissue. The active ingredient selected by the screening method of the present invention exhibits pharmacological effect in a tissue different from the tissue to which the candidate substance is administered in the screening system.

[0037] In a preferred embodiment of the present invention, the animal is a transgenic animal into which a reporter gene that is specifically expressed in the undifferentiated cells and / or the differentiated cells has been introduced, which is preferred because the undifferentiated cells and / or the differentiated cells can be easily observed by observing the reporter.

[0038] In a preferred embodiment of the present invention, the animal is a transgenic animal into which a reporter gene that is specifically expressed in undifferentiated cells has been introduced, and the expression of the reporter gene that is specifically expressed in undifferentiated cells is observed in step B. Such a configuration allows for easy observation of undifferentiated cells.

[0039] In a preferred embodiment of the present invention, the animal is a transgenic animal into which a reporter gene that is specifically expressed in differentiated cells has been introduced, In the step C, the expression of a reporter gene that is specifically expressed in the differentiated cells is observed. Such a configuration allows for easy observation of differentiated cells.

[0040] In a preferred embodiment of the present invention, the reporter gene encodes a fluorescent protein or a fusion protein of a fluorescent protein and another protein. By observing the fluorescence of the fluorescent protein, the expression of the reporter gene can be easily observed.

[0041] In a preferred embodiment of the present invention, the expression of the reporter gene is observed by observing the fluorescence of the fluorescent protein.

[0042] In a preferred embodiment of the present invention, undifferentiated cell markers are observed in step B. Such a configuration allows for easy observation of undifferentiated cells.

[0043] In a preferred embodiment of the present invention, differentiated cell markers are observed in step C. Such a configuration allows for easy observation of differentiated cells.

[0044] In a preferred embodiment of the present invention, the means for observing the undifferentiated cell marker and / or the differentiated cell marker is immunostaining or in situ hybridization. By employing these observation means, the markers can be easily observed.

[0045] In a preferred embodiment of the present invention, the animal is a transgenic animal into which a reporter gene that is specifically expressed in the undifferentiated cells has been introduced, and the expression of the reporter gene that is specifically expressed in the undifferentiated cells is observed in step B, and differentiated cell markers are observed in step C. Undifferentiated and differentiated cells can be observed by different means.

[0046] In a preferred embodiment of the present invention, the animal is a transgenic animal into which a reporter gene that is specifically expressed in the differentiated cells has been introduced, and undifferentiated cell markers are observed in step B, and the expression of the reporter gene that is specifically expressed in the differentiated cells is observed in step C. Undifferentiated and differentiated cells can be observed by different means.

[0047] In a preferred embodiment of the present invention, the animal is a transgenic animal into which a reporter gene that is specifically expressed in undifferentiated cells and a reporter gene that is specifically expressed in differentiated cells have been introduced, and in step B, the expression of the reporter gene that is specifically expressed in undifferentiated cells is observed, and in step C, the expression of the reporter gene that is specifically expressed in differentiated cells is observed.

[0048] In a preferred embodiment of the present invention, the candidate substance is a 5-HT receptor inhibitor, an AChR inhibitor, an adenylate cyclase activator, an AhR activator, an Akt inhibitor, an ALK inhibitor, an ATPase inhibitor, an autophagy inhibitor, a calcium channel inhibitor, a carbonic anhydrase inhibitor, a Cdc42 inhibitor, a CDK inhibitor, a COX inhibitor, a dehydrogenase inhibitor, a DHFR inhibitor, an EGFR inhibitor, an ERK inhibitor, an estrogen / progesterone receptor inhibitor, a gamma secretase inhibitor, a glutamate receptor ligand (potentiator), a GSK-3 The inhibitors include one or more selected from inhibitors, HDAC activators, HDAC inhibitors, Hedgehog / Smoothened agonists, IGF-IR inhibitors, interleukin receptor inhibitors, IRAK inhibitors, JAK / STAT inhibitors, MAO inhibitors, MEK inhibitors, mitophagy inhibitors, NF-kB inhibitors, NF-kB-AMPK-tyrosine kinase pathway inhibitors, Notch-1 inhibitors, P450 inhibitors, PAEF inhibitors, PDE inhibitors, PPAR inhibitors, TGF-β receptor inhibitors, TGF-beta / Smad inhibitors, TNF receptor inhibitors, and Wnt / β-catenin pathway activators. By selecting substances having such primary effects as candidate substances, effective ingredients can be screened efficiently.

[0049] In a preferred embodiment of the present invention, the candidate substance is a 5-HT receptor inhibitor, an AChR inhibitor, an adenylate cyclase activator, an AhR activator, an Akt inhibitor, an ALK inhibitor, an ATPase inhibitor, an autophagy inhibitor, a calcium channel inhibitor, a carbonic anhydrase inhibitor, a Cdc42 inhibitor, a CDK inhibitor, a COX inhibitor, a dehydrogenase inhibitor, a DHFR inhibitor, an EGFR inhibitor, an ERK inhibitor, an estrogen / progesterone receptor inhibitor, a gamma secretase inhibitor, a glutamate receptor ligand (potentiator), a GSK-3 The present invention also includes substances that are predicted by in silico methods to be one or more of the following: inhibitors, HDAC activators, HDAC inhibitors, Hedgehog / Smoothened agonists, IGF-IR inhibitors, interleukin receptor inhibitors, IRAK inhibitors, JAK / STAT inhibitors, MAO inhibitors, MEK inhibitors, mitophagy inhibitors, NF-kB inhibitors, NF-kB-AMPK-tyrosine kinase pathway inhibitors, Notch-1 inhibitors, P450 inhibitors, PAEF inhibitors, PDE inhibitors, PPAR inhibitors, TGF-β receptor inhibitors, TGF-beta / Smad inhibitors, TNF receptor inhibitors, and Wnt / β-catenin pathway activators. Many compounds that exert the above-described primary action are known, and knowledge of the three-dimensional structures and pharmacophores of proteins that are the primary action sites of the compounds has also been accumulated. Therefore, compounds that are predicted to exert the above-described primary action can be easily searched for by computational processing on a computer. By subjecting such predicted compounds as candidate substances to the screening method of the present invention, more efficient screening can be achieved.

[0050] In a preferred embodiment of the present invention, the candidate substance is a substance that positively regulates the expression of one or more proteins selected from c-Myc, Sox2, Klf4, and Oct4. These proteins are known to mediate self-renewal and pluripotency, and by selecting substances that positively regulate their expression as candidate substances, we can efficiently screen for effective ingredients.

[0051] In a preferred embodiment of the present invention, the candidate substance is a substance predicted by an in silico method to positively regulate the expression of one or more proteins selected from c-Myc, Sox2, Klf4, and Oct4. Compounds that positively regulate the expression of the four proteins mentioned above have been primarily searched for in vitro, and many have been discovered, with knowledge accumulating, including their primary site of action. Therefore, compounds predicted to exert the expression-regulating or primary action mentioned above can be easily searched for by computer processing. By subjecting such predicted compounds as candidate substances to the screening method of the present invention, more efficient screening can be achieved.

[0052] In a preferred embodiment of the present invention, the candidate substance is a substance that acts on one or more signal pathways selected from the Notch signal pathway, the PI3K / AKT / mTOR signal pathway, the JAK / STAT signal pathway, the MAPK signal pathway, the TGFβ / SMAD signal pathway, and the Wnt signal pathway. Substances that act on these signaling pathways are highly likely to increase the number of undifferentiated and differentiated cells in vivo, and therefore screening efficiency can be improved by using these substances as candidate substances.

[0053] In a preferred embodiment of the present invention, the candidate substance is a substance predicted by in silico methods to act on one or more signal pathways selected from the Notch signal pathway, the PI3K / AKT / mTOR signal pathway, the JAK / STAT signal pathway, the MAPK signal pathway, the TGFβ / SMAD signal pathway, and the Wnt signal pathway. Many antagonists and agonists of these signaling pathways are known, and a vast amount of knowledge has been accumulated, including their primary sites of action. Therefore, compounds predicted to exert the above-mentioned effects can be easily searched for by computer processing. By subjecting such predicted compounds as candidate substances to the screening method of the present invention, more efficient screening can be achieved.

[0054] In a preferred embodiment of the present invention, the in silico method is the SDBB ​​method and / or the LBDD method. Software for realizing the SDBB ​​method and the LDBB method is provided for a fee or free of charge, and is easy to implement.

[0055] In a preferred embodiment of the present invention, step A comprises locally administering the candidate substance to or near a region in the animal embryo where a specific tissue develops, and step C comprises observing the function of the specific tissue. By observing the effect of improving function, highly accurate screening can be performed.

[0056] the step A comprises locally administering the candidate substance to or near a region in the animal embryo that develops into a specific tissue; The step C includes observing the motor function of the specific tissue or the amount of a substance secreted by the specific tissue.

[0057] In a preferred embodiment of the present invention, the step A comprises locally administering the candidate substance to or near a region where the heart will develop in the animal embryo, and the step C comprises observing the motor function of the heart. According to this aspect of the present invention, active ingredients that are effective in treating heart diseases can be screened efficiently.

[0058] In a preferred embodiment of the present invention, step A comprises locally administering the candidate substance to or near a region in the animal embryo that will develop into a pancreas, and step C comprises observing the amount of insulin produced or secreted by β cells. According to this aspect of the present invention, active ingredients that are effective in treating pancreatic diseases such as diabetes can be efficiently screened.

[0059] In a preferred embodiment of the present invention, the animal is a model animal for a nervous system disorder, disorder, or disease. By using a model animal for a disease or the like, it is possible to efficiently screen for an active ingredient that is effective against the disease.

[0060] A preferred embodiment of the present invention is a method for screening for an active ingredient for treating or preventing glaucoma.

[0061] In a preferred embodiment of the present invention, step A is a step of administering a candidate substance to the retina of the animal, step B is a step of observing precursor cells of retinal ganglion cells or neural stem cells that differentiate into these cells, and step C is a step of observing retinal ganglion cells. By using such a form, it is possible to accurately screen for active ingredients that are effective in treating or preventing glaucoma.

[0062] In a preferred embodiment of the present invention, the animal is a glaucoma model. By using a glaucoma model as a screening tool, active ingredients having therapeutic or preventive effects on glaucoma can be efficiently screened.

[0063] A preferred embodiment of the present invention is a method for screening for an active ingredient for the treatment of spinal cord injury.

[0064] In a preferred embodiment of the present invention, step A is a step of administering a candidate substance to the spinal cord of the animal, step B is a step of observing undifferentiated cells in the spinal cord, and step C is a step of observing differentiated cells in the spinal cord. By using such a form, it is possible to accurately screen for active ingredients that have a therapeutic effect on spinal cord damage.

[0065] In a preferred embodiment of the present invention, the animal is a spinal cord injury model. By using a spinal cord injury model as a screening tool, it is possible to efficiently screen for active ingredients that have therapeutic effects on spinal cord injury.

[0066] The present invention also relates to a method for screening for an active ingredient for treating or preventing a disease, disorder, or illness, or a symptom thereof, comprising the following steps E, F, and G: [Step E] A step of administering a candidate substance to a model animal (excluding humans) of a disease, disorder, or illness. [Step F] A step of determining the therapeutic effect of the treatment on the nervous system disease, disorder, or illness in the animal that has undergone Step E. [Step G] A step of selecting a candidate substance having a therapeutic effect as the active ingredient. By directly observing the therapeutic effect in a model animal of a disease or the like, it is possible to accurately screen for an active ingredient that has a therapeutic effect on the disease or the like.

[0067] A preferred embodiment of the present invention is a method for screening active ingredients of regenerative medicines and / or anticancer agents.

[0068] The present invention is a method for screening for an active ingredient for treating or preventing a disease, disorder, or illness of the nervous system, heart, or pancreas, or a symptom thereof.

[0069] In a preferred embodiment of the present invention, in the step E, a candidate substance is administered to the retina of a glaucoma model animal, and in the step F, the therapeutic effect on glaucoma is determined. By using such a form, it is possible to efficiently screen for active ingredients that have a therapeutic effect on glaucoma.

[0070] In a preferred embodiment of the present invention, the presence or absence of an increase in retinal ganglion cells is observed in step F. By adopting such an embodiment, it is possible to efficiently screen for an active ingredient that has a therapeutic effect on glaucoma by regenerating retinal ganglion cells that are damaged or have a reduced function due to glaucoma.

[0071] In a preferred embodiment of the present invention, in the step F, the therapeutic effect on glaucoma is determined by observing the behavior of the animal to determine whether or not the visual acuity has recovered. In a preferred embodiment of the present invention, active ingredients having therapeutic effects on glaucoma can be efficiently screened.

[0072] In a preferred embodiment of the present invention, in the step E, a candidate substance is administered to a spinal cord injury site in a spinal cord injury model animal; In the step F, the therapeutic effect of spinal cord injury is evaluated. By using such a form, it is possible to efficiently screen for active ingredients that have a therapeutic effect on spinal cord injury.

[0073] In a preferred embodiment of the present invention, in step F, the regenerative effect on the spinal cord at the spinal cord injury site is assessed. By using such a form, active ingredients having therapeutic effects on spinal cord injury can be screened more efficiently.

[0074] In a preferred embodiment of the present invention, in the step F, the therapeutic effect on spinal cord injury is determined by observing the behavior of the animal to determine whether or not motor ability has recovered. By using such a form, it is possible to efficiently screen for active ingredients that have a therapeutic effect on movement disorders associated with spinal cord injury.

[0075] In a preferred embodiment of the present invention, in the step E, a candidate substance is injected into the blood vessels of a cancer model animal, and in the step F, the therapeutic effect on cancer is determined. Such a form allows for efficient screening of anticancer drugs.

[0076] In a preferred embodiment of the present invention, in the step E, a candidate substance is administered to a leukemia model animal, and in the step F, the presence or absence of vascular endothelial thickening or tumor formation is observed. By using such a form, it is possible to screen for active ingredients that exert anti-cancer effects by forcing cancer cells to differentiate into normal vascular endothelial cells.

[0077] In a preferred embodiment of the present invention, in the step E, a candidate substance is administered to a leukemia model animal, and in the step F, the survival rate of the animal is observed. Such a form allows for efficient screening of active ingredients of anticancer drugs.

[0078] The present invention also relates to a method for screening for active ingredients for treating or preventing diseases, disorders, or illnesses, or symptoms thereof, which comprises performing a primary screening using a screening method comprising the above-mentioned steps A, B and / or C and D, and subjecting candidate substances selected as active ingredients in the primary screening to a secondary screening using a screening method comprising the above-mentioned steps E, F, and G. By carrying out primary and secondary screening in this way, it is possible to more accurately screen for active ingredients that have therapeutic effects on diseases, etc.

[0079] In a preferred embodiment of the present invention, in step A of the primary screening, a candidate substance is administered to normal animals. In this way, by using normal animals in the primary screening and disease model animals in the secondary screening, efficient and highly accurate screening can be achieved.

[0080] In a preferred embodiment of the present invention, the candidate substance is a compound encompassed by the following general formula (I), general formula (II), or general formula (III), or a salt thereof, or a hydrate thereof.

[0081] [ka]

[0082] In general formula (I), Ring A is may contain 1 to 4 heteroatoms independently selected from oxygen atoms, nitrogen atoms and sulfur atoms; saturated or unsaturated, may further be substituted with a C1-3 alkyl group or a halogen atom, 3 to 8 members, is a monocyclic or fused bicyclic group; L is a substituent R 3 or L is absent, The substituent R 3 represents a C1-10 saturated or unsaturated hydrocarbon group which may have a cyclic structure and which may be substituted with a halogen atom; R 1 teeth, -C≡N, -COOH, -CHO, -COOCH3, -NO2, or a halogen atom; or may contain 1 to 4 heteroatoms independently selected from oxygen atoms, nitrogen atoms and sulfur atoms; saturated or unsaturated, may further be substituted with a C1-3 alkyl group or a halogen atom, 3 to 8 members, is a monocyclic or fused bicyclic group; R 2 teeth, may contain 1 to 6 heteroatoms independently selected from oxygen atoms, nitrogen atoms, and sulfur atoms; saturated or unsaturated, may further be substituted with a C1-3 alkyl group or a halogen atom, It is a 3- to 20-membered monocyclic or fused bicyclic group.

[0083] [ka]

[0084] In general formula (II), R 1 teeth, hydrogen atoms, a halogen atom, or a C1-20 hydrocarbon group which is saturated or unsaturated, linear or branched, and optionally has a cyclic structure, which is optionally substituted with a halogen atom, and which may contain 1 to 4 heteroatoms independently selected from oxygen atoms, nitrogen atoms, and sulfur atoms; L 1 is one of the following structures: [ka] ; L 2 teeth, saturated or unsaturated, optionally substituted with a group selected from a C1-3 hydrocarbon group, a hydroxyl group optionally substituted with a C1-3 hydrocarbon group, an amino group optionally substituted with a C1-3 hydrocarbon group, a sulfate group optionally substituted with a C1-3 hydrocarbon group, a phosphate group optionally substituted with a C1-3 hydrocarbon group, and a halogen atom; may contain 1 to 4 heteroatoms independently selected from oxygen atoms, nitrogen atoms and sulfur atoms; C1-30 hydrocarbon chain; R 2 is a carboxyl group which may be substituted with a C1-3 hydrocarbon group, a hydroxyl group which may be substituted with a C1-3 hydrocarbon group, a hydroxamic acid group which may be substituted with a C1-3 hydrocarbon group, a sulfo group which may be substituted with a C1-3 hydrocarbon group, a boronic acid group which may be substituted with a C1-3 hydrocarbon group, a carbamoyl group which may be substituted with a C1-3 hydrocarbon group, a sulfamoyl group which may be substituted with a C1-3 hydrocarbon group, a sulfoximine group which may be substituted with a C1-3 hydrocarbon group, a cyano group, or a tetrazolyl group.

[0085] [ka]

[0086] In general formula (III), Ring A, ring B and ring C each independently represent may contain 1 to 4 heteroatoms independently selected from oxygen atoms, nitrogen atoms and sulfur atoms; saturated or unsaturated, may further be substituted with a C1-3 hydrocarbon group, a halogen atom, a hydroxyl group optionally substituted with a C1-3 hydrocarbon group, an amino group optionally substituted with a C1-3 hydrocarbon group, a sulfate group optionally substituted with a C1-3 hydrocarbon group, or a phosphate group optionally substituted with a C1-3 hydrocarbon group; 3 to 8 members, It is a monocyclic ring; R 1 is a group selected from the following: [ka] R 3 ~R 6 are each independently a hydrogen atom, a C1-3 hydrocarbon group, a hydroxyl group optionally substituted with a C1-3 hydrocarbon group, or an amino group optionally substituted with a C1-3 hydrocarbon group, R 7 represents a hydrogen atom, a C1-3 hydrocarbon group, or an amino group optionally substituted with a C1-3 hydrocarbon group, n represents an integer of 1 to 4; L 1 , L 2 are each independently a C1-3 alkylene or alkenylene group, -N(H)-, or -O-; Among these divalent groups, groups other than -O- may be saturated or unsaturated, linear or branched, and may have a cyclic structure; may be substituted with a halogen atom; may contain 1 to 6 heteroatoms independently selected from oxygen atoms, nitrogen atoms, and sulfur atoms; or may be substituted with a C1 to C30 hydrocarbon group; R 2is a C1-30 hydrocarbon group which is saturated or unsaturated, linear or branched, and may have a cyclic structure, may be substituted with a halogen atom, and may contain 1 to 6 heteroatoms independently selected from oxygen atoms, nitrogen atoms, and sulfur atoms.

[0087] In this way, by using compounds encompassed by general formulas (I) to (III) as candidate substances for screening, it is possible to screen for active ingredients that are effective against nervous system diseases and the like with a high probability.

[0088] In a preferred embodiment of the present invention, the candidate substance is a derivative of any one of the following compounds 1 to 7, a salt thereof, or a hydrate thereof.

[0089] [ka]

[0090] The seven compounds listed above were selected as active ingredients using the screening method described above. By using these compounds as bases and creating derivatives with structural substitutions, additions, deletions, etc. as screening candidates, it is possible to screen for active ingredients with a high probability.

[0091] The present invention also relates to a method for producing a pharmaceutical composition, which comprises a formulation step of mixing a substance selected as an active ingredient by the above-mentioned screening method with pharmaceutical additives to form a formulation. According to the manufacturing method of the present invention, an effective pharmaceutical composition can be manufactured.

[0092] In a preferred embodiment of the present invention, the screening method is a screening method in which the compounds encompassed by the above-mentioned general formulas (I) to (III) are used as candidate substances, or a screening method in which derivatives of the above-mentioned seven compounds are used as candidate substances.

[0093] In a preferred embodiment of the present invention, the pharmaceutical composition is a regenerative medicine and / or an anticancer agent. According to the present invention, regenerative medicines and / or anticancer agents can be produced.

[0094] In a preferred embodiment of the present invention, the pharmaceutical composition is used for the treatment or prevention of diseases, disorders or conditions of the nervous system, heart or pancreas, or symptoms thereof.

[0095] In a preferred embodiment of the present invention, the pharmaceutical composition is used for the treatment or prevention of glaucoma.

[0096] In a preferred embodiment of the present invention, the pharmaceutical composition is used for treating spinal cord injury.

[0097] In a preferred embodiment of the present invention, the pharmaceutical composition is used for the treatment of blood cancer.

[0098] In a preferred embodiment of the present invention, the pharmaceutical composition is used for the treatment of leukemia.

[0099] In a preferred embodiment of the present invention, the pharmaceutical composition is a liquid formulation, The formulation step includes mixing the active ingredient with an aqueous medium.

[0100] In a preferred embodiment of the present invention, the pharmaceutical composition is an injection. In a preferred embodiment of the present invention, the pharmaceutical composition is an eye drop.

[0101] The pharmaceutical composition is a freeze-dried formulation, and the formulation step comprises dissolving the active ingredient in a solvent and freeze-drying the solution.

[0102] In a preferred embodiment of the present invention, the pharmaceutical composition is an ointment, and the formulation step comprises mixing the active ingredient with a base.

[0103] In a preferred embodiment of the invention, the pharmaceutical composition is an eye ointment.

[0104] In a preferred embodiment of the present invention, the pharmaceutical composition is a formulation for nasal administration.

[0105] In a preferred embodiment of the present invention, the pharmaceutical composition is a formulation for oral administration.

[0106] The present invention also relates to a method for designing a pharmaceutical composition, which comprises the step of selecting a pharmaceutical additive to be combined with a substance selected as an active ingredient by the above-mentioned screening method. According to the present invention, effective pharmaceutical compositions can be designed.

[0107] In a preferred embodiment of the present invention, the pharmaceutical composition is a regenerative medicine and / or an anticancer agent. According to this aspect of the present invention, regenerative medicines and / or anticancer drugs can be designed.

[0108] In a preferred embodiment of the present invention, the pharmaceutical composition is used for the treatment or prevention of diseases, disorders or conditions of the nervous system, heart or pancreas, or symptoms thereof. In accordance with the present invention, pharmaceutical compositions can be designed for the treatment or prevention of diseases, disorders or conditions of the nervous system, heart or pancreas, or symptoms thereof.

[0109] In a preferred embodiment of the present invention, the method includes a step of selecting an indication for the pharmaceutical composition, and pharmaceutical additives can be selected according to the selected indication. In a preferred form of the invention, the indication is a disease, disorder or illness of the nervous system, heart or pancreas, or a symptom thereof, or cancer.

[0110] In a preferred embodiment of the present invention, the method further comprises the step of selecting a dosage form of the pharmaceutical composition, and pharmaceutical additives can be selected according to the selected dosage form. A preferred embodiment of the present invention includes selecting a liquid formulation as the dosage form and selecting an aqueous medium to be mixed with the active ingredient. In a preferred embodiment of the present invention, an injection is selected as the dosage form. In a preferred embodiment of the present invention, eye drops are selected as the dosage form. In a preferred embodiment of the present invention, a freeze-dried preparation is selected as the dosage form.

[0111] In a preferred embodiment of the present invention, an ointment is selected as the dosage form, and a base material for mixing with the active ingredient is selected. In a preferred embodiment of the present invention, an eye ointment is selected as the dosage form.

[0112] In a preferred embodiment of the present invention, a formulation for nasal administration is selected as the dosage form. In a preferred embodiment of the present invention, a formulation for oral administration is selected as the dosage form.

[0113] The present invention also relates to a method comprising designing a pharmaceutical composition by the above-described method, preparing a substance selected as an active ingredient by the above-described screening method, producing a pharmaceutical composition by the above-described production method, and statistically processing a data set obtained from people to whom the pharmaceutical composition has been administered in a clinical trial. [Effects of the Invention]

[0114] According to the screening method of the present invention, it is possible to screen for active ingredients of pharmaceuticals, more specifically, active ingredients that are effective in treating or preventing diseases, disorders, or illnesses of the nervous system, heart, or pancreas, or symptoms thereof, or anticancer drugs. According to the production method of the present invention, it is possible to produce a pharmaceutical composition or an anti-cancer agent that is effective in treating or preventing diseases, disorders, or illnesses of the nervous system, heart, or pancreas, or symptoms thereof. [Brief explanation of the drawings]

[0115] [Figure 1] FIG. 1 is a diagram schematically illustrating the steps of the screening method of the present invention. [Figure 2] FIG. 1 is a diagram schematically illustrating a configuration including steps A, B, and D. [Figure 3]FIG. 1 is a diagram schematically illustrating a configuration including steps A, C, and D. [Figure 4] FIG. 1 is a diagram schematically illustrating a configuration including steps A, B, C, and D. [Figure 5] This is a diagram summarizing the configurations including steps A, B, C, and D. (a) This shows a configuration in which steps A, B, C, and D are performed in this order. (b) This shows a configuration in which steps A, C, B, and D are performed in this order. (c) This shows a configuration in which steps B and C are performed simultaneously after step A, and then step D is performed. [Figure 6] Photographs showing the results of Compounds 1 and 2 in Test Example 1. The photographs are superimposed fluorescent images of GFP fluorescence, which indicates midbrain neural stem cells, and bright-field images. The upper photograph is a planar view of the head of a zebrafish embryo, and the lower photograph is a lateral view of the head. [Figure 7] Photographs showing the results of Compounds 3 to 5 in Test Example 1. The photographs are a superposition of a fluorescent image of GFP fluorescence, which indicates midbrain neural stem cells, and a bright-field image. The top photograph is a planar photograph of the head of a zebrafish embryo, and the bottom photograph is a lateral photograph of the head. [Figure 8] Photographs showing the results of Compounds 6 and 7 in Test Example 1. The photographs are superimposed fluorescent images of GFP fluorescence, which indicates midbrain neural stem cells, and bright-field images. The upper photograph is a planar view of the head of a zebrafish embryo, and the lower photograph is a lateral view of the head. [Figure 9] FIG. 1 shows the results of quantitative PCR showing the expression levels of sox2 in the control, compounds 2 and 7 in Test Example 1. [Figure 10] This figure shows the results of quantitative PCR showing the expression levels of sox1α, sox2, sox3, and her6 when a control, compound 2, and a comparative compound in which no increase in undifferentiated neural cells or differentiated neural cells was observed in Test Example 1 were administered. [Figure 11] Photographs showing the results for Compounds 1 and 2 in Test Example 2. The upper row is a bright-field image. The lower row is a fluorescent image showing the fluorescence indicating neuronal cell body clusters (telencephalon and diencephalon) and the axon bundles connecting them. [Figure 12] 1 is a photograph showing the results of Compounds 3 to 5 in Test Example 2. The photograph is a superimposition of a fluorescent image and a bright-field image, which observed the fluorescence showing the cell body clusters (telencephalon and diencephalon) and the axon bundles connecting them. [Figure 13] Photographs showing the results for Compounds 6 and 7 in Test Example 2. The upper row is a bright-field image. The lower row is a fluorescent image showing the fluorescence indicating neuronal cell body clusters (telencephalon and diencephalon) and the axon bundles connecting them. [Figure 14] Photographs showing the results of Compound 7 in Test Example 5. The upper and lower images are stained images before and after treatment, respectively. The areas indicated by the black arrows (areas where green fluorescence is observed) are retinal ganglion cells. [Figure 15] 1 is a photograph showing the results of Compound 2 in Test Example 6. The right side shows a stained image, and the left side shows a bar graph showing the results of counting the number of Purkinje cells. [Figure 16] 1 is a photograph showing the results of Compound 5 in Test Example 6. The right side shows a stained image, and the left side shows a bar graph showing the results of counting the number of Purkinje cells. [Figure 17] 1 is a photograph showing the results of Compound 7 in Test Example 6. The right side shows a stained image, and the left side shows a bar graph showing the results of counting the number of Purkinje cells. [Figure 18] 1 is a photograph showing fluorescence of midbrain neurons in zebrafish injected with Compound 5 in Test Example 7. [Figure 19] 1 shows photographs of stained cardiomyocytes in zebrafish injected with Compounds 2, 5, and 7 in Test Example 8. [Figure 20] 1 shows the results of counting the stroke volume of blood cells in zebrafish injected with Compound 7 in Test Example 8. [Figure 21] 1 shows a captured image of a video of the heartbeat of a zebrafish injected with Compound 7 in Test Example 8. [Figure 22] 10 is a fluorescent photograph of a transgenic zebrafish embryo expressing GFP in pancreatic β cells in Test Example 9. [Figure 23]1 shows photographs of insulin staining by in situ hybridization of zebrafish injected with a test compound in Test Example 9. The results of injection of compounds 2, 5, 6, and 7 are shown. [Figure 24] Photographs of immunostaining with anti-insulin antibody in Test Example 9. The results are shown for compounds 2 and 6 injected at concentrations of 100 nM and 5 nM, respectively. The results are shown for three tests for each condition. [Figure 25] Bar graph quantitating the results of Figure 24. The vertical axis is the mean fluorescence intensity. [Figure 26] This figure shows the results of fluorescent observation of vascular endothelial cells in a zebrafish model of T-cell acute lymphoblastic leukemia injected with Compound 7 or E3 Ringer (control) in Test Example 10. The lower figure is a low-magnification photograph, and the upper figure is an enlarged photograph of the boxed area in the lower figure. The arrowheads indicate cells that are morphologically hemocyte-like but are fusing with the vascular endothelium. The dotted circle indicates a thickened blood cell or a similarly fused blood cell with the vascular endothelium. [Figure 27] 1 is a graph showing the survival rates of control and drug-injected individuals in Test Example 10. DETAILED DESCRIPTION OF THE INVENTION

[0116] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, where appropriate. However, the scope of the present invention is not limited to the embodiments described below.

[0117] [1] First screening method The screening method of the present invention was completed based on the fact discovered as a result of the inventor's intensive research efforts, namely, the fact that there are multiple compounds that can induce self-replication and differentiation of undifferentiated cells in the body as a single agent. The screening method of the present invention is based on the discovery of the present inventors and uses animals as a screening tool to screen for active pharmaceutical ingredients using the effect of promoting the proliferation or function of undifferentiated cells and / or differentiated cells as an index.

[0118] The term "active pharmaceutical ingredient" as used herein is not limited to active ingredients of "drugs" that require manufacturing and sales approval from regulatory authorities (such as the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration in the United States), but is a concept that broadly encompasses ingredients that have useful physiological activity. For example, the term "active pharmaceutical ingredient" as used in this specification broadly includes not only active ingredients of pharmaceuticals based on a Western medical approach, but also active ingredients of pharmaceuticals such as herbal medicines based on an Eastern medical approach, health foods, supplements, and the like.

[0119] In one embodiment of the present invention, the present invention is a method for screening active ingredients of regenerative medicines and / or anticancer drugs. The term "regenerative medicine" as used herein refers to pharmaceuticals that act on stem cells, progenitor cells, etc. to promote their proliferation and differentiation, thereby regenerating the function of damaged tissues or organs. Furthermore, anticancer drugs are not limited to pharmaceuticals that inhibit the proliferation of cancer cells or induce cell death. Anticancer drugs also include pharmaceuticals that act on cancer cells and induce their differentiation into harmless normal cells.

[0120] One embodiment of the present invention is a method for screening for active ingredients for the treatment or prevention of diseases, disorders or conditions of the nervous system, heart or pancreas or symptoms thereof. Hereinafter, "disease, disorder, or illness" will also be referred to as "disease, etc."

[0121] Nervous system disorders include Parkinson's disease, Alzheimer's disease, Creutzfeldt-Jakob disease, prion diseases, corticobasal degeneration, amyotrophic lateral sclerosis, multiple sclerosis, progressive motor weakness, immune-mediated neuropathies, central nervous system injuries such as brain injury, spinal cord injury, optic nerve injury, and olfactory nerve injury, Alzheimer's disease with parkinsonism, bradykinesia, akinesia, movement disorders that impair fine motor control and manual dexterity, dysphonia, monotonous speech, rigidity, dystonia, inflammation associated with Parkinson's disease, facial, jaw, tongue, and postural tremors, and tremors associated with Parkinson's disease. Parkinsonian gait; shuffling gait; shuffling gait; accelerated gait; mood, cognitive, sensory, or sleep disorders; dementia; depression; drug-induced Parkinsonism; vascular Parkinsonism; multiple system atrophy; progressive supranuclear palsy; disorders with primary tau pathology; corticobasal ganglionic degeneration; Parkinsonism with dementia; hyperactivity disorder; chorea; Huntington's disease; dystonia; Wilson's disease; Tourette's syndrome; essential tremor; myoclonus; tardive dyskinesia; schizophrenia; bipolar disorder, and autism spectrum disorder. Heart diseases include angina pectoris, myocardial infarction, valvular disease, cardiomyopathy, atrial septal defect, cardiac tumor, heart failure, arrhythmia, and the like. Pancreatic diseases include type 1 diabetes, type 2 diabetes, acute pancreatitis, chronic pancreatitis, pancreatic cancer, and mucus-producing tumors.

[0122] One embodiment of the present invention is a method for screening for an active ingredient for treating blood cancer. Examples of blood cancer include leukemias such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia; Hodgkin lymphomas such as classical Hodgkin lymphoma and nodular lymphocyte-predominant Hodgkin lymphoma; B-cell lymphoblastic leukemia / lymphoma; T-cell lymphoblastic leukemia / lymphoma; chronic lymphocytic leukemia / small lymphocytic lymphoma; follicular lymphoma; MALT lymphoma; and lymphoplasmacytic lymphoma. Malignant lymphomas such as idiopathic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, and lymphoma of the skin (mycosis fungoides, Sézary syndrome); multiple myelomas such as symptomatic multiple myeloma, benign monoclonal hypergammaglobulinemia, asymptomatic myeloma, non-secretory myeloma, and solitary plasmacytoma of bone. The active ingredients selected by the screening method of the present invention exert their detoxifying effect by forcibly differentiating cancer cells into normal cells (vascular endothelial cells). The present invention is effective in that it allows screening of active ingredients that exert anti-cancer effects through such a new mechanism of action.

[0123] The present invention is characterized by comprising the following step A, step B and / or step C, and step D (FIG. 1). [Step A] A step of administering a candidate substance to an animal (excluding humans). [Step B] A step of observing undifferentiated cells in the animal that has undergone Step A. [Step C] A step of observing differentiated cells in the animal that has undergone Step A. [Step D] A step of selecting as the active ingredient a candidate substance that increases the amount of undifferentiated cells, or a candidate substance that increases the amount of differentiated cells or the function of tissue composed of differentiated cells, compared to when the candidate substance is not administered.

[0124] In the present invention, it is sufficient to carry out either step B or step C. Therefore, an embodiment including steps A, B, and D (FIG. 2) is also acceptable, as is an embodiment including steps A, C, and D (FIG. 3).

[0125] In the embodiment shown in FIG. 2, in step D, a candidate substance that increases the amount of undifferentiated cells compared to when the candidate substance is not administered is selected as the active ingredient (FIG. 2). On the other hand, in the embodiment shown in Figure 3, in step D, a candidate substance that increases the amount of differentiated cells or improves the function of tissue composed of differentiated cells compared to when the candidate substance is not administered is selected as the active ingredient (Figure 3).

[0126] Of course, an embodiment in which both steps B and C are executed may also be used (FIG. 4). In this case, in step D, a candidate substance that increases the amount of undifferentiated neural cells and differentiated neural cells compared to when the candidate substance is not administered is selected as the active ingredient (Figure 4).

[0127] In an embodiment in which both steps B and C are performed, the order of these two steps is not critical. That is, the steps may be executed in the order of A, B, C, and D (FIG. 5(a)), or in the order of A, C, B, and D (FIG. 5(b)). Moreover, an embodiment in which the steps B and C are carried out simultaneously may also be used (FIG. 5(c)).

[0128] Each step will be described in detail below.

[0129] [1-1]A process [1-1-1]Animals In step A, the candidate substance is administered to an animal. Because using humans as a screening tool is ethically problematic, the candidate substance is administered to a non-human animal. The animal to which the candidate substance is administered in step A is not particularly limited as long as it is not a human, and may be an animal classified as any animal, such as a Platypoda, a Crest-Tentacator, a Cyclophora, a Nematoda, a Psilocordata, a Panarthropod, an Echinoderm, a Hemichordata, a Cephalochordata, a Tunicate, a Vertebrate, or a Coelota. Among these, an embodiment in which a vertebrate having a central nervous system consisting of a brain and a spinal cord is used as a screening tool is preferred.

[0130] The vertebrate may be any of mammals, fish, birds, reptiles, and amphibians. Among these, it is preferable to use animals that are commonly used as model organisms. Specific examples of mammals include mice, guinea pigs, rats, rabbits, monkeys, dogs, and cats. Examples of fish include zebrafish, medaka, and tiger pufferfish. Examples of birds include chickens and quails. Examples of reptiles include geckos, and examples of amphibians include frogs and newts.

[0131] From the viewpoint of simple and rapid screening of active ingredients applicable to humans, it is preferable to use zebrafish, which are prolific, have a short life cycle, have transparent to translucent bodies, and allow observation of the fluorescence of fluorescent proteins expressed in the body. Furthermore, from the viewpoint of easily screening for active ingredients that are more applicable to humans, it is preferable to use mice, which are prolific mammals with a short life cycle.

[0132] When zebrafish (including adults and embryos) are used as a screening tool, it is preferable to use a multiwell plate. Zebrafish administered with different candidate substances are placed in each well. By cultivating zebrafish administered with multiple candidate substances on a multiwell plate in this way, the subsequent steps B and / or C can be easily performed. In other words, high-throughput screening is possible.

[0133] A particularly preferred embodiment is to use transgenic zebrafish embryos genetically incorporating a reporter gene for a fluorescent protein and grow them on a multiwell plate, which enables simultaneous observation of undifferentiated cells and / or differentiated cells using a fluorescent plate reader in step B and / or step C.

[0134] The number of wells in a multiwell plate is not particularly limited, but is preferably 6 or more, more preferably 12 or more, even more preferably 24 or more, even more preferably 48 or more, and even more preferably 96 or more.

[0135] There are no particular limitations on the growth stage of the animal used in step A. It is preferable to use an embryo in which cell division and differentiation are active. Hereinafter, an embodiment in which an animal embryo is used as a screening tool will be described.

[0136] [1-1-1-1] Screening using embryos The animal species of the embryos used is not particularly limited, but since embryos of viviparous animals exist within the mother's womb and are difficult to handle, it is preferable to use embryos of oviparous animals. Specifically, it is preferable to use embryos of fish, reptiles, amphibians, and birds. It is preferable to use embryos contained in soft eggs with transparent eggshells as screening tools. Because of the advantages of being transparent, easy to handle, and able to be prepared in large quantities, it is preferable to use fish embryos, and it is particularly preferable to use zebrafish embryos.

[0137] The method for administering the candidate substance to the embryo is not particularly limited. In one embodiment, the embryo is immersed in a solution in which the candidate substance is dissolved or dispersed, and the entire embryo is exposed to the candidate substance. In a more preferred embodiment, the candidate substance is administered locally to a portion of the embryo, in which case the candidate substance is administered locally to or near a region that will develop into a particular tissue in the animal embryo. The term "vicinity" refers to a range within which the candidate substance can reach the target region by diffusion or dispersion through body fluids.

[0138] Local administration into animal embryos can be performed by standard methods using a micromanipulator. Furthermore, the in vivo lipofection method developed by the present inventors can be effective for local administration of molecules that are negatively charged due to ionization or other factors (Non-Patent Document 12).

[0139] The above-mentioned "specific tissue" is not particularly limited, and may be tissue derived from any of the ectoderm, endoderm, and mesoderm. The candidate substance may be administered to or near the area where any ectodermal tissue develops, such as the skin epidermis, hair, nails, skin glands (including mammary glands and sweat glands), sensory organs (including the epithelium of the oral cavity, pharynx, nose, and distal rectum), salivary glands, lens, brain, or spinal cord. Candidate substances may be administered to or near areas that develop into any endodermal tissue, such as the digestive tract from the esophagus to the large intestine (excluding the oral cavity, pharynx, and terminal part of the rectum), the lungs, thyroid gland, pancreas, liver, cells of secretory glands that open into the digestive tract, the peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, and urinary tract (bladder, most part of the urethra, and part of the ureter). Candidate substances may be administered to or near areas where any endodermal tissue develops, such as body cavities and the mesothelium lining them, muscles, skeletons, skin dermis, connective tissue, heart, blood vessels (including vascular endothelium), blood (including blood cells), lymphatic vessels, spleen, kidneys and ureters, and gonads (testes, uterus, and gonadal epithelium).

[0140] In addition, the active ingredient selected in the embodiment in which the candidate substance is administered to an area occurring in or near a specific tissue is not an ingredient that is effective only in that specific tissue. The active ingredient selected by the screening method of this embodiment is an ingredient that is also effective in tissues other than the specific tissue. In other words, the active ingredient selected by the screening method of the present invention can be used as a regenerative medicine and / or anticancer agent with no restrictions on the scope of application. Therefore, the present invention can take the following embodiments. One embodiment of the present invention is a method for screening for an effective ingredient for treating or preventing a disease, disorder, or illness or symptoms thereof in the same tissue as the specific tissue into which a candidate substance is locally injected. One embodiment of the present invention is a method for screening for an effective ingredient for treating or preventing a disease, disorder, or illness or symptoms thereof in a tissue different from the specific tissue into which the candidate substance is locally injected.

[0141] For example, if a candidate substance is locally administered to or near the area where the nervous system develops in an embryo and an increase in undifferentiated neural cells / differentiated neural cells is observed, the candidate substance should not be considered an ingredient effective only for diseases of the nervous system. In this case, the candidate substance is understood to be an ingredient effective as a regenerative medicine for tissues and organs other than the nervous system. The candidate substance is also understood to be an ingredient effective as an anticancer agent. This is supported by the results of the test examples described below.

[0142] Below, embodiments in which candidate substances are administered to or near areas of development in the nervous system, heart, and pancreas are each described in detail.

[0143] [1-1-1-1-1] Local administration to the area where the disease occurs in the nervous system or nearby In one embodiment of the present invention, the candidate substance is locally administered to or near a region in the animal embryo that will develop into the nervous system. This embodiment will be described below after a brief description of the process of nervous system development during embryonic development.

[0144] Nearly all animals, including sea urchins, Drosophila, fish, frogs, mice, and humans, undergo a process called gastrulation during early embryonic development. During gastrulation, pluripotent cells invaginate toward the center of the embryo (gastrulation). These migrating cells subsequently give rise to the mesodermal and endoderm layers, which are the more differentiated embryonic cells. The mesoderm will give rise to the vascular and musculoskeletal systems, while the endoderm will give rise to the digestive tract and associated internal organs. Meanwhile, the ectoderm region is committed to epidermal differentiation through the activity of bone morphogenetic protein 4 (BMP4). However, the ectoderm region, which is affected by organizer-derived BMP inhibitors during gastrulation, is committed to the neural tissue fate, and the neural plate is formed from this region.

[0145] The neural plate contains a map of the future brain. Cell behavior is determined according to this map, determining the location of each brain region, such as the cerebrum, diencephalon, midbrain, and cerebellum, and the brain is then formed. This region-dependent fate determination mechanism is called "regionalization" or "pattern formation." This phenomenon involves the expression of specific transcription factors induced by molecules derived from local organizers, mechanisms for silencing the expression of transcription factors, and a cell selection mechanism dependent on the function of cell surface molecules induced by transcription factors.

[0146] The midline of the neural plate is depressed to form a neural groove, and both sides of this groove bulge toward the amniotic cavity, eventually fusing at their tips to form the neural tube. Neural tube formation begins in the cervical region of the embryo and progresses in both the anterior and posterior directions. Closure of the anterior portion also begins at the cephalic end. During this process, the open portion that connects to the amniotic cavity is called the cranial neuropore, and this neuropore gradually shrinks. At this stage, the anterior portion of the neural tube appears larger than the posterior portion and appears as a bulge. This bulge is called the brain vesicle (primary brain vesicle), and because it has three bulges, this developmental stage is also called the triplasynthetic stage (primary brain vesicle stage). From anterior to posterior, the bulges consist of the forebrain vesicle, mesencephalon vesicle, and rhombencephalon (or hindbrain vesicle). The walls of the bulges differentiate into neural tissue, and the lumen becomes the ventricles. Later, the forebrain vesicle differentiates into the telencephalon and diencephalon vesicles, and the rhombencephalon vesicle differentiates into the hindbrain and myelencephalon vesicles, forming secondary vesicles. The telencephalon vesicle bulges left and right, and its inner cavity becomes the lateral ventricles. The inner cavities of the diencephalon vesicle and hindbrain vesicle become the third and fourth ventricles, respectively. The inner cavities of the mesencephalon vesicle become the aqueduct without undergoing any major morphological changes. The dorsal side of the telencephalon becomes the cerebral cortex, and the basal ganglia and other components differentiate from the ventral side. The epithalamus, thalamus, and hypothalamus differentiate from the diencephalon vesicle region. The cerebellum develops from the dorsal side of the hindbrain vesicle region, and the pons forms on the ventral side. The myelencephalon region becomes the medulla oblongata, and is connected to the spinal cord, which forms posteriorly from the neural tube. During the early stages of morphogenesis, a pair of cavities begin to develop on the sides of the forebrain. These cavities begin to form the optic vesicles, the primordium of the eyes, before the anterior end of the neural tube closes, and the optic vesicles are complete after the neural tube closes.

[0147] In light of the above, one form of the "region that will develop into the nervous system in an animal embryo" is the region that will form the neural plate at the gastrulation stage. That is, in step A, the candidate substance may be locally administered to the region that will form the neural plate at the gastrulation stage or in its vicinity.

[0148] Furthermore, the neural plate is one example of the "region that develops into the nervous system in an animal embryo." That is, in step A, the candidate substance may be locally administered to the neural plate or its vicinity.

[0149] Furthermore, the neural tube is one example of the "region that develops into the nervous system in an animal embryo." That is, in step A, the candidate substance may be locally administered to the neural tube or its vicinity. In this case, local administration may be performed to a neural tube that has a neural hole, ie, that is, that is, that is, that is, that is, a neural tube that is not completely closed, or to a neural tube that is completely closed.

[0150] In addition, in the case of local administration to the neural tube, local administration may be performed to the region of the brain expected to be located anterior to the neural tube, or to the region of the spinal cord expected to be located posterior to the neural tube.

[0151] Furthermore, the primary brain vesicle can be cited as one form of the "region that develops into the nervous system in an animal embryo." That is, in step A, the candidate substance may be locally administered to the primary brain vesicle or in the vicinity thereof. The primary brain vesicles to be locally administered may be any of the forebrain vesicles, mesencephalic vesicles, and rhombencephalic vesicles.Also, the compound may be locally administered into the lumen (ventricle) of a primary brain vesicle.

[0152] Furthermore, the secondary brain vesicle can be cited as one form of the "region that develops into the nervous system in an animal embryo." That is, in step A, the candidate substance may be locally administered to the secondary brain vesicle or in the vicinity thereof. The secondary vesicles to be locally administered may be any of telencephalic vesicles, diencephalic vesicles, hindbrain vesicles, and myelencephalic vesicles.Also, the compound may be locally administered to the lumen of a secondary vesicle, such as the lateral ventricle, the third ventricle, the fourth ventricle, or the cerebral aqueduct.

[0153] The optic vesicle is one example of the "region that develops into the nervous system in an animal embryo." That is, in step A, the candidate substance may be locally administered to the optic vesicle or its vicinity.

[0154] [1-1-1-1-2] Local administration to the area where the heart occurs or its vicinity In one embodiment of the present invention, the candidate substance is locally administered to or near the region where the heart will develop in the animal embryo. This embodiment will be described below after a brief description of the process of heart development during embryonic development.

[0155] Heart development begins with the determination of the left-right axis at the primitive node, and this information is transmitted to the left and right lateral plate mesoderm. In the cephalad cardiac primordium (cardiogenic region), immature mesodermal cells differentiate into cardiac progenitor cells that express cardiac muscle-specific transcription factors. These cardiac progenitor cells further differentiate and gradually migrate to the center of the embryo, becoming cardiac myocytes and forming a single primitive heart tube at the midline. The primitive heart tube contracts in a peristaltic manner, gradually initiating rhythmic contractions and bending and looping toward the right side of the embryo to form the outline of the heart. Within the primitive heart tube, each component is determined segmentally from caudal to cephalad: sinus venosus, primitive atrium, primitive ventricle, bulbus cordis, and truncus arteriosus. As the cardiac loops form, these anterior-posterior segments, particularly in the ventricular region, change position to a left-right relationship. At the same time, as the heart tube elongates, the ventricles sag downward, and the upper and lower positions of the atrioventricular chambers are reversed. Meanwhile, within the primitive heart tube, four endocardial cushions (top, bottom, left, and right) develop in the atrioventricular canal, forming two atrioventricular valves and part of the ventricular septum. In the outflow tract, two (initially four) conotruncal swellings develop, dividing the pulmonary artery and aorta in a spiral. The atrial and ventricular septa then complete their development.

[0156] Previous studies have revealed that some mesodermal cells expressing the transcription factor MESP1 differentiate into cardiac muscle. However, it has been shown that MESP1-expressing cells differentiate not only into cardiac muscle cells but also into non-cardiac cells, including vascular endothelial cells, vascular smooth muscle cells, and extraembryonic mesoderm, which constitutes the placenta. It has been found that cardiac muscle cells subsequently develop from a cell population expressing the transcription factor NKX2.5 among these MESP1-expressing mesodermal cells. Because this cell population contributes almost exclusively to the future heart, these NKX2.5-positive mesodermal components are thought to be cardiac progenitor cells. Furthermore, it has become clear that many transcription factors, such as TBX5, GAT A family, ISLET-1, HAND1 / 2, and MEF2C, play important roles in cardiac development and interact with each other to induce cardiac differentiation.

[0157] In light of the above, one form of the "region that will develop into the heart in an animal embryo" is the cardiac primordium formed in the early stages of development. That is, in step A, the candidate substance may be locally administered to the cardiac primordium or its vicinity.

[0158] Furthermore, one form of the "region that will develop into the heart in an animal embryo" is a region where immature mesodermal cells that differentiate into cardiac progenitor cells are localized, or a region where cardiac progenitor cells are localized. That is, in step A, an embodiment may be adopted in which the candidate substance is locally administered to or near the region where immature mesodermal cells that differentiate into cardiac progenitor cells are localized, or the region where cardiac progenitor cells are localized.

[0159] Furthermore, a primitive heart tube can be given as one form of the "region that will develop into the heart in an animal embryo." That is, in step A, the candidate substance may be locally administered to the primitive heart tube or its vicinity.

[0160] Furthermore, a primitive heart tube can be given as one form of the "region that will develop into the heart in an animal embryo." That is, in step A, the candidate substance may be locally administered to the primitive heart tube or its vicinity.

[0161] [1-1-1-1-3] Local administration to the area of ​​the pancreas where the disease occurs or in its vicinity In one embodiment of the present invention, the candidate substance is locally administered to or near the region in the animal embryo that will develop into the pancreas. This embodiment will be described below after a brief description of the process of pancreatic development during embryonic development.

[0162] The pancreas is composed of two types of tissue with completely different functions. One is the exocrine tissue, which produces and secretes digestive enzymes and releases them into the digestive tract through ducts, and the other is the endocrine tissue, which produces and secretes hormones and releases them into the circulating blood. Each tissue contains multiple types of cells, but it is known that they all differentiate from common precursor cells during development.

[0163] The tubular primitive gut is formed from the endoderm, a single sheet of cells that appears early in development. All cells that make up the pancreas are derived from Pdx1-positive cells. More specifically, all cells that make up the pancreas are derived from the pancreatic bud (pancreatic primordium), which forms from endodermal epithelial cells present in the posterior foregut of the primitive gut (primitive foregut). The pancreatic bud (pancreatic primordium) emerges from the dorsal side of the posterior foregut and, slightly later, protrudes from the ventral side. Pancreatic bud cells proliferate and form a highly branched pancreatic epithelium. The pancreatic epithelium then differentiates into endocrine cells (alpha cells, beta cells, delta cells, PP cells, etc.), pancreatic duct cells, and exocrine cells (acinar cells). The pancreatic buds that emerge from the dorsal and ventral sides of the posterior foregut rotate and fuse to eventually become a single pancreas.

[0164] In light of the above, the pancreatic bud (pancreatic primordium) can be cited as one form of the "region that will develop into the pancreas in an animal embryo." That is, in step A, the candidate substance may be locally administered to the pancreatic bud or its vicinity.

[0165] Furthermore, one form of the "region that will develop into the pancreas in an animal embryo" is the pancreatic epithelium. That is, in step A, the candidate substance may be locally administered to the pancreatic epithelium or its vicinity.

[0166] [1-1-1-2] Screening using adults, etc. In one embodiment of the present invention, late developmental to neonatal stages of animal embryos that have completed the full developmental process, and juvenile to adult individuals of the animals, are used as screening tools. In this case, in step A, the candidate substance is administered to the animal. Examples of the administration route include local administration by injection or the like, oral administration, transdermal administration, enteral administration, etc. In a preferred embodiment, the candidate substance is administered locally to the animal.

[0167] When a candidate substance is administered locally, the administration site is not particularly limited. Preferred embodiments include local administration to the nervous system, heart, or pancreas. Hereinafter, local administration to the nervous system will be particularly described.

[0168] The nervous system may be either the central nervous system or the peripheral nervous system. Once the central nervous system is damaged, it rarely regenerates, and the damage persists. For the purpose of screening for regenerative medicines for the central nervous system, which has traditionally been thought to be unable to regenerate, it is preferable to locally administer a candidate substance to the central nervous system in step A.

[0169] The central nervous system to which the candidate substance is locally administered in step A may be any of the brain, spinal cord, optic nerve, and olfactory nerve. Preferably, the candidate substance is locally administered to the brain, spinal cord, or optic nerve, or in the vicinity thereof.

[0170] In addition, when administering locally to the brain, more specifically, local administration into the ventricles is preferred. Local administration to the brain is preferably by injection. Although injection may be performed manually, it is preferable to use an infusion pump to control the dosage before injection. Furthermore, when the animal to be injected is small, a micromanipulator may be used.

[0171] Local administration to the spinal cord is preferably by injection. Although injection may be performed manually, it is preferable to use an infusion pump to control the dose. Furthermore, when the animal to be injected is small, a micromanipulator may be used.

[0172] When administering locally to the optic nerve, it is more preferable to administer locally to retinal ganglion cells or their vicinity. Although the candidate substance may be administered to retinal ganglion cells or their vicinity by injection into the eyeball, it is preferable to administer the candidate substance by dropping or applying it to the eyeball. When the animal to which the candidate substance is administered is a terrestrial animal (more specifically, a mammal, more specifically, a mouse), administration by eye drop is preferable.

[0173] [1-1-1-3]Disease model In one embodiment of the present invention, a candidate substance is administered to a normal animal that is not affected by a disease, etc. Normal animals are easily and inexpensively available in large quantities compared to the disease models described below, and are therefore advantageous when screening a large group of candidate substances.

[0174] In one embodiment of the present invention, the animal may be a model animal of a disease, etc. The disease model animal may be a genetic model, a drug-induced model, or an injury model in which tissues, organs, or cells are physically damaged. By using disease model animals, it is possible to efficiently screen for active ingredients that are effective when applied to the disease.

[0175] Animal models of central nervous system diseases include depression models such as the learned helplessness model (mouse) and the olfactory bulbectomy depression model (rat); dementia models such as the Tg2576 mouse, the APP & PS1 double transgenic (PSAPP) mouse, the methamphetamine / chlordiazepoxide-induced hyperactivity model (mouse), the cholinergic nucleus lesion model (mouse), the drug-induced memory impairment model (mouse), and the focal brain lesion model (rat); and schizophrenia models such as neonatal phencyclidine (PCP)-administered mice, NR1 knockdown mice, G protein-coupled receptor 2 (SREB2) knockout mice, SREB2-overexpressing mice, and drug-induced catalepsy (rat). These include animal models of Parkinson's disease such as the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease model (mouse); epilepsy models such as the convulsion model (mouse) and the amygdala or hippocampal kindling model (rats); nerve injury models such as spinal cord injury models caused by spinal cord crush (rats, mice, zebrafish) and nerve injury models caused by sciatic nerve crush or transection (rats, mice); and cerebral infarction models caused by middle cerebral artery occlusion (mouse).

[0176] Other animal models of optic nerve diseases include glaucoma model animals such as a Vav gene-deficient model (mouse), a GLAST-deficient normal-tension glaucoma model (mouse), and a high intraocular pressure model (rat) caused by cauterization of the episcleral vein.

[0177] Spinal cord injury model animals can be easily created by applying physical pressure to the spinal cord of normal animals or by piercing the spinal cord.

[0178] [1-1-2] Candidate substance There are no limitations on the candidate substance to be administered to animals in step A. The candidate substance may be any of a low molecular weight compound, a protein, a peptide, or a nucleic acid.

[0179] In one embodiment of the present invention, the candidate substance is a low molecular weight compound. As used herein, the term "low molecular weight compound" refers to a compound having a molecular weight of 2000 or less, more preferably a compound having a molecular weight of 1500 or less, and even more preferably a compound having a molecular weight of 1000 or less.

[0180] In one embodiment of the present invention, the candidate substance is a protein. The type of protein is not particularly limited. The protein may be a protein extracted from a cell or a protein produced by genetic engineering techniques. The candidate substance may also be a fusion protein in which multiple proteins are fused together, or a protein containing an unnatural amino acid as a component.

[0181] In one embodiment, the candidate substance is an antibody. The antibody may be a polyclonal antibody or a monoclonal antibody. This embodiment allows screening of antibody drugs.

[0182] In one embodiment of the present invention, the candidate substance is a peptide. The number of amino acids constituting the peptide is not particularly limited, and examples thereof include peptides composed of 2 to 100 amino acids. Peptides may be extracted from cells, or may be prepared by genetic engineering or chemical synthesis, and may contain unnatural amino acids as constituent amino acids of the peptide.

[0183] In one embodiment of the present invention, the candidate substance is a nucleic acid, which may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a chimeric nucleic acid thereof. In one embodiment of the present invention, the candidate substance is an expression vector that expresses a protein or RNA. In one embodiment of the present invention, the candidate substance is a knockdown vector that suppresses the expression of a specific protein in cells by RNA interference.

[0184] In one embodiment of the invention, the candidate agent is a peptide nucleic acid (PNA).

[0185] In one embodiment, the candidate substance does not comprise a nucleic acid, in one embodiment, the candidate substance does not comprise a protein, and in one embodiment, the candidate substance does not comprise a peptide nucleic acid.

[0186] In one embodiment of the present invention, the candidate substance comprises one or more substances selected from Group A below. [Group A] 5-HT receptor inhibitors, AChR inhibitors, adenylate cyclase activators, AhR activators, Akt inhibitors, ALK inhibitors, ATPase inhibitors, autophagy inhibitors, calcium channel inhibitors, carbonic anhydrase inhibitors, Cdc42 inhibitors, CDK inhibitors, COX inhibitors, dehydrogenase inhibitors, DHFR inhibitors, EGFR inhibitors, ERK inhibitors, estrogen / progesterone receptor inhibitors, gamma-secretase inhibitors, glutamate receptor ligands (potentiators), GSK-3 inhibitors, HDAC activators, HDAC inhibitors, Hedgehog / Smoothened agonists, IGF-IR inhibitors, interleukin receptor inhibitors, IRAK inhibitors, JAK / STAT inhibitors, MAO inhibitors, MEK inhibitors, mitophagy inhibitors, NF-kB inhibitors, NF-kB-AMPK-tyrosine kinase pathway inhibitors, Notch-1 inhibitors, P450 inhibitors, PAEF inhibitors, PDE inhibitors, PPAR inhibitors, TGF-β receptor inhibitors, TGF-beta / Smad inhibitors, TNF receptor inhibitors, Wnt / β-catenin pathway activators

[0187] As shown in the test examples of the present specification, compounds selected as active ingredients by the screening method of the present invention have the above-mentioned primary action. If a compound has a primary action in common with the compound selected as effective in the test examples of the present specification, it is highly likely to be selected as an effective compound by the screening method of the present invention. Therefore, it is preferable to subject a compound having the above-mentioned primary action as a candidate substance to step A in order to achieve highly efficient screening.

[0188] Known compounds with the above-mentioned primary action may be used as candidate substances. The primary action sites (targets) of compounds are organized in various databases and registered in a searchable format, so it is preferable to use these. Examples of such compound databases include PubChem (http: / / pubchem.ncbi.nlm.nih.gov / ), ChemBank (http: / / chembank.broadinstitute.org / welcome.htm), myPresto (http: / / presto.protein.osaka-u.ac.jp / myPresto4 / ), LigandBox (http: / / ligandbox.protein.osaka-u.ac.jp / ligandbox / ), ZINC (http: / / zinc.docking.org / ), ChEBI (http: / / www.ebi.ac.uk / chebi / ), ChEMBL (https: / / www.ebi.ac.uk / chembl / ), and CTD Comparative Toxicogenomics. Database (http: / / ctd.mdibl.org / ), ChemSpider (http: / / www.chemspider.com / ), ChemCupid (http: / / www.namiki-s.co.jp / chemcupid / ), Nikkaji (http: / / nikkajiweb.jst.go.jp / nikkaji_web / pages / top.html), KEGG LIGAND (http: / / www.genome.jp / kegg / ligand.html), DrugBank (http: / / www.drugbank.ca / ), SuperDrug (http: / / bioinformatics.charite.de / superdrug2 / ), SuperTarget (h ttp: / / bioinf-apache.charite.de / supertarget / ), SuperNatural (http: / / bioinformatics.charite.de / supernatural2 / ), PharmGKB (http: / / www.pharmgkb.org / ), Het-PDB Navi, STITCH (http: / / hetpdbnavi.nagahama-i-bio.ac.jp / ), GLIDA (http: / / pharminfo.pharm.kyoto-u.ac.jp / services / glida / ), HIC-Up (http: / / xray.bmc.uu.se / hicup / ), and Dundee Prodrg2 (http: / / davapc1.bioch.dundee.ac.uk / prodrg / ).

[0189] In one embodiment of the present invention, the candidate substance includes a substance that is predicted by an in silico method to be one or more substances selected from Group A above. Screening using cultured cells or laboratory animals is costly and time-consuming, so it is not realistic to use all available compound libraries as candidate substances. Therefore, in silico screening using computers is widely used in modern drug discovery. In the present invention, it is also preferable to use compounds that are predicted in silico to have the above-mentioned primary action as candidate substances.

[0190] This section details in silico predictions. In silico drug discovery requires the use of some theory to model the real-world phenomena occurring in vivo, within cells, or in test tubes. Based on the type of modeling theory used, these methods can be broadly divided into informatics-based methods and simulation-based methods.

[0191] Informatics techniques are also called LBDD (Ligand-Based Drug Discovery) methods because they primarily use structure-activity relationship information on the drug (ligand) side, and are modeled using machine learning based on similarity indices and statistical theory. Machine learning is also a fundamental technology of artificial intelligence.

[0192] The simulation method uses structural information of proteins to simulate the interactions between proteins and drugs on a computer based on theories such as classical molecular force fields, quantum mechanics, and molecular dynamics. Because it is based on protein structure, it is called SBDD (Structure-Based Drug Discovery). Information on the three-dimensional structure of proteins is registered in databases such as PDBj (https: / / pdbj.org / ), RCSB DB (https: / / www.rcsb.org / ), BMRB (https: / / bmrb.io / ), and PDBe (https: / / www.ebi.ac.uk / pdbe / ), and can be used. Alternatively, the three-dimensional structure can be analyzed by NMR or X-ray crystallography. Alternatively, the three-dimensional structure of a protein can be predicted from the amino acid sequence and used in the SBDD method.

[0193] In one embodiment of the present invention, the candidate substance includes a substance predicted by the LBDD method to be one or more compounds selected from Group A. That is, by referring to structure-activity relationship information based on multiple compounds (ligands) already known to have the primary activity of Group A, compounds having a structure predicted to have the same primary activity are selected as candidate substances. The structure-activity relationship information can be modeled using various similarity indices (molecular weight, functional group structure, electron density, hydrophobicity, hydrophilicity, skeleton, etc.) or machine learning based on statistical theory. Drug discovery using the LBDD method is widely practiced, and various software programs available free of charge or for a fee can be used in carrying out the present invention.

[0194] In one embodiment of the present invention, the candidate substance comprises a substance predicted by SBDD to be one or more substances selected from Group A. Specifically, the three-dimensional structural coordinates of the protein that is the target of the primary action of Group A are prepared, and the three-dimensional structural information is used by a computer to analyze the pocket, and compounds predicted to bind therein are selected as candidate substances. Since extremely high-performance supercomputers are currently available, it is preferable to use such computers to perform SBDD.

[0195] In one embodiment of the present invention, the candidate substance includes a substance that is predicted by the LBDD method and the SBDD method to be one or more substances selected from Group A above. The LBDD method (an informatics-based method) and the SBDD method (a simulation-based method) are mutually complementary. Informatics-based design has the advantage of short calculation times and stable accuracy, such as hit rates, that is not very dependent on the expertise of the modeling researcher. On the other hand, it has the disadvantage that only compounds with the same binding site and binding mode as the known drug used will be hit. Simulation has the advantage of being able to predict precise binding free energy in accordance with physical laws and can expand drug discovery to all possible binding sites of proteins, and has the potential to compensate for the shortcomings of informatics. However, simulation has the disadvantage of requiring very long calculation times depending on the level, and there is a risk of complete failure if the calculation settings are inappropriate. For the above reasons, an embodiment in which candidate substances are selected using both the LBDD method and the SBDD method is preferred.

[0196] By introducing transcription factors such as Oct3 / 4, Klf4, c-Myc, and Sox2 into somatic cells, induced pluripotent stem cells (iPS cells) can be obtained from the somatic cells. These four factors play an extremely important role in maintaining the self-renewal ability and pluripotent differentiation properties of stem cells. Compounds that can enhance the expression of these four factors are highly likely to be useful as regenerative medicines. In fact, as shown in the test examples described below, active ingredients selected by the screening method of the present invention have been confirmed to enhance the expression of these transcription factors.

[0197] In view of the above, in the present invention, it is preferable that the candidate substance is a substance that positively regulates the expression of one or more selected from c-Myc, Sox2, Klf4, and Oct4, which enables efficient screening. In this embodiment, candidate substances can be substances known to positively regulate the expression of one or more of c-Myc, Sox2, Klf4, and Oct4. Substances having such expression-regulating activity can be selected by referring to a compound database. In the present invention, a substance that is known to positively regulate in vitro the expression of one or more of c-Myc, Sox2, Klf4, and Oct4 can be used as a candidate substance.

[0198] In one embodiment of the present invention, a substance predicted by an in silico method to positively regulate the expression of one or more of c-Myc, Sox2, Klf4, and Oct4 is used as a candidate substance. As mentioned above, many compounds are known to positively regulate the expression of one or more of c-Myc, Sox2, Klf4, and Oct4, and information on their primary sites of action has been accumulated in compound databases. Furthermore, many of the three-dimensional structures of the proteins that serve as primary sites of action are also registered in protein databases. By applying in silico methods such as SBDD and LBDD based on this information, it is possible to easily search for substances that are suspected to exert the above-mentioned expression-regulating effects.

[0199] In one embodiment of the present invention, the candidate substance is an agonist of a factor that positively regulates the expression of one or more factors selected from c-Myc, Sox2, Klf4, and Oct4. The factor can be easily identified by referring to a protein database that organizes and stores the functions of known proteins, etc. Furthermore, agonists of the factor can be easily identified by referring to the compound database described above. By using such agonists as candidate substances, it is possible to efficiently screen for active ingredients in regenerative medicine.

[0200] In one embodiment of the present invention, the candidate substance is an antagonist of a factor that negatively regulates the expression of one or more selected from c-Myc, Sox2, Klf4, and Oct4. The factor can be easily identified by referring to a protein database that organizes and stores the functions of known proteins, etc. Antagonists of the factor can be easily identified by referring to the compound database described above. By using such antagonists as candidate substances, it is possible to efficiently screen for active ingredients in regenerative medicine.

[0201] Protein databases that organize and store information such as the functions of known proteins are publicly available. These databases contain information on protein-protein interactions automatically extracted by text mining from published academic papers, or manually recorded information. Massive amounts of microarray data are also collected, and information linking proteins related to their expression status is stored. Examples of such databases include ASEdb (http: / / nic.ucsf.edu / asedb / ), Bacteriome.org (http: / / www.compsysbio.org / bacteriome / ), BioGRID (http: / / www.thebiogrid.org / ), DACSIS (http: / / cib.cf.ocha.ac.jp / DACSIS / ), DIP (http: / / dip.doe-mbi.ucla.edu / dip / ), DroID (http: / / www.droidb.org / ), HCPIN (http: / / nesg.org:9090 / HCPIN / index.jsp), HPID (http: / / wilab.inha.ac.kr / hpid / ), HPRD (http: / / www.hprd.org / ), and HUGE ppi(http: / / www.kazusa.or.jp / huge / ppi / ), IntAct(http: / / www.ebi.ac.uk / intact / ), Pathguide(http: / / www.pathguide.org / ), POINT(ht tp: / / point.bioinformatics.tw / Welcome.do), ProNIT(http: / / gibk26.bio.kyutech.ac.jp / jouhou / pronit / pronit.html), Protein-Protein Examples include Interaction Panel (http: / / genome.gsc.riken.go.jp / ppi / ) and STRING (http: / / string.embl.de / ).

[0202] In one embodiment of the present invention, the candidate substance is a substance predicted by a computer to be an agonist of a factor that positively regulates the expression of one or more factors selected from c-Myc, Sox2, Klf4, and Oct4. This embodiment can be implemented by combining the above-mentioned database storing protein interaction information with the above-mentioned in silico method using the LBDD method or SBDD method. That is, by referring to the above-mentioned database storing protein interaction information, factors that positively regulate the expression of one or more selected from c-Myc, Sox2, Klf4, and Oct4 are identified. Next, compounds that are predicted to be agonists of the factors by the in silico method are selected as candidate substances. Such candidate substances have potential as regenerative medicines by upregulating one or more of c-Myc, Sox2, Klf4, and Oct4. Therefore, the screening method of this embodiment allows for efficient selection of active ingredients.

[0203] In one embodiment of the present invention, the candidate substance is a substance predicted by a computer to be an antagonist of a factor that negatively regulates the expression of one or more factors selected from c-Myc, Sox2, Klf4, and Oct4. This embodiment can be implemented by combining the above-mentioned database storing protein interaction information with the above-mentioned in silico method using the LBDD method or SBDD method. That is, by referring to the above-mentioned database storing protein interaction information, factors that negatively regulate the expression of one or more selected from c-Myc, Sox2, Klf4, and Oct4 are identified. Next, compounds predicted to be antagonists of the factors by the in silico method are selected as candidate substances. Such candidate substances have potential as regenerative medicines by upregulating one or more of c-Myc, Sox2, Klf4, and Oct4. Therefore, the screening method of this embodiment allows for efficient selection of active ingredients.

[0204] In one embodiment of the present invention, the candidate substance is a substance predicted by a computer to have low toxicity to living organisms. In modern drug discovery, safety evaluation of compounds is widely performed using in silico methods. In this embodiment, substances that have undergone safety evaluation using in silico methods are used as candidate substances, which increases the possibility that active ingredients selected as a result of screening can be provided as pharmaceuticals.

[0205] Notch signaling is an evolutionarily well-conserved pathway in multicellular organisms that determines cell fate during development and maintains homeostasis of adult tissues. The Notch pathway mediates cell-contact signal transduction. Within this pathway, both signal-sending and signal-receiving cells are affected by ligand-receptor crosstalk, thereby controlling a series of cell fate determination mechanisms in the development of the nervous, cardiac, immune, and endocrine systems. Notch receptors are single-pass transmembrane proteins consisting of a functional extracellular domain (NECD), a transmembrane domain (TM), and an intracellular domain (NICD). There are four classes of Notch receptors, 1 to 4. Notch receptors undergo processing such as cleavage (S1 cleavage) and glycosylation in the endoplasmic reticulum or Golgi apparatus of signal-receiving cells, resulting in the upregulation of Ca. 2+ This results in a heterodimer stabilized by TM-NICD, which is composed of a membrane-inserted TM-NICD and a noncovalently bound NECD. The processed Notch receptor is transported to the plasma membrane by endosomes, where it becomes available for ligand binding through regulation by Deltex and inhibition by NUMB.

[0206] Delta-like members (DLL1, DLL3, and DLL4) and members of the Jagged family (JAG1 and JAG2) function as ligands for Notch signaling receptors. Upon ligand binding, the NECD is cleaved from the TM-NICD domain by TACE (TNF-α ADAM metalloproteinase-converting enzyme) (S2 cleavage). The NECD remains bound to the ligand and undergoes endocytosis and recycling via Mib ubiquitination in the signal-sending cell. In the signal-receiving cell, γ-secretase releases NICD from the TM (S3 cleavage). This release allows NICD to translocate to the nucleus, where it associates with the CSL (CBF1 / Su(H) / Lag-1) transcription factor complex and induces the activation of Notch canonical target genes, including Myc, p21, and the HES family.

[0207] Inhibition of Notch signaling has been reported to inhibit the self-renewal and maintenance of stemness of cancer stem cells (Non-Patent Document 13, Non-Patent Document 14). In addition, the Notch signaling pathway plays various roles during cardiac development, but there is also seemingly contradictory evidence that it promotes or impairs cardiomyogenesis in vitro (Non-Patent Document 15).

[0208] Notch signaling is believed to be essential for maintaining neural progenitor cells (NPCs) in the developing brain. Activation of the Notch signaling pathway maintains NPCs in a proliferative state, whereas loss-of-function mutations in key components of the pathway cause precocious neuronal differentiation and NPC depletion (Non-Patent Document 16). Modulators of Notch signaling, such as the Numb protein, can antagonize the effects of Notch, resulting in cell cycle arrest and NPC differentiation (Non-Patent Document 17, Non-Patent Document 18). Thus, the Notch signaling pathway controls NPC self-renewal and cell fate.

[0209] It is also known that hyperactivating mutations in Notch signaling lead to T-cell acute lymphoblastic leukemia, and that hypoactivating mutations in Notch signaling lead to basal cell carcinoma, a type of skin cancer. Thus, Notch signaling has dual properties, acting as both an oncogene and a tumor suppressor gene, depending on the tissue in which it acts (Non-Patent Document 19). As mentioned above, activation of Notch signaling is known to promote proliferation and suppress differentiation of NPCs. It is also known that activation of Notch signaling in blood cells is important for the self-renewal and maintenance of undifferentiated potential of hematopoietic stem cells. However, activation of Notch signaling is also known to induce differentiation of epidermal cells (Non-Patent Document 20). In other words, Notch signaling has a dual role in inducing self-renewal and maintenance of undifferentiated potential of undifferentiated cells, or conversely, inducing differentiation, depending on the circumstances surrounding the cells. It has also been reported that Notch signaling activates both the expression of genes and the expression of inhibitors of those genes. This report suggests that the response capacity after Notch signaling activation may be transient. Furthermore, since the targets of the Notch signaling pathway consist of both positive and negative regulatory factors, cells can be prepared for either outcome. This is thought to be the reason for the dual nature of Notch signaling (maintaining the self-renewal and undifferentiated potential of undifferentiated cells and inducing differentiation) (Non-Patent Document 21).

[0210] Thus, the Notch signaling pathway plays an important role in regulating the maintenance and differentiation of undifferentiated cells. It is particularly important to note that, depending on the cell's environment, Notch signaling has seemingly contradictory effects: maintaining the self-renewal and undifferentiated potential of undifferentiated cells and inducing differentiation. It is strongly speculated that the effects of the active ingredients discovered by the screening method of the present invention, which induce both self-amplification and differentiation of undifferentiated cells as a single agent, are attributable to the dual nature of Notch signaling. In fact, as shown in the test examples below, multiple inhibitors of the Notch signaling pathway (Notch-1 inhibitors, gamma-secretase inhibitors) have been screened as active ingredients using the screening method of the present invention. This suggests that targeting the Notch signaling pathway may enable more efficient screening of active ingredients. That is, in one embodiment of the present invention, a substance that acts on the Notch signal pathway or a substance that is suspected to act on the Notch signal pathway is used as a candidate substance.

[0211] In one embodiment of the present invention, a candidate substance is a substance that is or is suspected to be an inhibitor of the Notch signaling pathway. Such inhibitors include expression inhibitors of one or more Notch ligands selected from DLL1, DLL3, DLL4, JAG1, and JAG2; inhibitors of one or more Notch ligands selected from DLL1, DLL3, DLL4, JAG1, and JAG2; inhibitors of one or more Notch receptors selected from Notch-1, Notch-2, Notch-3, and Notch-4; inhibitors of S1 cleavage (Furin (PACE) inhibitors), inhibitors of S2 cleavage (TACE inhibitors), inhibitors of S3 cleavage (γ-secretase inhibitors), and inhibitors of the formation of transcription factor complexes in the NICD nucleus. Many of these inhibitors are known, but they can be identified by querying a compound database. Furthermore, compounds that are suspected to be inhibitors of the Notch signaling pathway can be easily identified by informatics techniques based on the known inhibitors. Furthermore, compounds that are suspected to be inhibitors of the Notch signaling pathway can be easily identified by simulation techniques based on the structures of the proteins that are the targets of the inhibitors. As will be shown in the test examples below, inhibitors of the Notch signal pathway have been screened as active ingredients by the screening method of the present invention.

[0212] In one embodiment of the present invention, the candidate substance is an agonist of the Notch signaling pathway or a substance suspected to be an agonist, such as an enhancer of expression of one or more Notch ligands selected from DLL1, DLL3, DLL4, JAG1, and JAG2, or an agonist of one or more Notch receptors selected from Notch-1, Notch-2, Notch-3, and Notch-4. Many of these agonists are known, but they can be identified by querying a compound database. Furthermore, compounds that are suspected to be agonists of the Notch signaling pathway can be easily identified by informatics techniques based on the known inhibitors. Furthermore, compounds that are suspected to be agonists of the Notch signaling pathway can be easily identified by simulation techniques based on the structures of the proteins that are targets of the inhibitors.

[0213] mTOR is a serine / threonine kinase that senses nutrients such as glucose and amino acids and plays a role in regulating cell growth, metabolism, and survival. Discovered as the target molecule of the antibiotic rapamycin, this enzyme has recently been revealed to form two distinct complexes, mTORC1, which consists of mTOR, Raptor, and mLST8 (GβL), and mTORC2, which consists of mTOR, Rictor, mLST8 (GβL), and Sin1, and to have independent networks.

[0214] The mTORC1 complex is activated by the aforementioned nutrients as well as growth factors, hormones, stress, etc., and phosphorylates molecules involved in protein synthesis and cell proliferation, such as 4EBP1 and p70S6K, and is involved in mRNA translation, autophagy inhibition, ribosome biogenesis, and other processes. On the other hand, the mTORC2 complex is not regulated by nutrients but is activated by PI3K upon stimulation with growth factors, phosphorylating Akt, SGK, and PKC, and is involved in the suppression of apoptosis, cell growth, and control of the cytoskeleton.

[0215] Among these, the PI3K / AKT / mTOR signaling pathway is activated in most cancers and has therefore been extensively studied as a potential target for anticancer drugs. PI3Ks are a unique family of intracellular lipid kinases that phosphorylate the 3'-hydroxyl group of the inositol ring of phosphatidylinositides (PtdIns). PI3Ks are classified into three classes. The most studied is class I PI3K, which promotes the conversion of membrane-bound phosphatidylinositol-(4,5)-bisphosphate (PIP2) to phosphatidylinositol-(3,4,5)-triphosphate (PIP3). PIP3 functions as a second messenger, promoting the recruitment and activation of PH domain-containing kinases, such as PI3K-dependent kinase-1 (PDK1). PIP3 signaling is regulated by PTEN, which antagonizes PI3K activity. The serine / threonine kinase AKT, also known as protein kinase B (PKB), contains a PH domain and is recruited to the plasma membrane together with PDK1. Full activation of AKT requires phosphorylation of amino acid residues T308 and S473 by PDK1 and mTORC2. Activated AKT phosphorylates many target proteins, notably glycogen synthase kinase 3 (GSK3), tuberous sclerosis complex 2 (TSC2), caspase 9, and PRAS40 (AKT1S1), exerting a relatively broad spectrum of downstream effects that promote cell proliferation, differentiation, apoptosis, angiogenesis, and metabolism.

[0216] Studying the PI3K / AKT / mTOR signaling pathway during development has been challenging because the deficiency of its components often results in embryonic lethality. However, the advent of ES and iPS cells has alleviated this problem to some extent. Consequently, it has become clear that the PI3K / AKT / mTOR signaling pathway is crucial for embryonic development and is essential for the self-renewal of iPS cells. Although human and mouse ES cells require significantly different signaling for their culture and growth, studies using kinase inhibitors and genetic approaches have revealed that activation of PI3K / AKT signaling is required for both types of ES cells to maintain their undifferentiated nature. Treatment of mouse ES cells with general PI3K inhibitors or p110β-specific inhibitors impairs their pluripotency, and deletion of the PI3K gene also abolishes pluripotency in mouse ES cells. Similarly, inhibition of PI3K in human ES cells simultaneously down-regulates pluripotency markers and up-regulates lineage-specific genes, both of which strongly suggest a loss of pluripotency. Supporting this, knocking out Pten in both mouse and human ES cells promotes the proliferation and survival of these cells (Non-Patent Document 22). In addition, LIF is added to the culture of ES cells, which is widely used in research, and it is known that the LIF receptor activates the PI3K / AKT signaling pathway. LIF is a cytokine that was originally identified as a member of the IL-6 family, but in ES cell culture it is used as a differentiation inhibitor (maintaining totipotency).

[0217] Thus, the PI3K / AKT / mTOR signaling pathway plays an important role in maintaining stem cell self-renewal and pluripotency and in controlling differentiation. Therefore, the screening method of the present invention may be embodied as targeting the PI3K / AKT / mTOR signaling pathway. That is, in one embodiment of the present invention, a substance that acts on the PI3K / AKT / mTOR signal pathway or a substance that is suspected to act on the PI3K / AKT / mTOR signal pathway is used as a candidate substance.

[0218] In one embodiment of the present invention, a substance that is an inhibitor of the PI3K / AKT / mTOR signaling pathway or is suspected to be an inhibitor is used as a candidate substance. Such inhibitors include PI3K inhibitors, AKT inhibitors, mTOR inhibitors, mTORRC1 inhibitors, mTORC2 inhibitors, mTORC1 / 2 inhibitors, and PI3K / mTOR inhibitors. Many of these inhibitors are known, but they can be identified by querying a compound database. Furthermore, compounds that are predicted to be inhibitors of the PI3K / AKT / mTOR signaling pathway can be easily identified using informatics techniques based on the known inhibitors. Furthermore, compounds that are predicted to be inhibitors of the PI3K / AKT / mTOR signaling pathway can be easily identified using simulation techniques based on the structures of the proteins targeted by the inhibitors. As will be shown in the test examples below, inhibitors of the PI3K / AKT / mTOR signal pathway have been screened as active ingredients by the screening method of the present invention.

[0219] In one embodiment of the present invention, the candidate substance is an agonist or a suspected agonist of the PI3K / AKT / mTOR signaling pathway, such as a PTEN inhibitor or a PI3K activator. Many of these inhibitors and activators are known, but they can be identified by querying compound databases. Furthermore, compounds that are suspected to be agonists of the PI3K / AKT / mTOR signaling pathway can be easily identified using informatics techniques based on the known inhibitors and activators. Furthermore, compounds that are suspected to be agonists of the PI3K / AKT / mTOR signaling pathway can be easily identified using simulation techniques based on the structures of the proteins that are targets of the inhibitors.

[0220] The JAK / STAT signaling pathway is a signaling system that transmits chemical signals from outside the cell to the cell nucleus, causing DNA transcription and expression. It is involved in immunity, cell proliferation, differentiation, apoptosis, carcinogenesis, etc. The JAK / STAT signaling cascade is mainly composed of three components: receptors for cytokines such as interferons, interleukins, and growth factors on the cell surface; JAK; and STAT. The mechanism is as follows: when a ligand (such as interferon, interleukin, or growth factor) binds, the receptor activates JAK, which then expresses its kinase activity. Activated JAK phosphorylates tyrosine residues on the receptor, creating binding sites for proteins with SH2 domains on the receptor. STATs with SH2 domains bind to the tyrosine residues phosphorylated by JAK, and then are themselves phosphorylated by JAK. Activated STATs phosphorylated on tyrosine residues form hetero- or homodimers, translocate into the cell nucleus, and initiate transcription of target genes. STATs can be tyrosine-phosphorylated directly by receptor tyrosine kinases (such as epidermal growth factor receptor) or by non-receptor cytoplasmic tyrosine kinases (such as c-src).

[0221] This JAK / STAT signaling pathway is negatively regulated at multiple stages. Cytokine receptors and activated STATs are dephosphorylated and inactivated by protein tyrosine phosphatases. It is also known that suppressors of cytokine signaling (SOCS) bind to JAKs, inhibiting their binding and phosphorylation of STATs and competitively inhibiting the phosphorylated tyrosines of cytokine receptors. STATs are negatively regulated in the nucleus by inhibitors of activated STATs (PIAS). For example, PIAS1 and PIAS3 bind to and inhibit DNA, thereby suppressing transcriptional activation by STAT1 and STAT3.

[0222] Recent studies have shown that JAK / STAT and MAPK signaling pathways regulate important homeostatic processes in germline and adult stem cells in Drosophila, as well as regenerative processes in several tissues, including the gonad, intestine, and appendages (Non-Patent Document 23). In addition, JAK / STAT and MAPK signaling pathways have been reported to regulate the stem cell properties of Müller glia in the mammalian retina (Non-Patent Document 24). Furthermore, LIF is added as a differentiation inhibitor in ES cell culture, which is widely used in research, and it is known that the LIF receptor activates the JAK / STAT signaling pathway.

[0223] Thus, the JAK / STAT signaling pathway plays an important role in the regulation of stem cells. Furthermore, as described in the test examples below, multiple JAK / STAT inhibitors have been screened using the screening method of the present invention. Therefore, the screening method of the present invention may be embodied as targeting the JAK / STAT signal pathway. That is, in one embodiment of the present invention, a substance that acts on the JAK / STAT signal pathway or a substance that is suspected to act on the JAK / STAT signal pathway is used as a candidate substance.

[0224] In one embodiment of the present invention, candidate substances are substances that are or are suspected to be inhibitors of the JAK / STAT signaling pathway. Examples of such inhibitors include JAK inhibitors and STAT inhibitors. Many of these inhibitors are known, but they can be identified by querying a compound database. Furthermore, compounds that are suspected to be inhibitors of the JAK / STAT signaling pathway can be easily identified using informatics techniques based on the known inhibitors. Furthermore, compounds that are suspected to be inhibitors of the JAK / STAT signaling pathway can be easily identified using simulation techniques based on the structures of the proteins that are the targets of the inhibitors. As will be shown in the test examples below, inhibitors of the JAT / STAT signal pathway have been screened as active ingredients by the screening method of the present invention.

[0225] In one embodiment of the present invention, candidate substances are substances that are or are suspected to be agonists of the JAK / STAT signaling pathway, including PIAS inhibitors, SOCS inhibitors, and PTP inhibitors. Many of these inhibitors are known, but they can be identified by querying compound databases. Furthermore, compounds that are suspected to be agonists of the JAK / STAT signaling pathway can be easily identified using informatics techniques based on the known inhibitors. Furthermore, compounds that are suspected to be agonists of the JAK / STAT signaling pathway can be easily identified using simulation techniques based on the structures of the proteins that are targets of the inhibitors.

[0226] Components of the mitogen-activated protein kinase (MAPK) signaling pathway transduce and regulate extracellular stimuli to control and fine-tune vital cellular functions, including growth, cell division, metabolism, motility, innate immunity, cellular stress responses, apoptosis, and survival in eukaryotes ranging from yeast to humans. There are four major branches of the MAPK signaling pathway, and over a dozen MAPK enzymes, classified into at least seven distinct groups. The MAPK cascade is characterized by the sequential activation of three protein kinases through dual-specificity serine / threonine protein kinases (MAPKs), MAPK activators (MEKs, MKKs, or MAPK kinases), and MEK activators (MEK kinases [MEKKs] or MAPK kinase kinases). Activation of the classical MAPK pathway begins at the plasma membrane, where small GTPases and various protein kinases phosphorylate and activate MAPKKKs. Subsequently, MAPKKKs directly phosphorylate MAPKKs, which then phosphorylate MAPKs. Activated MAPKs interact with and phosphorylate numerous cytoplasmic substrates, ultimately regulating transcription factors that induce context-specific gene expression.

[0227] In the pathways activated by growth factors, A-Raf, B-RAF, Mos, and Tpi-2 are known as MAPKKKs, MEK1 / 2 as MAPKKs, and ERK1 / 2 as MAPKs, and their downstream factors include Elk-1, Ets-2, RSK, MNK, MSK, and cPLA2. In pathways activated by stress, cytokines, growth factors, etc., MLK3, TAK1, MEKK4, and ASK1 are known as MAPKKKs, MKK3 / 6 as MAPKKs, and p38α / β / γ / 5 as MAPKs, and their downstream factors include CHOP, ATF2, MNK, MSK, MEF2, and Elk-1. In pathways activated by stress, cytokines, and growth factors, MEKK1 / 4, DLK, MLK1-4, LZK, TAK1, ASK1, and ZAK are known as MAPKKKs, MKK4 / 7 as MAPKKs, and JKK1, 2, and 3 as MAPKs, and their downstream factors include JUN, ATF2, RNPK, p53, NFAT4, and Shc. In the pathways activated by stress and growth factors, MEKK2 / 3, MEK5, and ERK5 are known as MAPKKKs, MAPKKs, and MAPKs, respectively, and MEF2 is known as a downstream factor thereof.

[0228] In addition, LIF is added as a differentiation inhibitor in ES cell cultures, which are widely used in research, and it is known that the LIF receptor activates the MAPK signaling pathway (RAS-RAF-MEK-ERK pathway), which is essential for maintaining self-renewal and totipotency.

[0229] Thus, the MAPK signaling pathway plays an important role in the regulation of stem cells. Furthermore, as described in the test examples below, multiple MAPK signaling pathway inhibitors have been screened using the screening method of the present invention. Therefore, the screening method of the present invention may be embodied as targeting the MAPK signal pathway. That is, in one embodiment of the present invention, a substance that acts on the MAPK signal pathway or a substance that is suspected to act on the MAPK signal pathway is used as a candidate substance.

[0230] In one embodiment of the present invention, the candidate substance is a substance that is or is suspected to be an inhibitor of the MAPK signaling pathway. Such inhibitors include A-Raf inhibitors, B-RAF inhibitors, Mos inhibitors, Tpi-2 inhibitors, MEK1 / 2 inhibitors, ERK1 / 2 inhibitors, Elk-1 inhibitors, Ets-2 inhibitors, RSK inhibitors, MNK inhibitors, MSK inhibitors, cPLA2 inhibitors, MLK3 inhibitors, TAK1 inhibitors, MEKK4 inhibitors, ASK1 inhibitors, MKK3 / 6 inhibitors, p38α / β / γ / 5 inhibitors, CHOP inhibitors, ATF2 inhibitors, MNK inhibitors, MSK inhibitors, M Examples of inhibitors include one or more selected from EF2 inhibitors, Elk-1 inhibitors, MEKK1 / 4 inhibitors, DLK inhibitors, MLK1-4 inhibitors, LZK inhibitors, TAK1 inhibitors, ASK1 inhibitors, ZAK inhibitors, MKK4 / 7 inhibitors, JKK1,2,3 inhibitors, JUN inhibitors, FOS inhibitors, ATF2 inhibitors, RNPK inhibitors, p53 inhibitors, NFAT4 inhibitors, Shc inhibitors, MEKK2 / 3 inhibitors, MEK5 inhibitors, ERK5 inhibitors, and MEF2 inhibitors. Many of these inhibitors are known, but they can be identified by querying a compound database. Furthermore, compounds predicted to be inhibitors of the MAPK signaling pathway can be easily identified using informatics techniques based on the known inhibitors. Furthermore, compounds predicted to be inhibitors of the MAPK signaling pathway can be easily identified using simulation techniques based on the structures of the proteins targeted by the inhibitors. As will be shown in the test examples below, inhibitors of the MAPK signal pathway have been screened as active ingredients by the screening method of the present invention.

[0231] In one embodiment of the present invention, the candidate substance is an agonist or a suspected agonist of the MAPK signaling pathway, such as an interleukin receptor activator. Many of these activators are known, but they can be identified by querying compound databases. Furthermore, compounds that are suspected to be agonists of the MAPK signaling pathway can be easily identified using informatics techniques based on the known inhibitors. Furthermore, compounds that are suspected to be agonists of the MAPK signaling pathway can be easily identified using simulation techniques based on the structures of the proteins that are targets of the inhibitors.

[0232] Transforming growth factor-β (TGF-β), Nodal, Activin, BMP, and other cytokines belonging to the TGF-β superfamily regulate various cellular functions. The TGF-β superfamily regulates a wide range of biological phenomena, including cell proliferation inhibition, differentiation, cell death, angiogenesis, immunity, extracellular matrix production, and aging. Cells stimulated by the TGF-β superfamily, which functions extracellularly, convert this stimulation into phosphorylation of the transcription factor SMAD on the cell membrane. The TGF-β superfamily is roughly divided into two groups: TGF-β / Nodal / Activin and BMP. TGF-β / Nodal / Activin stimulation is converted into phosphorylation of SMAD2 and SMAD3 (SMAD2 / 3), while BMP stimulation is converted into phosphorylation of SMAD1, SMAD5, and SMAD8 (SMAD1 / 5 / 8). These SMADs (SMAD1 / 2 / 3 / 5 / 8) are called R-SMADs (receptor-regulated SMADs). R-SMADs phosphorylated in response to TGF-β superfamily stimuli form heterotrimers with SMAD4 (common-mediator SMAD), known as Co-SMADs, which translocate into the nucleus and regulate (activate or suppress) the expression of various genes. In addition to R-SMADs and Co-SMADs, there are also I-SMADs (inhibitory SMADs, SMAD6 and SMAD7) that negatively regulate the function of the TGF-β superfamily.

[0233] In the field of stem cells, BMP-4 is an important serum-derived factor that, together with LIF, maintains mouse ES cells in an undifferentiated state, and it has been understood that this action requires the activation of a specific intracellular signaling pathway (the Smad pathway). However, as knowledge about other stem cells, including human ES cells, has accumulated, it has also been pointed out that BMPs often promote stem cell differentiation.

[0234] Thus, the TGFβ / SMAD signaling pathway plays an important role in the regulation of stem cells. Furthermore, as described in the test examples below, multiple TGFβ / SMAD inhibitors have been screened using the screening method of the present invention. Therefore, the screening method of the present invention may be embodied as targeting the TGFβ / SMAD signaling pathway. That is, in one embodiment of the present invention, a substance that acts on the TGFβ / SMAD signal pathway or a substance that is suspected to act on the TGFβ / SMAD signal pathway is used as a candidate substance.

[0235] In one embodiment of the present invention, candidate substances are substances that are or are suspected to be inhibitors of the TGFβ / SMAD signaling pathway. Examples of such inhibitors include Tβ receptor inhibitors, Nodal receptor inhibitors, Activin receptor inhibitors, BMP receptor inhibitors, and SMAD inhibitors. Many of these inhibitors are known, but they can be identified by querying a compound database. Furthermore, compounds that are predicted to be inhibitors of the TGFβ / SMAD signaling pathway can be easily identified using informatics techniques based on the known inhibitors. Furthermore, compounds that are predicted to be inhibitors of the TGFβ / SMAD signaling pathway can be easily identified using simulation techniques based on the structures of the proteins that are the targets of the inhibitors. As will be shown in the test examples below, inhibitors of the TGFβ / SMAD signal pathway have been screened as active ingredients by the screening method of the present invention.

[0236] In one embodiment of the present invention, candidate substances are substances that are or are suspected to be agonists of the TGFβ / SMAD signaling pathway, including Tβ receptor agonists, Nodal receptor agonists, Activin receptor agonists, BMP receptor agonists, and SMAD activators. Many of these agonists are known, but they can be identified by querying a compound database. Furthermore, compounds that are suspected to be agonists of the TGFβ / SMAD signaling pathway can be easily identified using informatics techniques based on the known agonists. Furthermore, compounds that are suspected to be agonists of the TGFβ / SMAD signaling pathway can be easily identified using simulation techniques based on the structure of the protein that is the target of the agonists.

[0237] To date, 19 WNT genes have been identified in humans. Some of these genes undergo alternative splicing, resulting in multiple isoforms of their gene products, the Wnt proteins. Wnt proteins primarily bind to Frizzled receptors, which are seven-transmembrane receptor proteins, and transmit signals intracellularly. Frizzled receptors consist of a family of approximately 10 proteins, and their expression varies depending on the cell type, contributing to the specificity of cell type and signal transduction. The intracellular level of β-catenin is maintained at a low level when Wnt signaling is off, and in this state, β-catenin is incorporated into a degradation complex composed of Adenomatous Polyposis Coli (APC) and other proteins, where it is phosphorylated by the serine / threonine kinases casein kinase 1 (CK1) and glycogen synthase 3b (GSK3β), subsequently ubiquitinated by β-transducin repeat-containing protein (bTrCP), and degraded by the proteasome.

[0238] On the other hand, under Wnt signaling activation, Dishevelled recruits GBP / Frat-1, which dissociates GSK3β from the degradation complex, protecting β-catenin. Frodo and β-arrestin act in concert with Dishevelled. Meanwhile, Dapper acts as a Dishevelled antagonist. Furthermore, the low-density lipoprotein receptor-related protein family, LRP5 / LRP6, acts as a co-receptor for the canonical Wnt signaling pathway via β-catenin.

[0239] In the canonical cascade mediated by β-catenin, activation of Wnt signaling stabilizes β-catenin and translocates it into the nucleus, where it activates the transcription of target genes such as TCF, Lymphoid Enhancer D1, PPARd, and Twin. On the other hand, when Wnt signaling is turned off, TCF / LEF binds to transcriptional regulators such as Groucho and is inactivated. The transcriptional regulatory activity of Groucho is mediated by histone deacetylases (HDACs), which regulate gene transcription.

[0240] ICAT and Duplin bind to β-catenin and inhibit the interaction between β-catenin and TCF / LEF, thereby negatively regulating the canonical pathway. β-catenin is also involved in cell adhesion, binding to the intracellular domain of type I classical cadherins such as E-cadherin and N-cadherin, and further forming a complex with α-catenin. This cadherin / catenin complex is thought to interact with the actin cytoskeleton via other molecules.

[0241] The PCP (Planar Cell Polarity) pathway, a non-canonical cascade, regulates cell polarity by controlling the actin cytoskeleton and asymmetric cytoskeletal formation. When Wnt binds to the Frizzled receptor, Dishevelled activates the small GTPases Rho and Rac. In Rho-mediated signaling, Daam-1 forms a complex with Dishevelled and Rho, activating the Rho kinase ROCK. In contrast, Rac-mediated signaling does not interact with Daam-1 and activates Jun kinase (JNK).

[0242] In the Wnt / Calcium pathway, another non-canonical cascade, Wnt binding to Frizzled receptors results in the release of calcium into the cytoplasm. This is mediated by Frizzled receptors Knypek and Ror2. Intracellular second messengers of this pathway include heterotrimeric G proteins, phospholipase C (PLC), and protein kinase C (PKC). The Wnt / Calcium pathway has been reported to be important for cell-cell adhesion and cell dynamics during gastrulation.

[0243] Wnt signaling antagonists are classified into two classes: secreted frizzled-related protein (sFRP) and Dickkopf (Dkk) classes. The sFRP class includes the sFRP family of sFRPs (sFRP1-5), Wnt inhibitory factor-1 (WIF-1), and Cerberus. These antagonists directly bind to Wnt and inhibit its binding to the receptor. On the other hand, Dkk1-4, which belong to the Dkk class, bind to the extracellular domain of LRP5 / LRP6 and inhibit Wnt signaling. Kremen binds to Dkk bound to LRP5 / LRP6 and endocytose the Dkk-LRP complex, thereby preventing the exposure of LRP on the cell surface. As a result, Wnt cannot bind to the receptor and signaling is inhibited. Therefore, theoretically, sFRP class antagonists inhibit both the canonical pathway and the non-canonical pathway of Wnt signaling mediated by β-catenin, whereas Dkk class antagonists selectively inhibit only the canonical pathway. Isolated Wnt proteins are active in various stem cells, including neural stem cells, mammary stem cells, and embryonic stem cells. In general, Wnt proteins act to maintain the undifferentiated state of stem cells (Non-Patent Document 25).

[0244] Thus, the Wnt signaling pathway plays an important role in the regulation of stem cells. Furthermore, as described in the test examples below, multiple Wnt / β-catenin genes have been screened using the screening method of the present invention. Therefore, the screening method of the present invention may be embodied as targeting the Wnt signal pathway. That is, in one embodiment of the present invention, a substance that acts on the Wnt signal pathway or a substance that is suspected to act on the Wnt signal pathway is used as a candidate substance.

[0245] In one embodiment of the present invention, a candidate substance is a substance that is or is suspected to be an inhibitor of the Wnt signaling pathway. Examples of such inhibitors include Wnt inhibitors and Frizzled receptor inhibitors. Many of these inhibitors are known, but they can be identified by querying a compound database. Furthermore, compounds that are suspected to be inhibitors of the Wnt signaling pathway can be easily identified using informatics techniques based on the known inhibitors. Furthermore, compounds that are suspected to be inhibitors of the Wnt signaling pathway can be easily identified using simulation techniques based on the structures of the proteins that are the targets of the inhibitors. As will be shown in the test examples below, inhibitors of the Wnt signal pathway have been screened as active ingredients by the screening method of the present invention.

[0246] In one embodiment of the present invention, a substance that is an agonist of the Wnt signaling pathway or a substance that is suspected to be an agonist is used as a candidate substance. Examples of such agonists include GSK-3 inhibitors, CK1 inhibitors, bTrCP inhibitors, and Pan-Proteasome inhibitors. Many of these inhibitors are known, but they can be identified by querying a compound database. Furthermore, compounds that are suspected to be agonists of the Wnt signaling pathway can be easily identified using informatics techniques based on the known inhibitors. Furthermore, compounds that are suspected to be agonists of the Wnt signaling pathway can be easily identified using simulation techniques based on the structure of the protein that is the target of the inhibitor.

[0247] The Notch signaling pathway, PI3K / AKT / mTOR signaling pathway, JAK / STAT signaling pathway, MAPK signaling pathway, and TGFβ / SMAD signaling pathway are generally known to promote stem cell self-renewal and maintenance of totipotency. However, as shown in the test examples below, the group of compounds that induce an increase in both undifferentiated and differentiated cells in vivo includes inhibitors and activators of these signaling pathways. Therefore, it can be said that any substance that acts on these pathways, whether it is an inhibitor or activator, is likely to induce an increase in both undifferentiated and differentiated cells in vivo. Therefore, in a preferred embodiment of the present invention, a substance that acts on (including inhibition and activation of) one or more signal pathways selected from the Notch signal pathway, the PI3K / AKT / mTOR signal pathway, the JAK / STAT signal pathway, the MAPK signal pathway, the TGFβ / SMAD signal pathway, and the Wnt signal pathway is used as a candidate substance for screening.

[0248] For the same reason, in a preferred embodiment of the present invention, a substance predicted by in silico methods to act (including inhibition and activation) on one or more signal pathways selected from the Notch signal pathway, the PI3K / AKT / mTOR signal pathway, the JAK / STAT signal pathway, the MAPK signal pathway, the TGFβ / SMAD signal pathway, and the Wnt signal pathway is used as a candidate substance for screening.

[0249] In one embodiment, a compound encompassed by general formula (I), general formula (II), or general formula (III) described below, or a salt or hydrate thereof, is administered to an animal as a candidate substance. The salt is preferably a pharmacologically acceptable salt, such as an inorganic acid salt, an organic acid salt, an inorganic base salt, an organic base salt, or an acidic or basic amino acid salt.

[0250] Preferred examples of inorganic acid salts include hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates, and preferred examples of organic acid salts include acetates, succinates, fumarates, maleates, tartrates, citrates, lactates, stearates, benzoates, mandelates, methanesulfonates, ethanesulfonates, p-toluenesulfonates, and benzenesulfonates.

[0251] Preferred examples of inorganic base salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, and ammonium salts. Preferred examples of organic base salts include diethylamine salts, diethanolamine salts, meglumine salts, and N,N'-dibenzylethylenediamine salts.

[0252] Preferred examples of acidic amino acid salts include aspartates and glutamates, and preferred examples of basic amino acid salts include arginine salts, lysine salts and ornithine salts.

[0253] General formulas (I) to (III) will be described in detail below.

[0254] [1-1-2-1]General formula (I) In one embodiment of the present invention, a compound encompassed by the following general formula (I), or a salt or hydrate thereof, is administered to an animal as a candidate substance.

[0255] [ka]

[0256] In general formula (I), ring A may be a monocyclic ring or a fused bicyclic ring. When ring A is a monocyclic ring, ring A is preferably a 3- to 8-membered ring, more preferably a 4- to 6-membered ring, more preferably a 5- to 6-membered ring, and more preferably a 5-membered ring. When ring A is a fused bicyclic ring, ring A is preferably a 4- to 8-membered ring, more preferably a 5- to 8-membered ring, more preferably a 5- to 7-membered ring, more preferably a 5- or 6-membered ring. In a preferred embodiment, ring A is a monocyclic ring.

[0257] Ring A may be saturated or unsaturated. When ring A is unsaturated, the number of double bonds contained in ring A is preferably 1 to 3, more preferably 1 to 2. Preferably, ring A is unsaturated. More preferably, ring A is an aromatic ring.

[0258] Ring A may contain a heteroatom. The number of heteroatoms contained in ring A is preferably 1 to 4, more preferably 1 to 3, more preferably 2 to 3, and more preferably 2. Examples of heteroatoms include oxygen atoms, nitrogen atoms, and sulfur atoms, and more preferably nitrogen atoms. The heteroatoms are independently selected from oxygen atoms, nitrogen atoms and sulfur atoms, and 1 to 4, more preferably 1 to 3, more preferably 2 to 3, and more preferably 2 heteroatoms are selected. Ring A is preferably an aromatic heterocycle containing a heteroatom, more preferably an aromatic heterocycle containing a nitrogen atom, and more preferably a monocyclic aromatic heterocycle containing a nitrogen atom.

[0259] Specific examples of ring A include aziridine, azirine, oxirane, oxirene, phosphirane, phosphirene, thiirane, thiylene, diaziridine, diazirine, oxaziridine, dioxirane, azetidine, azeto, oxetane, oxete, thietane, thiet, diazetidine, diazeto, dioxetane, dioxete, dithietane, dithiet, pyrrolidine, pyrrole, tetrahydrofuran, furan, tetrahydrothiophene, thiophene, and imidazolidine. , pyrazolidine, imidazole, imidazoline, pyrazole, oxazolidine, isoxazolidine, oxazole, oxazoline, isoxazole, thiazolidine, isothiazolidine, thiazole, isothiazole, dioxolane, dithiolane, triazole, furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, tetrazole, oxatetrazole, thiatetrazole, pentazole, piperidine, pyridine, pyridinium cation, tetrahydropyran, pyran, pyrylium cation, thiane, thiopyran, thiopyrylium cation, piperazine, diazine, morpholine, oxazine, thiomorpholine, thiazine, dioxane, dioxine, dithiane, dithiin, hexahydro-1,3,5-triazine, triazine, trioxane, trithiane, tetrazine, pentazine, azepane, azepine, oxepane, oxepine, thiepane, thiepine, diazepane, diazepine, thiazepine, azocane, azocine, oxocane, oxocine, thiocane, thiocine, azonane, azonin, oxonane, oxonin, thionane, and thionine.

[0260] More preferred embodiments of ring A include pyrrole, furan, thiophene, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, thiazole, thiazoline, isothiazole, triazole, furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, tetrazole, oxatetrazole, thiatetrazole, pentazole, pyridine, pyran, thiopyran, diazine, oxaxidine, thiazine, dioxin, dithiin, triazine, and tetrazine.

[0261] More preferred embodiments of ring A include pyrrole, imidazole, imidazoline, pyrazole, pyrazoline, triazole, pyridine, diazine, triazine, and tetrazine.

[0262] More preferred embodiments of ring A include imidazole, pyrazole, triazole, pyridine, diazine and triazine. More preferably, ring A is pyrazole.

[0263] When ring A is pyrazole, R 2 is bonded to preferably any one of the 3rd to 5th positions, more preferably the 4th position, of the pyrazole ring. Also, -LR 1 is preferably attached to the 1st, 3rd or 5th position of the pyrazole ring, more preferably to the 1st or 3rd position. The numbers of the pyrazole rings are as shown in the example below. [ka]

[0264] Ring A may be further substituted with a C1-3 alkyl group or a halogen atom. The C1-3 alkyl group may be linear or branched, and specific examples include a methyl group, an ethyl group, a propyl group, and a methylethyl group. The halogen atom referred to here preferably includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferably, 1 to 3, more preferably 1 to 2, and more preferably 1 hydrogen atom of ring A may be substituted.

[0265] In the general formula (I), L is a substituent R 3 It is a C1-10 saturated or unsaturated hydrocarbon chain which may be substituted with.

[0266] The hydrocarbon chain is preferably C1-9, more preferably C1-8, more preferably C1-7, more preferably C1-6, more preferably C1-5, more preferably C1-4, more preferably C1-3, more preferably C1-2.

[0267] Specific examples of the hydrocarbon chain include -CH2-, -CH2CH2-, -CH=CH-, -CH2CH2CH2-, -CH=CHCH2-, -CH2CH=CH-, -CH2CH2CH2CH2-, -CH=CHCH2CH2-, -CH2CH=CHCH2CH2-, -CH2CH=CHCH2-, -CH2CH2CH=CHCH2-, -CH2CH2CH=CH-, -CH=CHCH=CH-, and the like.

[0268] The hydrocarbon chain is a substituent R 3 may be substituted with. Substituent R 3 represents a C1-10 saturated or unsaturated hydrocarbon group which may have a cyclic structure and which may be substituted with a halogen atom. The halogen atom referred to here preferably includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Substituent R 3 is preferably C1 to C10, more preferably C2 to C9, more preferably C3 to C8, more preferably C4 to C6.

[0269] Substituent R 3 may be linear or branched. In addition, the substituent R 3 may have a cyclic structure, in which case the ring is preferably a 3- to 10-membered ring, more preferably a 4- to 8-membered ring, more preferably a 4- to 7-membered ring, more preferably a 4- to 6-membered ring, more preferably a 5- or 6-membered ring, and more preferably a 5-membered ring.

[0270] In addition, in general formula (I), L may not be present.

[0271] In general formula (I), R 1 is R defined below 1-1 or R 1-2 is. R1-1 : -C≡N, -COOH, -CHO, -COOCH3, -NO2, or a halogen atom R 1-2 may contain 1 to 4 heteroatoms independently selected from oxygen atoms, nitrogen atoms, and sulfur atoms, saturated or unsaturated, may further be substituted with a C1-3 alkyl group or a halogen atom, 3 to 8 members, Monocyclic or fused bicyclic groups.

[0272] R 1-1 is more preferably —C≡N or —NO 2 .

[0273] R 1-2 may be a monocyclic ring or a fused bicyclic ring. R 1-2 When is a monocyclic ring, R 1-2 is preferably a 3- to 8-membered ring, more preferably a 4- to 6-membered ring, more preferably a 5- or 6-membered ring, and more preferably a 6-membered ring. R 1-2 When is a fused bicyclic ring, R 1-2 is preferably a 4- to 8-membered ring, more preferably a 5- to 8-membered ring, more preferably a 5- to 7-membered ring, and more preferably a 5- or 6-membered ring. In a preferred embodiment, R 1-2 is a monocyclic ring.

[0274] R 1-2 may be saturated or unsaturated. If unsaturated, R 1-2 The number of double bonds contained in is preferably 1 to 3, and more preferably 2 to 3. Preferably, R 1-2 is unsaturated. More preferably, R 1-2 is an aromatic ring.

[0275] R 1-2 may contain heteroatoms. R 1-2The number of heteroatoms contained in is preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and more preferably 1. Examples of heteroatoms include oxygen atoms, nitrogen atoms, and sulfur atoms, and more preferably nitrogen atoms. The heteroatoms are independently selected from oxygen atoms, nitrogen atoms and sulfur atoms, and the number of the heteroatoms is preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and more preferably 1. R 1-2 is preferably an aromatic heterocycle containing a heteroatom, more preferably an aromatic heterocycle containing a nitrogen atom, and more preferably a monocyclic aromatic heterocycle containing a nitrogen atom.

[0276] R 1-2Specific examples of such compounds include aziridine, azirine, oxirane, oxirene, phosphirane, phosphirene, thiirane, thiylene, diaziridine, diazirine, oxaziridine, dioxirane, azetidine, azeto, oxetane, oxete, thietane, thiet, diazetidine, diazeto, dioxetane, dioxete, dithietane, dithiet, pyrrolidine, pyrrole, tetrahydrofuran, furan, tetrahydrothiophene, thiophene, imidazolidine, pyrazolidine, imidazole, imidazoline, pyrazole, oxazolidine, isoxazolidine, oxazole, oxazoline, isoxazole, thiazolidine, isothiazolidine, thiazole, isothiazole, dioxolane, dithiolane, triazole, furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, tetrazole, oxatetrazole, thiatetrazole, pentazole, piperidine, pyridine, and pyridinium. cation, tetrahydropyran, pyran, pyrylium cation, thiane, thiopyran, thiopyrylium cation, piperazine, diazine, morpholine, oxazine, thiomorpholine, thiazine, dioxane, dioxine, dithiane, dithiin, hexahydro-1,3,5-triazine, triazine, trioxane, trithiane, tetrazine, pentazine, azepane, azepine, oxepane, oxepine, thiepane, thiepine, diazepane, diazepine, thiazepine, azocane, azocine, oxocane, oxocine, thiocane, thiocine, azonane, azonin, oxonane, oxonin, thionane, and thionine.

[0277] R 1-2 More preferred embodiments of the above include pyrrole, furan, thiophene, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, thiazole, thiazoline, isothiazole, triazole, furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, tetrazole, oxatetrazole, thiatetrazole, pentazole, pyridine, pyran, thiopyran, diazine, oxaxidine, thiazine, dioxin, dithiin, triazine, and tetrazine.

[0278] R 1-2 More preferred embodiments include pyrrole, imidazole, imidazoline, pyrazole, pyrazoline, triazole, pyridine, diazine, triazine, and tetrazine.

[0279] R 1-2 More preferred embodiments include imidazole, pyrazole, triazole, pyridine, diazine, and triazine. More preferably, R 1-2 is pyridine.

[0280] R 1-2 may be further substituted with a C1-3 alkyl group or a halogen atom. The C1-3 alkyl group may be linear or branched, and specific examples include a methyl group, an ethyl group, a propyl group, and a methylethyl group. The halogen atom referred to here preferably includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferably, R 1-2 Of the hydrogen atoms in the above, 1 to 3, more preferably 1 to 2, and more preferably 1 may be substituted.

[0281] In a preferred embodiment, R 1 R 1-1 In this case, it is preferred that L is present. Also, R 1 R 1-2 In the case of 3 It is preferred that it is not substituted by

[0282] In general formula (I), R 2 is preferably a 3- to 20-membered ring, more preferably a 4- to 18-membered ring, more preferably a 6- to 16-membered ring, more preferably a 6- to 14-membered ring, more preferably a 6- to 12-membered ring, more preferably an 8- to 12-membered ring.

[0283] R 2may be monocyclic or fused bicyclic. R 2 When is a monocyclic ring, R 2 is preferably a 3- to 12-membered ring, more preferably a 4- to 10-membered ring, more preferably a 5- to 8-membered ring, and more preferably a 5- or 6-membered ring. R 2 When is a fused bicyclic ring, R 2 is preferably a 5- to 20-membered ring, more preferably a 6- to 18-membered ring, more preferably an 8- to 16-membered ring, more preferably an 8- to 12-membered ring, more preferably an 8- to 10-membered ring, and more preferably a 9- to 10-membered ring.

[0284] R 2 When is a fused bicyclic ring, R 2 is preferably a fused ring of two 4-membered rings, two 4-membered rings, two 5-membered rings, two 6-membered rings, two 5-membered rings, two 5-membered rings, two 6-membered rings, or two 6-membered rings.

[0285] R 2 may be saturated or unsaturated. If it is unsaturated, the number of double bonds is preferably 1 to 6, more preferably 2 to 6, more preferably 3 to 6, and more preferably 4 or 5. R 2 is preferably an aromatic ring, more preferably a fused bicyclic aromatic ring.

[0286] R 2 may contain heteroatoms. R 2 The number of heteroatoms contained in is preferably 1 to 6, more preferably 1 to 5, more preferably 1 to 4, and more preferably 1 to 3. Examples of heteroatoms include oxygen atoms, nitrogen atoms, and sulfur atoms, and more preferably nitrogen atoms. The heteroatoms are independently selected from oxygen atoms, nitrogen atoms and sulfur atoms, and the number of the heteroatoms is preferably 1 to 6, more preferably 1 to 5, more preferably 1 to 4, and more preferably 1 to 3. R 2is preferably an aromatic heterocycle containing a heteroatom, more preferably an aromatic heterocycle containing a nitrogen atom, and more preferably a fused bicyclic aromatic heterocycle containing a nitrogen atom.

[0287] R 2 is preferably one of the following structures:

[0288] [ka]

[0289] In the above formula, X represents a carbon atom or a nitrogen atom. Of the atoms represented by X, the total number of nitrogen atoms is 1 to 6, more preferably 1 to 5, more preferably 1 to 4, and more preferably 1 to 3, and the remainder are carbon atoms.

[0290] [1-1-2-2] General formula (II) [1] The compound in question In one embodiment of the present invention, a compound encompassed by the following general formula (II), or a salt or hydrate thereof, is administered to an animal as a candidate substance.

[0291] [ka]

[0292] In general formula (II), R 1 is a hydrogen atom, a halogen atom, or a C1-20 hydrocarbon group. Here, preferred examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Below, R 1 The case where is in the form of a hydrocarbon group will be described in more detail.

[0293] where R 1 is preferably C1 to C18, more preferably C1 to C16, more preferably C1 to C14, more preferably C1 to C12, and more preferably C1 to C10.

[0294] R 1 may be saturated or unsaturated. 1 R may be linear or branched. 1 may contain a heteroatom. The heteroatom is preferably selected from an oxygen atom, a nitrogen atom, and a sulfur atom. More preferably, R 1 contains a nitrogen atom. 1 may contain 1 to 4, more preferably 1 to 3, and more preferably 1 to 2 heteroatoms independently selected from oxygen atoms, nitrogen atoms, and sulfur atoms.

[0295] Also, R 1 may have a cyclic structure. The cyclic structure is preferably a 3- to 8-membered ring, more preferably a 4- to 7-membered ring, and more preferably a 5- or 6-membered ring.

[0296] R 1 The following R 1-1 or R 1-2 It is preferable that the form is: R 1-1 : A saturated or unsaturated, linear or branched C1-20 hydrocarbon group which may be substituted with a halogen atom. R 1-2 A saturated or unsaturated 3- to 8-membered ring which may contain 1 to 4 heteroatoms independently selected from oxygen atoms, nitrogen atoms, and sulfur atoms, and which may be substituted with a halogen atom or a C1-3 hydrocarbon group.

[0297] R 1-1 is more preferably C1 to 18, more preferably C1 to 16, more preferably C1 to 14, more preferably C1 to 12, more preferably C1 to 10, more preferably C1 to 8, more preferably C1 to 6, more preferably C1 to 4, more preferably C1 to 3, and more preferably C1 to 2.

[0298] Also, R 1-1may be substituted with a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. The number of substitutions with halogen atoms can be preferably 1 to 3, more preferably 1 to 2, and more preferably 1.

[0299] R 1-2 is preferably a monocyclic ring or a fused bicyclic ring, more preferably a monocyclic ring. R 1-2 is a monocyclic ring, then R 1-2 is preferably a 3- to 8-membered ring, more preferably a 4- to 6-membered ring, more preferably a 5- or 6-membered ring, and more preferably a 6-membered ring. R 1-2 When is a fused bicyclic ring, R 1-2 is preferably a 4- to 8-membered ring, more preferably a 5- to 8-membered ring, more preferably a 5- to 7-membered ring, and more preferably a 5- or 6-membered ring.

[0300] R 1-2 may be saturated or unsaturated. When it is unsaturated, the number of double bonds contained in the ring is preferably 1 to 3, more preferably 2 to 3, and more preferably 3. Preferably, R 1-2 is unsaturated. More preferably, R 1-2 is an aromatic ring.

[0301] R 1-2 may contain heteroatoms. R 1-2 The number of heteroatoms contained in is preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and more preferably 1. Examples of heteroatoms include oxygen atoms, nitrogen atoms, and sulfur atoms, and more preferably nitrogen atoms. The heteroatoms are independently selected from oxygen atoms, nitrogen atoms and sulfur atoms, and the number of the heteroatoms is preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and more preferably 1. R 1-2In one embodiment, is preferably an aromatic heterocycle containing a heteroatom, more preferably an aromatic heterocycle containing a nitrogen atom, and more preferably a monocyclic aromatic heterocycle containing a nitrogen atom. In another embodiment, R 1-2 is an aromatic ring containing no heteroatoms, and more preferably a monocyclic aromatic ring containing no heteroatoms.

[0302] R 1-2Specific examples of such aryl groups include cyclopropane, cyclopropene, cyclobutane, cyclobutene, cyclobutadiene, cyclopentene, cyclopentadiene, cyclohexene, cyclohexadiene, benzene, cyclopentane, cycloheptene, cycloheptadiene, cycloheptatriene, cyclooctane, cyclooctene, cyclooctadiene, cyclooctatriene, aziridine, azirine, oxirane, oxirene, phosphirane, phosphirene, thiirane, thiylene, diaziridine, diazirine, oxaziridine, dioxirane, azetidine, azeto, oxetane, oxete, thietane, thiet, diazetidine, and diazetidine. dioxetane, dioxetane, dithietane, dithietane, pyrrolidine, pyrrole, tetrahydrofuran, furan, tetrahydrothiophene, thiophene, imidazolidine, pyrazolidine, imidazole, imidazoline, pyrazole, oxazolidine, isoxazolidine, oxazole, oxazoline, isoxazole, thiazolidine, isothiazolidine, thiazole, isothiazole, dioxolane, dithiolane, triazole, furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, tetrazole, oxatetrazole, thiatetrazole, pentazole, piperidine, pyridine, pyridinium cation, tetrahydropyran, pyran, pyrylium cation, thiane, thiopyran, thiopyrylium cation, piperazine, diazine, morpholine, oxazine, thiomorpholine, thiazine, dioxane, dioxine, dithiane, dithiin, hexahydro-1,3,5-triazine, triazine, trioxane, trithiane, tetrazine, pentazine, azepane, azepine, oxepane, oxepine, thiepane, thiepine, diazepane, diazepine, thiazepine, azocane, azocine, oxocane, oxocine, thiocane, thiocine, azonane, azonin, oxonane, oxonin, thionane, and thionine.

[0303] R 1-2More preferred embodiments of the above include cyclopentene, cyclopentadiene, cyclohexene, cyclohexadiene, benzene, cyclopentane, cycloheptene, cycloheptadiene, cycloheptatriene, cyclooctane, cyclooctene, cyclooctadiene, cyclooctatriene, pyrrole, furan, thiophene, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, thiazole, thiazoline, isothiazole, triazole, furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, tetrazole, oxatetrazole, thiatetrazole, pentazole, pyridine, pyran, thiopyran, diazine, oxaxidine, thiazine, dioxin, dithiin, triazine, and tetrazine.

[0304] R 1-2 More preferred embodiments include benzene, pyrrole, imidazole, imidazoline, pyrazole, pyrazoline, triazole, pyridine, diazine, triazine, and tetrazine.

[0305] R 1-2 More preferred embodiments include benzene, imidazole, pyrazole, triazole, pyridine, diazine, and triazine. More preferably, R 1-2 is benzene or pyridine.

[0306] R 1-2 may be further substituted with a C1-3 alkyl group or a halogen atom. The C1-3 alkyl group may be linear or branched, and specific examples include a methyl group, an ethyl group, a propyl group, and a methylethyl group. The halogen atom referred to here preferably includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferably, R 1-2 Of the hydrogen atoms in the ring constituting the group, 1 to 3, more preferably 1 to 2, and more preferably 1 may be substituted.

[0307] In general formula (II), L 1 is one of the following divalent groups: [ka]

[0308] More preferably, L 1 is one of the divalent groups listed below, which are biologically equivalent to each other. [ka]

[0309] More preferably, L 1 is any divalent group listed below. [ka]

[0310] More preferably, L1 is the following divalent group: [ka]

[0311] In general formula (II), L 2 is a C1-30 hydrocarbon group. 2 is more preferably C1 to 28, more preferably C1 to 26, more preferably C1 to 24, more preferably C1 to 22, more preferably C1 to 20, more preferably C1 to 18, more preferably C1 to 16, more preferably C1 to 14, more preferably C1 to 12, more preferably C1 to 10, and more preferably C1 to 8.

[0312] L 2 is linear. 2 may be saturated or unsaturated. Also, L 2 may be substituted with one or more of the substituents listed below. C1-3 hydrocarbon groups A hydroxyl group that may be substituted with a C1-3 hydrocarbon group An amino group that may be substituted with a C1-3 hydrocarbon group Sulfate group optionally substituted with a C1-3 hydrocarbon group Phosphate group optionally substituted with a C1-3 hydrocarbon group Halogen atoms

[0313] More specifically, L 2 may be substituted with one or more of the substituents listed below. Methyl, ethyl, propyl, or isopropyl group Hydroxyl group, methoxy group, ethoxy group, propoxy group, or isopropoxy group An amino group, or an alkylamino group in which one or two hydrogen atoms of the amino group are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group. Sulfate group, methyl sulfate group, ethyl sulfate group, propyl sulfate group, or isopropyl sulfate group A phosphate group or an alkyl phosphate group in which one or two hydrogen atoms of the phosphate group are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group.

[0314] More preferably, L 2 may be substituted with one or more substituents selected from a methyl group, an ethyl group, a propyl group, an isopropyl group, a hydroxyl group, an amino group, a sulfate group, and a phosphate group. More preferably, L 2 may be substituted with one or more substituents selected from a methyl group, an ethyl group, a propyl group, an isopropyl group, a hydroxyl group, an amino group, a sulfate group, and a phosphate group. More preferably, L 2 may be substituted with one or more substituents selected from a methyl group, an ethyl group, a propyl group, an isopropyl group, a hydroxyl group, and an amino group. More preferably, L 2may be substituted with one or more substituents selected from a methyl group, an ethyl group, a propyl group, an isopropyl group, and an amino group. More preferably, L 2 may be substituted with an amino group. L 2 The number of substitutions is preferably 1 to 3, more preferably 1 to 2, and more preferably 1.

[0315] In general formula (II), R 2 is any of the groups listed below. A carboxyl group that may be substituted with a C1-3 hydrocarbon group A hydroxyl group that may be substituted with a C1-3 hydrocarbon group A hydroxamic acid group optionally substituted with a C1-3 hydrocarbon group A sulfo group optionally substituted with a C1-3 hydrocarbon group Boronic acid group optionally substituted with a C1-3 hydrocarbon group A carbamoyl group optionally substituted with a C1-3 hydrocarbon group A sulfamoyl group optionally substituted with a C1-3 hydrocarbon group A sulfoximine group optionally substituted with a C1-3 hydrocarbon group Cyano group Tetrazolyl group

[0316] More preferably, R 2 is any of the groups listed below. Carboxyl group or a group in which the hydrogen atom of a carboxyl group has been substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group Hydroxyl group, methoxy group, ethoxy group, propoxy group, or isopropoxy group Hydroxamic acid groups or groups in which the hydrogen atoms of a hydroxamic acid group have been substituted with methyl, ethyl, propyl or isopropyl groups A sulfo group, or a group in which the hydrogen atom of a sulfo group has been substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group A boronic acid group or a group in which one or two hydrogen atoms of a boronic acid group are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group. A carbamoyl group, or a carbamoyl group in which one or two hydrogen atoms are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group A sulfamoyl group, or a sulfamoyl group in which one or two hydrogen atoms are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group Sulfoximine group or a group in which the hydrogen atom of the sulfoximine group has been substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group Cyano group Tetrazolyl group

[0317] In one embodiment, R 2 is a carboxyl group, a hydroxyl group, a hydroxamic acid group, a sulfo group, a boronic acid group, a carbamoyl group, a sulfamoyl group, a sulfoximine group, a cyano group, or a tetrazolyl group. More preferably, R 2 is a carboxyl group or a hydroxyamic acid group.

[0318] In one embodiment, R 2 is a carboxyl group which may be substituted with a C1-3 hydrocarbon group, or a hydroxamic acid group which may be substituted with a C1-3 hydrocarbon group.

[0319] [1-1-2-3] General formula (III) In one embodiment of the present invention, a compound encompassed by the following general formula (III), or a salt or hydrate thereof, is administered to an animal as a candidate substance.

[0320] [ka]

[0321] In general formula (III), ring A, ring B and ring C are each a 3- to 8-membered ring, more preferably a 4- to 6-membered ring, more preferably a 5- or 6-membered ring, and more preferably a 6-membered ring.

[0322] Ring A, ring B and ring C may be saturated or unsaturated. When they are unsaturated, the number of double bonds contained in the ring is preferably 1 to 3, more preferably 2 to 3, and more preferably 3. Preferably, at least one, more preferably at least two, and more preferably all of ring A, ring B, and ring C are unsaturated. More preferably, ring A, ring B, and ring C are aromatic rings.

[0323] Ring A, ring B and ring C may be substituted with one or more of the substituents listed below. Halogen atoms C1-3 hydrocarbon groups A hydroxyl group that may be substituted with a C1-3 hydrocarbon group An amino group that may be substituted with a C1-3 hydrocarbon group Sulfate group optionally substituted with a C1-3 hydrocarbon group Phosphate group optionally substituted with a C1-3 hydrocarbon group

[0324] More specifically, ring A, ring B and ring C may be substituted with one or more of the substituents listed below. Fluorine, chlorine, bromine, or iodine atoms Methyl, ethyl, propyl, or isopropyl group Hydroxyl group, methoxy group, ethoxy group, propoxy group, or isopropoxy group An amino group, or an alkylamino group in which one or two hydrogen atoms of the amino group are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group. Sulfate group, methyl sulfate group, ethyl sulfate group, propyl sulfate group, or isopropyl sulfate group A phosphate group or an alkyl phosphate group in which one or two hydrogen atoms of the phosphate group are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group.

[0325] Ring A, ring B and ring C may contain heteroatoms. The number of heteroatoms contained in ring A, ring B and ring C is preferably 1 to 4, more preferably 1 to 3, more preferably 1 to 2, and more preferably 2. Examples of heteroatoms include oxygen atoms, nitrogen atoms, and sulfur atoms, and more preferably nitrogen atoms. The heteroatoms are independently selected from oxygen atoms, nitrogen atoms and sulfur atoms, and the number of the heteroatoms is preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and more preferably 1.

[0326] In one embodiment, any one of ring A, ring B, and ring C is preferably an aromatic heterocycle containing a heteroatom, more preferably an aromatic heterocycle containing a nitrogen atom. In another embodiment, any one of ring A, ring B, and ring C is an aromatic ring containing no heteroatoms.

[0327] In one embodiment, ring A, ring B, and ring C are all aromatic rings, of which ring B is a heteroaromatic heterocycle containing a heteroatom, and ring A and ring C are aromatic rings containing no heteroatom.

[0328] In one embodiment, ring A is cyclohexane, cyclohexene, cyclohexadiene, or benzene, which may be further substituted with a C1-3 hydrocarbon group or a halogen atom.

[0329] In one embodiment, ring B is a 6-membered monocyclic heterocycle containing 1 to 3 nitrogen atoms, which is saturated or unsaturated and optionally substituted with a C1-3 hydrocarbon group or a halogen atom.

[0330] In one embodiment, ring C is cyclohexane, cyclohexene, cyclohexadiene, or benzene optionally substituted with a C1-3 hydrocarbon group, a hydroxyl group optionally substituted with a halogen atom or a C1-3 hydrocarbon group.

[0331] Specific examples of ring A, ring B, and ring C include cyclopropane, cyclopropene, cyclobutane, cyclobutene, cyclobutadiene, cyclopentene, cyclopentadiene, cyclohexene, cyclohexadiene, benzene, cyclopentane, cycloheptene, cycloheptadiene, cycloheptatriene, cyclooctane, cyclooctene, cyclooctadiene, cyclooctatriene, aziridine, azirine, oxirane, oxirene, phosphirane, phosphirene, thiirane, thiylene, diaziridine, diazirine, oxaziridine, dioxirane, azetidine, azeto, oxetane, oxete, thietane, thiet, and diazetidine. , diazeto, dioxetane, dioxete, dithietane, dithiet, pyrrolidine, pyrrole, tetrahydrofuran, furan, tetrahydrothiophene, thiophene, imidazolidine, pyrazolidine, imidazole, imidazoline, pyrazole, oxazolidine, isoxazolidine, oxazole, oxazoline, isoxazole, thiazolidine, isothiazolidine, thiazole, isothiazole, dioxolane, dithiolane, triazole, furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, tetrazole, oxatetrazole, thiatetrazole, pentazole, piperidine, pyridine, pyridinium cation, tetrahydropyran, pyran, pyrylium cation, thiane, thiopyran, thiopyrylium cation, piperazine, diazine, morpholine, oxazine, thiomorpholine, thiazine, dioxane, dioxine, dithiane, dithiin, hexahydro-1,3,5-triazine, triazine, trioxane, trithiane, tetrazine, pentazine, azepane, azepine, oxepane, oxepine, thiepane, thiepine, diazepane, diazepine, thiazepine, azocane, azocine, oxocane, oxocine, thiocane, thiocine, azonane, azonin, oxonane, oxonin, thionane, and thionine.

[0332] More preferred embodiments of ring A, ring B, and ring C include cyclopentene, cyclopentadiene, cyclohexene, cyclohexadiene, benzene, cyclopentane, cycloheptene, cycloheptadiene, cycloheptatriene, cyclooctane, cyclooctene, cyclooctadiene, cyclooctatriene, pyrrole, furan, thiophene, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, thiazole, thiazoline, isothiazole, triazole, furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, tetrazole, oxatetrazole, thiatetrazole, pentazole, pyridine, pyran, thiopyran, diazine, oxaxidine, thiazine, dioxin, dithiin, triazine, and tetrazine.

[0333] More preferred embodiments of ring A, ring B and ring C include benzene, pyrrole, imidazole, imidazoline, pyrazole, pyrazoline, triazole, pyridine, diazine, triazine and tetrazine.

[0334] More preferred embodiments of ring A, ring B and ring C include benzene, imidazole, pyrazole, triazole, pyridine, diazine and triazine. As the diazine, pyrimidine is preferred. More preferably, ring A, ring B and ring C are benzene or pyrimidine.More preferably, ring A and ring C are benzene, and ring B is pyrimidine.

[0335] In one embodiment, ring A is cyclohexane, cyclohexene, cyclohexadiene or benzene. More preferably, ring A is benzene. In this case, R is attached to the first and second carbon atoms of ring A, respectively. 1 and L 1 is preferably bonded to the

[0336] In one embodiment, Ring B is a 6-membered monocyclic heterocycle containing 1 to 3 nitrogen atoms, which is saturated or unsaturated and substituted with a halogen atom. More preferably, Ring B is a pyrimidine substituted with a halogen atom. More preferably, Ring B is a pyrimidine substituted with a chlorine atom. In this case, L is attached to the 4th carbon atom of ring B. 1 , 2nd place: L 2 It is preferable that a chlorine atom is bonded to each of the 1st and 5th positions.

[0337] In one embodiment, ring C is cyclohexane, cyclohexene, cyclohexadiene, or benzene substituted with a C1-3 alkoxy group. More preferably, ring C is cyclohexane, cyclohexene, cyclohexadiene, or benzene substituted with a methoxy group. More preferably, ring C is benzene substituted with a methoxy group. In this case, L is placed at the first position of ring C. 2 , 4th place: R 2 Preferably, a methoxy group is bonded to the first and second positions.

[0338] In general formula (III), R 1 is a group selected from the following: [ka]

[0339] R 1 is more preferably a group selected from the following: [ka]

[0340] where R 3 ~R 6 are each independently any of the following groups: Hydrogen atoms C1-3 hydrocarbon groups A hydroxyl group that may be substituted with a C1-3 hydrocarbon group An amino group that may be substituted with a C1-3 hydrocarbon group

[0341] More preferably, R 3 ~R 6 are each independently any of the following groups: Hydrogen atoms Methyl, ethyl, propyl, or isopropyl group Hydroxyl group, methoxy group, ethoxy group, propoxy group, or isopropoxy group An amino group, or an alkylamino group in which one or two hydrogen atoms of the amino group are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group.

[0342] More preferably, R 3 ~R 6 are each independently any of the following groups: Methyl, ethyl, propyl, or isopropyl group An amino group, or an alkylamino group in which one or two hydrogen atoms of the amino group are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group.

[0343] More preferably, R 3 ~R 6 are each independently a methyl group or a monomethylamino group.

[0344] R 7 represents a hydrogen atom, a C1-3 hydrocarbon group, or an amino group which may be substituted with a C1-3 hydrocarbon group. R 7 is more preferably a methyl group, an ethyl group, a propyl group, an isopropyl group, a monomethylamino group, a monoethylamino group, a monopropylamino group or a monoisopropylamino group. R 7 is more preferably a methyl group or a monomethylamino group.

[0345] n represents an integer of 1 to 4, more preferably an integer of 1 to 3, more preferably an integer of 1 or 2, and more preferably 1.

[0346] In general formula (III), L 1 , L 2 are each independently a C1-3 alkylene or alkenylene group, -N(H)- or -O-. More preferably, L 1 , L 2 are each independently a C1-2 alkylene or alkenylene group, -N(H)- or -O-. More preferably, L 1 , L 2 are each independently a C1-2 alkylene or alkenylene group, or -N(H)-.

[0347] Among the above-mentioned divalent groups, groups other than -O-, i.e., C1-3 alkylene or alkenylene groups and -N(H)-, may be further substituted with a C1-30 hydrocarbon group. The C1-30 hydrocarbon group may be saturated or unsaturated. The C1-30 hydrocarbon group may be linear or branched. The C1-30 hydrocarbon group may have a cyclic structure or may be substituted with a halogen atom. Furthermore, the C1-30 hydrocarbon group may contain 1 to 6 heteroatoms independently selected from oxygen, nitrogen, and sulfur atoms.

[0348] In general formula (III), R 2 is a C1 to C30, more preferably C2 to C28, more preferably C2 to C26, more preferably C2 to C24, more preferably C2 to C22, more preferably C2 to C20 hydrocarbon group.

[0349] R 2 may be saturated or unsaturated. 2 may be a straight chain or a branched chain.

[0350] R 2 R may have a cyclic structure. 2The number of ring structures contained in this structure is preferably 1 to 3, more preferably 1 to 2. The ring structure can be a 3- to 8-membered ring, preferably a 4- to 7-membered ring, more preferably a 5- or 6-membered ring.

[0351] R 2 may be substituted with a halogen atom. Preferred examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. The number of halogen atoms to be substituted is preferably 1 to 4, more preferably 1 to 3, more preferably 1 to 2, and more preferably 1.

[0352] R 2 may contain 1 to 6, preferably 1 to 5, more preferably 1 to 4 heteroatoms independently selected from oxygen atoms, nitrogen atoms, and sulfur atoms. A preferred example of the heteroatom is a nitrogen atom.

[0353] R 2 is preferably the structure shown below. [ka]

[0354] Ring D is a 3- to 8-membered ring, more preferably a 4- to 6-membered ring, more preferably a 5- or 6-membered ring, and more preferably a 6-membered ring.

[0355] Ring D may be saturated or unsaturated. When unsaturated, the number of double bonds contained in the ring is preferably 1 to 3, more preferably 2 to 3, and more preferably 3. Preferably, ring D is saturated.

[0356] Ring D may be substituted with one or more of the substituents listed below. Halogen atoms C1-3 hydrocarbon groups A hydroxyl group that may be substituted with a C1-3 hydrocarbon group An amino group that may be substituted with a C1-3 hydrocarbon group Sulfate group optionally substituted with a C1-3 hydrocarbon group Phosphate group optionally substituted with a C1-3 hydrocarbon group

[0357] More specifically, ring D may be substituted with one or more of the substituents listed below. Fluorine, chlorine, bromine, or iodine atoms Methyl, ethyl, propyl, or isopropyl group Hydroxyl group, methoxy group, ethoxy group, propoxy group, or isopropoxy group An amino group, or an alkylamino group in which one or two hydrogen atoms of the amino group are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group. Sulfate group, methyl sulfate group, ethyl sulfate group, propyl sulfate group, or isopropyl sulfate group A phosphate group or an alkyl phosphate group in which one or two hydrogen atoms of the phosphate group are each independently substituted with a methyl group, an ethyl group, a propyl group, or an isopropyl group.

[0358] Ring D may contain a heteroatom. The number of heteroatoms contained in ring D is preferably 1 to 4, more preferably 1 to 3, more preferably 1 to 2, and more preferably 2. Examples of heteroatoms include oxygen atoms, nitrogen atoms, and sulfur atoms, and more preferably nitrogen atoms. The heteroatoms are independently selected from oxygen atoms, nitrogen atoms and sulfur atoms, and the number of the heteroatoms is preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and more preferably 1.

[0359] Specific examples of ring D include cyclopropane, cyclopropene, cyclobutane, cyclobutene, cyclobutadiene, cyclopentene, cyclopentadiene, cyclohexene, cyclohexadiene, benzene, cyclopentane, cycloheptene, cycloheptadiene, cycloheptatriene, cyclooctane, cyclooctene, cyclooctadiene, cyclooctatriene, aziridine, azirine, oxirane, oxirene, phosphirane, phosphirene, thiirane, thiylene, diaziridine, diazirine, oxaziridine, dioxirane, azetidine, azeto, oxetane, oxete, thietane, thiet, diazetidine ... Zeto, dioxetane, dioxete, dithietane, dithiet, pyrrolidine, pyrrole, tetrahydrofuran, furan, tetrahydrothiophene, thiophene, imidazolidine, pyrazolidine, imidazole, imidazoline, pyrazole, oxazolidine, isoxazolidine, oxazole, oxazoline, isoxazole, thiazolidine, isothiazolidine, thiazole, isothiazole, dioxolane, dithiolane, triazole, furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, tetrazole, oxatetrazole, thiatetrazole, pentazole, piperidine, pyridine, pyridinium cation, tetrahydropyran, pyran, pyrylium cation, thiane, thiopyran, thiopyrylium cation, piperazine, diazine, morpholine, oxazine, thiomorpholine, thiazine, dioxane, dioxine, dithiane, dithiin, hexahydro-1,3,5-triazine, triazine, trioxane, trithiane, tetrazine, pentazine, azepane, azepine, oxepane, oxepine, thiepane, thiepine, diazepane, diazepine, thiazepine, azocane, azocine, oxocane, oxocine, thiocane, thiocine, azonane, azonin, oxonane, oxonin, thionane, and thionine.

[0360] Ring D is preferably piperazine substituted with a C1-3 hydrocarbon group, more preferably piperazine substituted with a methyl group.

[0361] L 3is a C1-20 divalent hydrocarbon group, more preferably a C1-18, more preferably a C1-16, more preferably a C1-14, more preferably a C1-12, more preferably a C1-10, more preferably a C1-8 divalent hydrocarbon group.

[0362] L 3 may be saturated or unsaturated. 3 may be a straight chain or a branched chain.

[0363] L 3 may have a cyclic structure. 3 The number of ring structures contained in this structure is preferably 1 to 2, and more preferably 1. The ring structure may be a 3- to 8-membered ring, preferably a 4- to 7-membered ring, more preferably a 5- or 6-membered ring, and more preferably a 6-membered ring.

[0364] L 3 may be substituted with a halogen atom. Preferred examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. The number of halogen atoms to be substituted is preferably 1 to 4, more preferably 1 to 3, more preferably 1 to 2, and more preferably 1.

[0365] L 3 may contain 1 to 6, preferably 1 to 5, more preferably 1 to 4 heteroatoms independently selected from oxygen atoms, nitrogen atoms, and sulfur atoms. A preferred example of the heteroatom is a nitrogen atom.

[0366] L 3 is preferably a divalent group having the structure shown below.

[0367] [ka]

[0368] Here, m and l each independently represent an integer of 1 to 4, preferably 1 to 3, more preferably 1 or 2. More preferably, m and l are 1.

[0369] Ring E is a 3- to 8-membered ring, more preferably a 4- to 6-membered ring, more preferably a 5- or 6-membered ring, and more preferably a 6-membered ring.

[0370] Ring E may be saturated or unsaturated. If it is unsaturated, the number of double bonds contained in the ring is preferably 1 to 3, more preferably 1 to 2, and more preferably 1. Preferably, ring E is a saturated ring.

[0371] Ring E may be a heterocycle. In this case, the number of heteroatoms contained in ring E is preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and more preferably 1. Examples of heteroatoms include oxygen atoms, nitrogen atoms, and sulfur atoms, and more preferably nitrogen atoms. The heteroatoms are independently selected from oxygen atoms, nitrogen atoms and sulfur atoms, and the number of the heteroatoms is preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and more preferably 1.

[0372] Specific examples of ring E include cyclopropane, cyclopropene, cyclobutane, cyclobutene, cyclobutadiene, cyclopentene, cyclopentadiene, cyclohexene, cyclohexadiene, benzene, cyclopentane, cycloheptene, cycloheptadiene, cycloheptatriene, cyclooctane, cyclooctene, cyclooctadiene, cyclooctatriene, aziridine, azirine, oxirane, oxirene, phosphirane, phosphirene, thiirane, thiylene, diaziridine, diazirine, oxaziridine, dioxirane, azetidine, azeto, oxetane, oxete, thietane, thiet, diazetidine, and diazetidine. Zeto, dioxetane, dioxete, dithietane, dithiet, pyrrolidine, pyrrole, tetrahydrofuran, furan, tetrahydrothiophene, thiophene, imidazolidine, pyrazolidine, imidazole, imidazoline, pyrazole, oxazolidine, isoxazolidine, oxazole, oxazoline, isoxazole, thiazolidine, isothiazolidine, thiazole, isothiazole, dioxolane, dithiolane, triazole, furazan, oxadiazole, thiadiazole, dioxazole, dithiazole, tetrazole, oxatetrazole, thiatetrazole, pentazole, piperidine, pyridine, pyridinium cation, tetrahydropyran, pyran, pyrylium cation, thiane, thiopyran, thiopyrylium cation, piperazine, diazine, morpholine, oxazine, thiomorpholine, thiazine, dioxane, dioxine, dithiane, dithiin, hexahydro-1,3,5-triazine, triazine, trioxane, trithiane, tetrazine, pentazine, azepane, azepine, oxepane, oxepine, thiepane, thiepine, diazepane, diazepine, thiazepine, azocane, azocine, oxocane, oxocine, thiocane, thiocine, azonane, azonin, oxonane, oxonin, thionane, and thionine.

[0373] Preferably, ring E is cyclohexane or piperidine, more preferably piperidine.

[0374] Also, in one embodiment, L 3does not exist.

[0375] [1-1-2-4] Derivatives of specific compounds In one embodiment of the present invention, derivatives of the following compounds 1 to 7, or salts thereof, or hydrates thereof are administered to animals as candidate substances. Compound 1: 3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile Compound 2: 4-[3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoline Compound 3: 2-amino-5-(ethylamino)-5-oxopentanoic acid Compound 4:N 1 -hydroxy-N 8 -phenyloctanediamino Compound 5: 4-[(5-bromopyridin-2-yl)amino]-4-oxobutyric acid Compound 6: 5-chloro-2-N-{4-[4-(dimethylamino)piperidin-1-yl]-2-methoxyphenyl}-4-N-[2-(dimethylphosphoryl)phenyl]pyrimidine-2,4-diamine Compound 7: 2-[[5-chloro-2-[2-methoxy-4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]amino]-N-methylbenzenesulfonamide The structures of these seven compounds are shown below.

[0376] [ka]

[0377] These seven compounds have been confirmed to have therapeutic or preventive effects against nervous system diseases, etc. in the Examples described below. Therefore, derivatives obtained by adding any group to the structure of these seven compounds, substituting a part of the structure with any structure, or deleting a part of the structure are likely to have therapeutic or preventive effects against nervous system diseases, etc., similar to these seven compounds. Therefore, by using derivatives of these seven compounds as candidate substances, it is possible to efficiently screen for active ingredients that have therapeutic or preventive effects on nervous system diseases and the like.

[0378] When derivatives of these seven compounds are used as candidate substances, the embodiment may include the following Step A', Step B and / or Step C, and Step D'. [Step A'] A step of selecting any of the seven compounds as a lead compound and administering a derivative of the lead compound as a candidate substance to an animal (excluding humans). [Step B] A step of observing undifferentiated neural cells in the animal that has undergone Step A. [Step C] A step of observing differentiated cells in the nervous system in the animal that has undergone Step A. [Step D'] A step of selecting the candidate substance as the active ingredient if it has a greater proliferation effect on undifferentiated neural cells and / or differentiated neural cells than administration of the lead compound. By adopting such an embodiment, the seven compounds can be used as lead compounds and optimized.

[0379] [1-2]B process Step B is a step of observing undifferentiated cells in the animal that has undergone step A. In this specification, the term "undifferentiated cells" includes both stem cells and precursor cells.

[0380] The means for observing undifferentiated cells is not particularly limited. Suitable examples include a means for observing a marker for undifferentiated cells and a means for observing a reporter gene that is specifically expressed in undifferentiated cells. Each of these means is described in detail below.

[0381] First, we will explain the methods for observing markers of undifferentiated cells. Cells have different morphological and functional characteristics at each stage of development. These changes in characteristics during development are due to the temporal and spatial control of the expression of various genes at each stage of development. Specifically, in the most undifferentiated stem cells, many genes necessary for self-renewal while suppressing differentiation and maintaining totipotency are expressed. As differentiation progresses, the expression of genes that maintain totipotency is suppressed, while many genes necessary for the cell to take on a specific morphology and perform a specific function are expressed. In embryology, a common method for tracing this developmental process is to observe the expression of "marker genes" that are expressed specifically at certain developmental stages. In a preferred embodiment of the present invention, markers that are specifically expressed in undifferentiated cells are observed in step B. The types of marker genes are outlined below.

[0382] Markers for neural stem cells (neuroepithelial cells) include Nestin, SOX2, Notch1, HES1, HES3, Occludin, E-cadherin, and SOX10. When zebrafish are used as a screening tool, Her-5 is a preferred example of a neural stem cell marker (Development 133(21):4293-303·December 2006).

[0383] Preferred examples of Schwann precursor cell markers include SOX10, GAP43, BLBP, MPZ, Dhh, and P75NTR.

[0384] Markers of radial glial cells include Vimentin, PAX6, HES1, HES5, GFAP, EAAT1 / GLAST, BLBP, TN-C, N-cadherin, Nestin, and SOX2.

[0385] Markers for oligodendrocyte precursor cells include PDGFRA and NG2.

[0386] Markers of intermediate progenitor cells include TBR2 and MASH1.

[0387] Markers of retinal stem cells or retinal progenitor cells include Pax6, Sox2, Nestin, vimentin, musashi, and Chx10 (Vsx2).

[0388] Markers of undifferentiated cardiac cells include Mesp1 and Nkx2.5.

[0389] Markers for undifferentiated pancreatic cells include Pdx1 and Ptfla.

[0390] Some of the markers mentioned above are also expressed in differentiated cells. The present invention may be embodied in such a way that genes expressed in both undifferentiated and differentiated cells are used as markers. In this case, it corresponds to an embodiment in which steps B and C are performed simultaneously (FIG. 5(c)).

[0391] A preferred example of a means for observing these markers is immunostaining using antibodies against proteins that are the products of the respective genes. The specific method of immunostaining is not particularly limited, and can be carried out by conventional methods.

[0392] Alternatively, the markers may be observed by in situ hybridization, which utilizes hybridization between mRNA, the transcription product of each gene, and a single-stranded nucleic acid molecule (probe) having a base sequence complementary to the mRNA. The specific method of in situ hybridization is not particularly limited, and can be performed by a conventional method.

[0393] Next, a method using a transgenic animal into which a reporter gene that is specifically expressed in undifferentiated cells has been introduced will be described. A reporter gene is an exogenous gene used to visualize the expression of a gene within a cell. By inserting a base sequence encoding a fluorescent protein or other protein into the expression control region of the gene whose expression you want to visualize, the cell is designed to emit fluorescence in synchronization with the expression of that gene. In one embodiment of the present invention, a transgenic animal into which a reporter gene that is specifically expressed in undifferentiated cells has been introduced is used as a screening tool. In this case, the expression of the reporter gene that is specifically expressed in undifferentiated cells is observed in step B.

[0394] Such transgenic animals can be produced by known gene editing techniques that are conventionally used. When a promoter that specifically regulates expression in undifferentiated cells is known, a base sequence in which a reporter gene sequence is linked downstream of the promoter can be inserted into the genome to produce a transgenic animal that specifically expresses in a particular cell. Furthermore, since the marker genes of undifferentiated cells are known as described above, transgenic animals can be produced by introducing a reporter gene into the locus of the marker gene, which results in the expression of the reporter gene synchronized with the expression of the marker gene.

[0395] Reporter genes include Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP, GFP, TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, HyPer, TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, KusabiraOrange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, and AsRed2. Examples of such genes include genes encoding fluorescent proteins such as Red, mStrawberry, TurboFP602, mRFP1, JRed, KillerRed, mCherry, KeimaRed, mRasberry, mPlum, PS-CFP, Dendra2, Kaede, EosFP, and KikumeGR, and proteins that catalyze color reactions such as LacZ.

[0396] Although the specific design method for introducing a reporter gene is not limited, it is preferable to use a transgenic animal designed to introduce the cDNA sequence of a reporter gene into any position in the open reading frame of a marker gene, more preferably any position that does not disrupt the function or topology of the protein that is the product of the marker gene, and to express a fusion protein of the marker gene and the reporter gene.

[0397] Various transgenic lines have been established in which reporter genes that are specifically expressed in undifferentiated cells have been introduced, and any available lines may be used. Below, several examples of transgenic lines in which reporter genes that are specifically expressed in undifferentiated neural cells have been introduced are shown, but it goes without saying that the embodiments of the present invention are not limited to these.

[0398] Her5-GFP transgenic zebrafish line (Development. 2003 Sep;130(18):4307-23.) Nestin-GFP transgenic zebrafish line (Developmental Dynamics 2009 Feb;238(2):475-86.) Gfap-GFP transgenic zebrafish line (Developmental Dynamics 2009 Feb;238(2):475-86.) Nestin-GFP transgenic mouse line (The Journal of Comparative Neurology, Volume 469, Issue 39, February 2004, Pages 311-324)

[0399] When the reporter gene is a fluorescent protein, the transgenic animal is irradiated with light of the excitation wavelength of the fluorescent protein, and the emitted fluorescence is observed. The fluorescence can be observed under a fluorescence microscope. Based on the brightness and area of ​​the fluorescent area, the proliferation of undifferentiated cells in the animal's body can be quantified.

[0400] When the reporter gene is a fluorescent protein, it is preferable because undifferentiated cells can be visualized dynamically and over time in a living transgenic animal that serves as a screening tool. To maximize such effects, the transgenic animal is preferably one with a light body color or a transparent to translucent body color, such as zebrafish.

[0401] When zebrafish embryos are used, it is preferable to add a melanin production inhibitor (for example, phenylthiourea) to the culture medium in step A in order to suppress the production of melanin pigment.

[0402] [1-3]C process Step C is a step of observing differentiated cells in the animal that has undergone step A. More specifically, quantitative and qualitative parameters of the differentiated cells are observed. Quantitative parameters include the number of cells and the size and extent of the tissue. Qualitative parameters include the function and shape of the tissue composed of the differentiated cells. When observing tissue function, the function to be observed is selected according to the type of tissue being observed. For example, when observing the heart, cardiac function can be evaluated by observing the stroke volume, etc. When observing the pancreas, pancreatic function can be evaluated by observing the amount of insulin, a pancreatic secretion, etc.

[0403] In this specification, the term "differentiated cells" refers to cells that do not have the ability to self-replicate. The term "neuronally differentiated cells" includes various types of neurons and glial cells. In this specification, immature neurons are classified into dividing neural progenitor cells and non-dividing neural progenitor cells, with the former being included in undifferentiated neural cells and the latter being included in differentiated neural cells.

[0404] The means for observing differentiated cells is not particularly limited. Suitable examples include means for observing markers of differentiated cells and means for observing reporter genes that are specifically expressed in differentiated cells. Each of these means is described in detail below.

[0405] In a preferred embodiment of the present invention, markers that are specifically expressed in differentiated cells are observed in step C. The types of marker genes are outlined below.

[0406] Among immature neurons, those classified as non-dividing neural progenitor cells do not undergo further cell division, unlike neural stem cells and radial glial cells, which are also immature nervous system cells. After migrating through the nervous system and reaching their destination, these cells extend neurites, form synaptic connections, and ultimately become part of the neural circuit network. Markers of immature neurons classified as non-dividing neural progenitor cells include Doublecortin, NeuroD1, TBR1, Beta III tubulin, and Stathmin 1.

[0407] Acetylated tubulin is a marker for stabilized microtubules, and in neurons, the microtubules located near the cell body in the extending axon are composed of stable, long-lived acetylated tubulin. Therefore, acetylated tubulin is useful as a marker for mature neurons that are extending axons.

[0408] Markers of mature neurons include NeuN, MAP2, Beta III tubulin, 160 kD neurofilament, 200 kD neurofilament, NSE, PSD93, and PSD95.

[0409] More specifically, examples of markers for glutamatergic neurons include vGluT1, vGluT2, glutaminase, glutamine synthetase, NMDAR1, and NMDAR2B.

[0410] Markers of GABAergic neurons include GABA transporter 1, GABAB receptor 1, GABAB receptor 2, GAD65, GAD67, and ABAT.

[0411] Markers of dopaminergic neurons include tyrosine hydroxylase, dopamine transporter, FOXA2, GIRK2, LMX1B, and Nurr1.

[0412] Markers of serotonergic neurons include tryptophan hydroxylase, serotonin transporter, and Pet1.

[0413] Markers of cholinergic neurons include acetylcholinesterase, ChAT, and VAChT.

[0414] Specific glial cell markers include the following: Markers of myelinating Schwann cells include SOX10, S100, EGR2, MBP, and MPZ.

[0415] Markers of non-myelinating Schwann cells include SOX10, S100, GAP43, NCAM, and p75NTR.

[0416] Oligodendrocyte markers include Olig1, Olig2, Olig3, OSP, MBP, MOG, and SOX10.

[0417] Astrocyte markers include GFAP, EAAT1 / GLAST, EAAT2 / GLT-1, Glutamine synthetase, S100 beta, and ALDH1L1.

[0418] Markers for retinal ganglion cells include Thy-1, Sdk1, Sdk2, and the like.

[0419] Markers of cardiomyocytes include GATA-4, α-sarcomeric actin, α-sarcomeric actinin, slow myosin heavy chain, troponin TC, and MYL-2.

[0420] Mature pancreatic β cell markers include insulin, C-peptide, MAFA, NKX6.1, PDX1, and Fltp.

[0421] Some of the markers mentioned above are also expressed in undifferentiated cells. The present invention may be embodied in such a way that genes expressed in both undifferentiated and differentiated cells are used as markers. In this case, it corresponds to an embodiment in which steps B and C are performed simultaneously (FIG. 5(c)).

[0422] Suitable methods for observing these markers include immunostaining and in situ hybridization. Observation of differentiated cells by these observation methods can be carried out by conventional methods.

[0423] In one embodiment of the present invention, a transgenic animal into which a reporter gene that is specifically expressed in differentiated cells has been introduced is used as a screening tool. In this case, the expression of the reporter gene that is specifically expressed in differentiated cells is observed in step C.

[0424] Such transgenic animals can be produced by conventionally known gene editing techniques, as described above in the explanation section for Step B. The types of reporter genes are also the same as those described above in the explanation section for Step B.

[0425] Various transgenic lines have been established in which a reporter gene that is specifically expressed in differentiated cells has been introduced, and any available line may be used. Some examples of transgenic lines in which a reporter gene that is specifically expressed in differentiated neural cells has been introduced are shown below, but it goes without saying that the embodiments of the present invention are not limited to these.

[0426] Huc-GFP transgenic zebrafish line (Developmental Biology 227, 279-293 (2000)) Ggaf-GFP transgenic zebrafish line (Developmental Dynamics 2009 Feb;238(2):475-86.)

[0427] When the reporter gene is a fluorescent protein, the transgenic animal is irradiated with light of the excitation wavelength of the fluorescent protein, and the emitted fluorescence is observed. The fluorescence can be observed under a fluorescence microscope, and based on the intensity of the fluorescence, the proliferation of differentiated cells in the animal can be quantified.

[0428] When the reporter gene is a fluorescent protein, it is preferable because differentiated cells can be visualized dynamically and over time in a living transgenic animal that serves as a screening tool. To maximize such effects, the transgenic animal is preferably one with a light body color or a transparent to translucent body color, such as zebrafish.

[0429] When zebrafish embryos are used, it is preferable to add a melanin production inhibitor (for example, phenylthiourea) to the culture medium in step A in order to suppress the production of melanin pigment.

[0430] [1-4] Form in which both Process B and Process C are implemented This section further explains an embodiment (FIG. 4) in which both steps B and C are performed. As explained with reference to FIG. 5, the order of steps B and C is not particularly limited, and these steps may be performed simultaneously. Steps B and C may be performed on the same animal or on different animals. In the latter case, the experimental conditions for the animals subjected to Steps B and C are made as consistent as possible.

[0431] In a preferred embodiment of the present invention, steps B and C are performed on the same individual animal. An embodiment in which steps B and C are performed on the same individual animal will be described below.

[0432] In one embodiment of the present invention, undifferentiated cell markers are observed in step B, and differentiated cell markers are also observed in step C. In such an embodiment, it is preferable to fix the animal that has undergone step A for staining and perform multiple staining with antibodies / probes against undifferentiated cell markers and differentiated cell markers. By performing multiple staining, undifferentiated cells and differentiated cells can be observed simultaneously. When performing multiple staining, follow standard procedures to design the antibody / probe labels for undifferentiated cell markers and differentiated cell markers so that they do not get mixed up. In the case of fluorescent labels, use labels with different excitation and emission wavelengths.

[0433] In one embodiment of the present invention, a transgenic animal is used into which a reporter gene that is specifically expressed in undifferentiated cells has been introduced, and the expression of the reporter gene is observed in step B, and the differentiated cell markers are observed in step C. In this case, it is preferable to carry out step B (such as fluorescent observation) first, which allows observation of the animal in a living state, and then carry out step C (such as immunostaining). By adopting such a configuration, it is possible to follow and observe the self-replication of undifferentiated cells over time, and to evaluate the effect of proliferation of differentiated cells as a result of proliferation of undifferentiated cells.

[0434] In one embodiment of the present invention, a transgenic animal is used into which a reporter gene that is specifically expressed in differentiated cells has been introduced, and undifferentiated cell markers are observed in step B, and the expression of the reporter gene that is specifically expressed in differentiated cells is observed in step C. In this case, it is preferable to carry out step C (such as fluorescent observation) first, which allows observation of the animal in a living state, and then carry out step B (such as immunostaining). By adopting such a configuration, it is possible to follow up and observe the proliferation of differentiated cells over time, and to evaluate the effect of self-replication of undifferentiated cells as a cause of proliferation of differentiated cells.

[0435] In another embodiment of the present invention, the animal used as a screening tool is a transgenic animal into which both a reporter gene specifically expressed in undifferentiated cells and a reporter gene specifically expressed in differentiated cells have been introduced. Then, in step B, the expression of the reporter gene specifically expressed in undifferentiated cells is observed, and in step C, the expression of the reporter gene specifically expressed in differentiated cells is observed.

[0436] By using transgenic animals in which two reporter genes, one for undifferentiated cells and one for differentiated cells, are introduced, it is possible to carry out both steps B and C dynamically and over time while the animal is alive. Furthermore, since steps B and C can be carried out simultaneously, the burden on the animal is reduced. When the reporter gene is a fluorescent protein gene, the reporter gene for the undifferentiated cell and the reporter gene for the differentiated cell are preferably genes encoding fluorescent proteins having different excitation wavelengths and fluorescence wavelengths.

[0437] [1-5]D process Step D is a step of selecting an active ingredient by evaluating the observation results of Step B and / or Step C. Specifically, Step D is a step of selecting, as an active ingredient, a candidate substance that brings about a quantitative and / or qualitative improvement in undifferentiated cells and / or differentiated cells compared to when the candidate substance is not administered.

[0438] When step B is carried out, a candidate substance that increases the amount of undifferentiated cells compared to when the candidate substance is not administered is selected as an active ingredient in step D. When step C is carried out, a candidate substance that increases the amount of differentiated cells compared to when the candidate substance is not administered is selected as the active ingredient in step D. Also, a candidate substance that improves the function of tissues composed of differentiated cells compared to when the candidate substance is not administered is selected as the active ingredient.

[0439] The effect of promoting the proliferation of undifferentiated cells and / or differentiated cells can be evaluated appropriately in accordance with the specific embodiments of steps B and C. For example, when immunostaining or in situ hybridization is performed in step B and / or step C, the cell proliferation effect can be evaluated by using the intensity and range of fluorescence or color in the stained image as an index. That is, when the intensity and range of fluorescence or color in the stained image is strong or wide, it can be determined that the candidate substance has the effect of promoting the proliferation of undifferentiated cells and / or differentiated cells.

[0440] The proliferation effect on undifferentiated cells and / or differentiated cells is preferably evaluated by comparing the results of a control experiment conducted under the same conditions except that the candidate substance is not administered. The control experiment may be carried out simultaneously with steps A to D, or the results of a control experiment may be recorded and used as a reference for evaluation.

[0441] [1-6] Two-stage screening As described above, the present invention comprises step A, step B and / or step C, and step D. Steps A to D may be performed two or more times in some embodiments. That is, active ingredients selected by performing steps A to D as primary screening may be subjected as candidate substances to steps A to D as secondary screening. In this case, it is preferable to change the conditions for the primary screening and the secondary screening. For example, in one embodiment, the primary screen uses embryos and the secondary screen uses adults. Furthermore, animals of a higher level than those used in the primary screening may be used in the secondary screening, specifically, fish may be used in the primary screening and mammals may be used in the secondary screening.

[0442] In one embodiment of the present invention, step A includes the following steps A1 and A2, step B includes the following steps B1 and B2, step C includes the following steps C1 and C2, and step D includes the following steps D1 and D2. Either or both of step B1 and step C1 may be performed, and either or both of step B2 and step C2 may be performed. [Step A1] A step of administering a candidate substance to an animal embryo. [Step B1] A step of observing undifferentiated cells in the animal embryo that has undergone Step A1. [Step C1] A step of observing differentiated cells in the animal embryo that has undergone Step A1. [Step D1] A step of selecting, as an active ingredient, a candidate substance that, as a result of Step B1 and / or Step C1, increases the amount of the undifferentiated cells and / or the differentiated cells compared to when the candidate substance is not administered. [Step A2] A step of administering the active ingredient selected in the step D1 to an adult animal. [Step B2] A step of observing undifferentiated cells in the adult animal that has undergone Step A2. [Step C2] A step of observing differentiated cells in the adult animal that has undergone Step A2. [Step D2] A step of selecting, as an active ingredient, a candidate substance that, as a result of Step B2 and / or Step C2, increases the amount of the undifferentiated cells and / or the differentiated cells compared to when the candidate substance is not administered.

[0443] Steps A1 to D1 are primary screening using embryos, and the latter steps A2 to D2 are secondary screening using adult animals. By performing two-stage screening in this way, active ingredients can be screened more accurately.

[0444] Preferably, steps A1 to D1 are primary screening using fish embryos, and the latter steps A2 to D2 are secondary screening using adult mammals. The use of fish embryos in the primary screening allows for convenient large-scale primary screening. Furthermore, the use of adult mammals in the secondary screening allows for the selection of active ingredients that are likely to be effective in humans.

[0445] Preferably, steps A1 to D1 are primary screening using zebrafish embryos, and the latter steps A2 to D2 are secondary screening using adult mice. The use of zebrafish embryos in the primary screening allows for convenient large-scale primary screening. Furthermore, the use of adult mice in the secondary screening allows for the selection of active ingredients that are likely to be effective in humans.

[0446] In one embodiment of the present invention, step A includes the following steps A1 and A2, step B includes the following steps B1 and B2, step C includes the following steps C1 and C2, and step D includes the following steps D1 and D2. Either or both of step B1 and step C1 may be performed, and either or both of step B2 and step C2 may be performed. [Step A1] A step of administering a candidate substance to a fish embryo. [Step B1] A step of observing undifferentiated cells in the animal embryo that has undergone Step A1. [Step C1] A step of observing differentiated cells in the animal embryo that has undergone Step A1. [Step D1] A step of selecting, as an active ingredient, a candidate substance that, as a result of Step B1 and / or Step C1, increases the amount of the undifferentiated cells and / or the differentiated cells compared to when the candidate substance is not administered. [Step A2] A step of administering the active ingredient selected in the step D1 to a mammal. [Step B2] A step of observing undifferentiated cells in the mammal that have undergone Step A2. [Step C2] A step of observing differentiated cells in the mammal that have undergone Step A2. [Step D2] A step of selecting, as an active ingredient, a candidate substance that, as a result of Step B2 and / or Step C2, increases the amount of the undifferentiated cells and / or the differentiated cells compared to when the candidate substance is not administered.

[0447] Steps A1 to D1 are primary screening using fish embryos, and the latter steps A2 to D2 are secondary screening using mammals. By performing two-stage screening in this manner, active ingredients can be screened more accurately. In step A2, either adult mammals or embryos may be used. The use of fish embryos in primary screening allows for convenient large-scale primary screening, and the use of mammals in secondary screening allows for the selection of active ingredients that are likely to be effective in humans.

[0448] [2] Second screening method Next, an embodiment of the second screening method will be described. The second screening method comprises the following steps E to G. [Step E] A step of administering a candidate substance to a model animal (excluding humans) of a disease, disorder, or illness. [Step F] A step of determining the therapeutic effect of the disease, disorder, or illness in the animal that has undergone Step E. [Step G] A step of selecting a candidate substance having a therapeutic effect as the active ingredient.

[0449] [2-1]E process The second screening method is characterized by using an animal model of a disease or the like (step E). Preferably, an animal model of a disease of the nervous system, heart, or pancreas is used. It is also preferable to use an animal model of cancer. For specific embodiments of step E, the explanation of step A above is applicable, and in particular the explanation regarding model animals for diseases etc. is applicable as is.

[0450] The cancer model animal is not particularly limited, and can be one in which a tumor suppressor gene has been deleted by genetic engineering techniques, or one produced by transplanting tumor cells or tumor tissue into an immunodeficient animal. Various types of cancer model animals are available for use, either for a fee or free of charge, and can be used without restriction depending on the purpose.

[0451] Animal models of blood cancer, particularly leukemia, include, for example, a mouse model of T-cell acute lymphoblastic leukemia (p53null, Ezh2 conditional KO) (Non-Patent Document 26) and a Cre / lox-regulated transgenic zebrafish model with conditional myc-induced T-cell acute lymphoblastic leukemia (Non-Patent Document 27).

[0452] As animal models of diabetes, there are type 1 diabetes model mice such as NOD / ShiJcl, KK / TaJcl, and KK-A y / TaJcl, BKS.Cg-m + / + Lepr db Examples of such type 2 diabetes model mice include GK / Jcl, GK / Jcl, SDT / Jcl, and SDT fatty / Jcl. Drug-induced diabetes model animals, such as streptozotocin (STZ)-induced type 1 diabetes models in rats, mice, and zebrafish, may also be used.

[0453] Animal models of myocardial injury include mice, rats, and zebrafish in which myocardial injury is induced by doxorubicin (DOX), which causes cardiotoxicity, particularly left ventricular dysfunction.

[0454] In step E, it is preferable to administer the candidate substance locally to or near the site of damage or functional impairment due to the disease. Alternatively, the candidate substance may be administered orally, transdermally, enterally, or the like. The administration form can be appropriately designed depending on the type of disease.

[0455] When administering to a glaucoma model, the candidate substance is preferably instilled or painted onto the retina. When administering to a spinal cord injury model, the candidate substance is preferably administered to the site of spinal cord injury. When administering to a blood cancer model, a diabetes model, or a myocardial injury model, it is preferable to administer the candidate substance by injection into a blood vessel.

[0456] [2-2]F process In step F, the therapeutic effect on the disease, disorder, or illness in the animal that has undergone step E is evaluated. Methods for assessing therapeutic efficacy include observation of the appearance and behavior of disease model animals, anatomical and pathological diagnosis, etc., and can be designed appropriately according to the nature of the disease suffered by the disease model animal.

[0457] Specific examples of observations of the appearance and behavior of disease models include the following: For example, the therapeutic effect in a depression model can be determined by the forced swimming test (mice, rats) or the tail suspension test (mice). The therapeutic effect in an anxiety disorder model can be determined by a Vogel-type conflict test (rats) and a social interaction test (rats). The therapeutic effect in a spinal cord injury model can be assessed using as an indicator whether or not motor function lost due to spinal cord injury is restored. The therapeutic effects in glaucoma models can be assessed using a two-choice visual discrimination task using water as an indicator (Prusky, West, & Douglas (2000)), a discrimination task in an appetitive situation using food as a reward (Gianfranceschi, Fiorentini, & Maffei (1999)), and a test using optokinetic responses (Douglas, Alam, Silver, Mcgill, Tschetter, & Prusky, 2005). The therapeutic effect in a diabetic model can be assessed based on indicators such as measurement of insulin secretion amount and blood glucose level. The therapeutic effect in a myocardial injury model can be assessed based on the recovery of myocardial function weakened by injury, that is, the recovery of cardiac function, based on indicators such as stroke volume.

[0458] The effectiveness of cancer treatment can be determined using indicators such as tumor shrinkage and tumor markers leaked into the blood. Furthermore, the active ingredients selected by the first screening method described above exert their anticancer effects by forcibly differentiating cancer cells into normal cells, and therefore, the therapeutic effect can be assessed based on whether or not differentiation of cancer cells into normal cells is observed.

[0459] When the active ingredient selected by the first screening method described above is applied to blood cancers such as leukemia, it induces the forced differentiation of cancer cells into vascular endothelial cells, thereby neutralizing harmful cancer cells. Therefore, the therapeutic effect of blood cancers, such as leukemia, is particularly evaluated by observing the presence or absence of vascular endothelial thickening or tumor formation. Furthermore, for cancers showing liquid metastasis, such as hematogenous or lymphatic metastasis, the presence or absence of vascular or lymphatic endothelial thickening or tumor formation is also observed.

[0460] Specific examples of anatomical and pathological diagnosis include the following: The therapeutic effect in a glaucoma model can be assessed by observing whether or not the number of damaged retinal ganglion cells has increased. Specific examples of the method for observing whether or not the number of retinal ganglion cells has increased are as described above in the first screening method. The therapeutic effect in a spinal cord injury model can be assessed by observing the crushed spinal cord with a microscope or MRI, and using the regenerative effect as an indicator.

[0461] In order to more accurately assess the therapeutic effect, it is preferable to conduct a control experiment. The control experiment is preferably carried out under the same conditions except that the candidate substance is not administered. The control experiment may be carried out simultaneously with steps E and F, or the results of the control experiment may be recorded once obtained and used as a reference to assess the therapeutic effect in step F.

[0462] [2-3]G process Step G is a step of selecting the candidate substance determined to have a therapeutic effect in step F as an active ingredient.

[0463] [3] Two-stage screening method The present invention also relates to a two-stage screening method in which a primary screening is performed by a first screening method, and candidate substances selected as active ingredients in the primary screening are subjected to a second screening method. The embodiments of the first screening method and the second screening method are as described above.

[0464] Preferably, in step A of the first screening method, normal animals are used instead of disease model animals. That is, in the first screening method, primary screening is performed on a large number of candidate substances at low cost using normal animals, which can be obtained in large quantities at low cost. After the number of candidate substances is narrowed down by the primary screening, secondary screening is performed using disease models. By performing such two-stage screening, active ingredients that are effective against nervous system diseases and the like can be screened efficiently and economically.

[0465] In one embodiment of the present invention, the first screen uses embryos as a screening tool. In one embodiment of the present invention, the first screen uses fish embryos, more preferably zebrafish embryos, as a screening tool, and the second screen uses mammals, more preferably mice or rats, as a screening tool.

[0466] In one embodiment of the present invention, a candidate substance selected as an active ingredient as a result of the first screening may be subjected to a second screening method. In this case, the second screening method may be performed based on the results of the first screening performed by another institution. That is, the practitioner of the present invention may perform only the second screening method.

[0467] [4] Manufacturing method The present invention also relates to a method for producing a pharmaceutical composition, which comprises a formulation step of mixing a substance selected as an active ingredient by the above-described screening method with pharmaceutical additives to form a formulation.

[0468] The person who implements the manufacturing method of the present invention may implement the above-mentioned screening method himself or herself, or may implement the invention of the manufacturing method based on the results of another person implementing the above-mentioned screening method. That is, one embodiment of the present invention includes a screening step of screening for an active ingredient by the above-mentioned screening method. The embodiment of the screening step is as described above. In one embodiment of the present invention, a manufacturing method is carried out using a substance that has already been determined to be an active ingredient by the above-described screening method.

[0469] The formulation process in which a substance selected as an active ingredient in screening is mixed with pharmaceutical additives to form a formulation will be described in detail below.

[0470] The dosage form and composition of the pharmaceutical composition can be appropriately designed depending on the disease to be treated or prevented. The pharmaceutical composition contains the above-mentioned active ingredient and any pharmaceutical additives. When the pharmaceutical composition is in the form of a solid preparation, the dosage form can be appropriately selected from granules, dry syrup, fine granules, tablets, powders, capsules, and pills. These dosage forms can be prepared by conventional methods.

[0471] When the pharmaceutical composition is in the form of a solid preparation, it may contain a stabilizer, such as sodium chloride and potassium chloride, formic acid, oxalic acid, acetic acid, citric acid, ascorbic acid and fumaric acid, miglyol (medium-chain fatty acid triglyceride), triethyl citrate and polyoxyethylene sorbitan monooleate, triacetin (glyceryl triacetate), glycerin fatty acid ester, acylglycerol, monoglyceride derivatives, and polyglycerin fatty acid ester.

[0472] When the pharmaceutical composition is in the form of a solid preparation, it may contain an excipient, such as isomalt, erythritol, D-mannitol, xylitol, sorbitol, reduced maltose syrup (maltitol), lactitol, oligosaccharide alcohol, xylose, glucose, fructose, maltose, lactose, sucrose, fructose, trehalose, isomerized sugar, starch syrup, refined white sugar, white sugar, refined white sugar spherical granules, anhydrous lactose, white sugar-starch spherical granules, oligosaccharide, dextrin, starch, etc. Polysaccharides such as semi-digested starch, glucose hydrate, crystalline cellulose, microcrystalline cellulose, pullulan, β-cyclodextrin, aminoethyl sulfonic acid, candy powder, sodium chloride, citric acid, sodium citrate, glycine, calcium gluconate, L-glutamine, tartaric acid, potassium hydrogen tartrate, ammonium carbonate, dextran 40, dextrin, calcium lactate, povidone, macrogol (polyethylene glycol) 1500, macrogol 1540, macrogol Examples of suitable granules include cellulose 4000, macrogol 6000, anhydrous citric acid, DL-malic acid, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, L-aspartic acid, alginic acid, carmellose sodium, hydrated silicon dioxide, crospovidone, calcium glycerophosphate, magnesium aluminosilicate, calcium silicate, magnesium silicate, light anhydrous silicic acid, synthetic aluminum silicate, wheat flour, wheat starch, wheat germ flour, wheat germ oil, rice flour, rice starch, cellulose acetate phthalate, titanium oxide, magnesium oxide, dihydroxyaluminum aminoacetate, tricalcium phosphate, talc, calcium carbonate, magnesium carbonate, precipitated calcium carbonate, natural aluminum silicate, corn starch, corn starch granules, potato starch, hydroxypropyl cellulose, hydroxypropyl starch, anhydrous calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate granules, and calcium dihydrogen phosphate.

[0473] The pharmaceutical composition may be in the form of a solid preparation, which is suspended in water and taken as a suspension. In this case, suspending agents include cellulose-based polymers such as carmellose, carmellose sodium, crystalline cellulose-carmellose sodium, hydroxypropyl cellulose, hypromellose (hydroxypropyl methylcellulose), methylcellulose, carboxymethylethylcellulose, hydroxyethyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose phthalate, fumaric acid-stearic acid-polyvinyl acetal diethylaminoacetate-hydroxypropyl methylcellulose mixture, ethyl acrylate-methyl methacrylate copolymer dispersion, aminoalkyl methacrylate copolymer, methacrylic acid copolymer, 2-methyl-5-vinylpyridine methylacrylate-methacrylic acid copolymer, dry methacrylic acid copolymer, dimethyl methacrylate copolymer, methyl ... Examples of suitable surfactants include acrylic polymers such as aminoethyl methacrylate-methyl methacrylate copolymer, vinyl polymers such as polyvinylpyrrolidone, crospovidone, carboxyvinyl polymer, polyvinyl acetal diethylaminoacetate, polyvinyl alcohol, polyvinyl alcohol-methyl methacrylate-acrylic acid copolymer, and polyvinyl alcohol copolymer, sodium alginate, carrageenan, carboxyvinyl polymer, dried aluminum hydroxide gel, xanthan gum, magnesium aluminum silicate, sodium polyphosphate, macrogol 4000, and macrogol 6000. Preferred are carmellose, carmellose sodium, crystalline cellulose-carmellose sodium, hydroxypropyl cellulose, hypromellose, and polyvinylpyrrolidone, and more preferably hypromellose.

[0474] When the pharmaceutical composition is in the form of a solid preparation, a lubricant may be added to improve lubricity, such as light anhydrous silicic acid, hydrated silicon dioxide, sucrose fatty acid ester, stearyl alcohol, stearic acid, magnesium stearate, calcium stearate, sodium stearyl fumarate, and talc.

[0475] When the pharmaceutical composition is formulated into a solid preparation, a flavoring agent may be added to a drug having an unpleasant taste, such as a bitter taste, to correct the taste. Examples of flavoring agents include ascorbic acid, aspartic acid, aspartame, sucralose, glycine, sodium chloride, magnesium chloride, hydrochloric acid, dilute hydrochloric acid, citric acid and its salts, anhydrous citric acid, L-glutamic acid and its salts, succinic acid and its salts, acetic acid, tartaric acid and its salts, sodium bicarbonate, fumaric acid and its salts, malic acid and its salts, glacial acetic acid, disodium inosinate, honey, reduced maltose syrup (maltitol), and licorice powder.

[0476] When the pharmaceutical composition is made into a solid preparation, it may contain a binder, such as hydroxypropyl cellulose, corn starch, pregelatinized starch, partially pregelatinized starch, gum arabic, powdered gum arabic, gelatin, agar, dextrin, pullulan, polyvinylpyrrolidone, polyvinyl alcohol, crystalline cellulose, methyl cellulose, ethyl cellulose, carboxymethyl ethyl cellulose, carmellose, carmellose sodium, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose, and hypromellose.

[0477] When the pharmaceutical composition is prepared as a solid preparation, it may contain a disintegrant, such as croscarmellose sodium, crospovidone, carmellose calcium, sodium carboxymethyl starch, or low-substituted hydroxypropyl cellulose.

[0478] When the pharmaceutical composition is formulated as a solid preparation, it may contain a coloring agent. Examples of coloring agents include iron oxide, tar dyes, and natural dyes. Examples of iron oxide include iron sesquioxide, yellow iron oxide, yellow iron sesquioxide, and black iron oxide.

[0479] When the pharmaceutical composition is made into a solid preparation, a flavoring agent or a sweetener may be further added, if necessary. Specific examples of flavorings include orange essence, orange oil, caramel, camphor, cinnamon oil, spearmint oil, strawberry essence, chocolate essence, cherry flavor, spruce oil, pine oil, peppermint oil, vanilla flavor, strawberry flavor, bitter essence, fruit flavor, peppermint essence, mixed flavor, mint flavor, menthol, lemon powder, lemon oil, and rose oil. Specific examples of sweeteners include aspartame, reduced maltose syrup (maltitol), licorice, xylitol, glycerin, saccharin, sucralose, D-sorbitol, acesulfame potassium, stevia, thaumatin, and adpantame.

[0480] The pharmaceutical composition may also be in the form of a parenteral dosage form. For example, it may be in the form of a fluid dosage form such as intravenous injection, intracerebral injection, intrathecal injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric-coated drug, eye drops, eye ointment solution or ointment. Such a fluid dosage form is advantageous when the active ingredient is to be administered directly to the affected area. The liquid preparation is preferably in the form of an injection or eye drops, and the ointment is preferably in the form of an eye ointment.

[0481] When the pharmaceutical composition is used as an injection, the aqueous medium may be water (i.e., water for injection) or a mixture of water and a water-miscible solvent. Examples of such water-miscible solvents include alcohols (e.g., aliphatic alcohols (e.g., methanol, ethanol, etc.) or aryl alcohols), polyols, esters, alkyl halides, ethers, cyanides / nitriles, ketones, aldehydes, amines, amides, formamides, sulfides, sulfoxides, and carboxylic acids.

[0482] When the pharmaceutical composition is used as an injection, a pH adjuster may be added. pH adjusters commonly used in injections can be used as the pH adjuster. Specifically, to shift the pH toward the acidic side, acidic substances such as acetic acid, lactic acid, phosphoric acid, tartaric acid, citric acid, ascorbic acid, hydrochloric acid, gluconic acid, and sulfuric acid can be used. To shift the pH toward the basic side, basic substances such as potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, monoethanolamine, diethanolamine, and triethanolamine can be used.

[0483] When the pharmaceutical composition is prepared as an injection, an isotonicity agent can be added, for example, sugars and sugar alcohols such as glucose, maltose, α-trehalose, sorbitol, and mannitol, polyhydric alcohols such as glycerin, propylene glycol, and polyethylene glycol, and electrolytes such as sodium chloride.

[0484] When the pharmaceutical composition is used as an injection, a buffering agent can be added, such as a citrate buffer, an acetate buffer (e.g., sodium acetate hydrate), a phosphate buffer, or a tartrate buffer.

[0485] When the pharmaceutical composition is prepared as an injection, ingredients commonly used in injections can be added, such as diluents, soothing agents, and preservatives.

[0486] The pharmaceutical composition may be in the form of a freeze-dried injection, which can be prepared into an injection by adding an aqueous medium. The freeze-dried pharmaceutical composition for injection can be produced by freeze-drying the above-described pharmaceutical composition for injection in the form of an aqueous solution in a conventional manner.

[0487] More specifically, the pharmaceutical composition is preferably in the form of an injection for intracerebral administration, an injection for intraventricular administration, or an injection for spinal administration.

[0488] The pharmaceutical composition may be in the form of eye drops, which may contain sugars such as glucose, fructose, galactose, mannose, ribose, ribulose, arabinose, xylose, lyxose, deoxyribose, maltose, trehalose, sucrose, cellobiose, lactose, pullulan, lactulose, raffinose, and maltitol.

[0489] When the pharmaceutical composition is in the form of eye drops, the following may be used: polyoxyethylene-polyoxypropylene block copolymer adducts of ethylenediamine (e.g., poloxamine); POE sorbitan fatty acid esters such as POE(20) sorbitan monolaurate (polysorbate 20) and POE(20) sorbitan monooleate (polysorbate 80); POE hydrogenated castor oils such as POE(60) hydrogenated castor oil; POE alkyl ethers such as POE(9) lauryl ether; POE(20)POP(4); Nonionic surfactants such as POE·POP alkyl ethers such as cetyl ether, POE alkyl phenyl ethers such as POE (10) nonylphenyl ether; glycine types such as alkyldiaminoethylglycine, acetate betaine types such as lauryldimethylaminoacetic acid betaine, and imidazoline types of amphoteric surfactants; POE alkyl ether phosphates and their salts such as sodium POE (10) lauryl ether phosphate, N-acyl amino acid salts such as sodium lauroylmethylalanine, alkyl ether carboxylates, N-acyltaurine salts such as sodium N-cocoylmethyltaurine, sulfonates such as sodium tetradecene sulfonate, alkyl sulfates such as sodium lauryl sulfate, POE alkyl ether sulfates such as sodium POE (3) lauryl ether sulfate, and anionic surfactants such as α-olefin sulfonates; alkylamine salts, alkyl quaternary ammonium salts (benzalkonium chloride, benzethonium chloride, etc.), and alkylpyridinium salts (cetylpyridinium chloride, cetylpyridinium bromide, etc.) may also be added.

[0490] When the pharmaceutical composition is formulated as an eye drop, a preservative, a disinfectant, or an antibacterial agent may be added. Specific examples include sorbic acid or its salts (sorbic acid, potassium sorbate, sodium sorbate, triclocarban sorbate, etc.), parahydroxybenzoic acid esters (methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, etc.), acrinol, methylrosaniline chloride, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, cetylpyridinium bromide, chlorhexidine, polyhexamethylene biguanide, alkylpolyaminoethylglycine, benzyl alcohol, phenethyl alcohol, chlorobutanol, and isopropanol. , ethanol, phenoxyethanol, carriers such as silver zirconium phosphate, thimerosal, dehydroacetic acid, chloroxylenol, chlorophene, resorcinol, thymol, hinokitiol, sulfamine, lysozyme, lactoferrin, triclosan, 8-hydroxyquinoline, undecylenic acid, caprylic acid, propionic acid, benzoic acid, propionic acid, halocarban, thiabendazole, polymyxin B, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, polylysine, hydrogen peroxide, polydronium chloride, Glokill (trade name e.g. Glokill PQ, manufactured by Rhodia), polydiallyldimethylammonium chloride, poly[oxyethylene(dimethyliminio)ethylene-(dimethyliminio)ethylene dichloride], polyethylenepolyamine-dimethylamine epichlorohydrin polycondensate (trade name, for example, Busan 1157, manufactured by Bachmann), etc.

[0491] When the pharmaceutical composition is in the form of eye drops, a pH adjuster may be added. Examples of pH adjusters include inorganic acids (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, polyphosphoric acid, boric acid, etc.), organic acids (e.g., lactic acid, acetic acid, citric acid, tartaric acid, malic acid, succinic acid, oxalic acid, gluconic acid, fumaric acid, propionic acid, acetic acid, aspartic acid, epsilon-aminocaproic acid, glutamic acid, aminoethylsulfonic acid, etc.), gluconolactone, ammonium acetate, inorganic bases (e.g., sodium bicarbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide, etc.), organic bases (e.g., monoethanolamine, triethanolamine, diisopropanolamine, triisopropanolamine, lysine, etc.), and borax.

[0492] When the pharmaceutical composition is formulated as an eye drop, an isotonic agent may be added, such as sugars such as glucose, mannitol, and sorbitol, and inorganic salts such as sodium chloride, potassium chloride, sodium carbonate, sodium bicarbonate, calcium chloride, magnesium sulfate, sodium hydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium thiosulfate, and sodium acetate.

[0493] When the pharmaceutical composition is made into an eye ointment, any ointment base can be used. The ointment base is not particularly limited, but generally, hydrophobic bases such as oils and fats, waxes, and hydrocarbon compounds can be used. Specific examples include mineral bases such as yellow petrolatum, white petrolatum, paraffin, liquid paraffin, plastibase, and silicone, and animal and vegetable bases such as beeswax and animal and vegetable oils and fats.

[0494] It is also known that administered substances can pass directly into the cerebrospinal fluid (CSF) or brain through the nasal mucosa without passing through the bloodstream (Illum L., Eur. J. Pharm. Sci., 11, 1-18 (2000). Frey WH, Drug Deliv. Technol., 2, 46-49 (2002). Lochhead JJ, Thorne G., Adv. Drug Deliv. Rev., 64, 614-628 (2012). Djupesland PG, Messina JC, Mahmoud RA, Ther. Deliv., 5, 709-733 (2014).) Therefore, in order to allow the active ingredient to reach the brain, the pharmaceutical composition can be in the form of a nasal administration preparation, such as nasal drops, liquid nasal sprays, powder nasal sprays, nasal mucosal injections, gel preparations, and ointments.

[0495] When the pharmaceutical composition is used as a nasal spray, a surfactant may be added. Examples of the surfactant include nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene polypropylene alkyl ethers, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyethylene glycol fatty acid esters, sucrose fatty acid esters, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, and polyoxyethylene polyoxypropylene polymers; amphoteric surfactants such as alkyl betaines, alkylamido betaines, alkyl sulfobetaines, and imidazolines; and anionic surfactants such as saturated higher fatty acid salts, alkyl sulfonates, alkyl ether sulfonates, alkyl ether sulfonates, and polyoxyethylene alkyl ether phosphates.

[0496] When the pharmaceutical composition is formulated as a nasal spray, a thickener may be added, such as celluloses such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carmellose, croscarmellose, and methyl cellulose, modified starches such as partially pregelatinized starch, polyvinyl alcohol, polyvinylpyrrolidone, crospovidone, polyethylene glycol, carboxyvinyl polymer, acrylic acid / alkyl methacrylate copolymer, xanthan gum, carrageenan, alginic acid and its salts, gum arabic, guar gum, locust bean gum, pullulan, gelatin, and sodium polyacrylate.

[0497] When the pharmaceutical composition is used as a nasal spray, an oily component may be added. Examples of the oily component include unsaturated fatty alcohols such as palmitoleyl alcohol, oleyl alcohol, eicosonyl alcohol, elaidyl alcohol, and linoleyl alcohol, unsaturated fatty acids such as oleic acid, elaidic acid, linoleic acid, undecylenic acid, myristoleic acid, palmitoleic acid, linderic acid, lauroleic acid, tsuzuic acid, petroselinic acid, vacenic acid, and gondoic acid, unsaturated fatty acid esters such as glycerin monooleate, glycerin dioleate, octyldodecyl oleate, and oleyl oleate, cetyl octanoate, myristyl alcohol, and the like. saturated fatty acid esters such as isopropyl phosphate and myristyl myristate; hydrocarbons such as oleyl alcohol, elaidyl alcohol, liquid paraffin, petrolatum and microcrystalline wax; silicone oils such as methyl polysiloxane, methylphenyl polysiloxane and dimethyl cyclopolysiloxane; waxes such as beeswax; higher alcohols such as cetyl alcohol and stearyl alcohol; sterols such as cholesterol; metal soaps such as aluminum stearate and magnesium stearate; avocado oil, palm oil, beef tallow and jojoba oil.

[0498] When the pharmaceutical composition is formulated as a nasal drop, an isotonic agent may be added, such as sugars such as sorbitol, glucose, and mannitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, and inorganic salts such as sodium chloride and potassium chloride.

[0499] When the pharmaceutical composition is used as a nasal drop, a pH adjuster may be added. Examples of pH adjusters include acetic acid, formic acid, lactic acid, tartaric acid, oxalic acid, glycolic acid, malic acid, citric acid, succinic acid, fumaric acid, phosphoric acid, hydrochloric acid, nitric acid, sulfuric acid and their salts, sodium hydroxide, potassium hydroxide, calcium hydroxide, arginine, methylamine, ethylamine, propylamine, dimethylamine, diethylamine, dipropylamine, trimethylamine, triethylamine, tripropylamine, monomethanolamine, monoethanolamine, monopropanolamine, dimethanolamine, diethanolamine, dipropanolamine, trimethanolamine, triethanolamine, isopropanolamine, diisopropanolamine, tripropanolamine, ammonia water, guanidine carbonate, sodium bicarbonate, ammonium carbonate, etc.

[0500] When the pharmaceutical composition is formulated as a nasal spray, a buffering agent may be added, such as boric acid and its salts, phosphates, acetates, and amino acid salts.

[0501] When the pharmaceutical composition of the present invention is formulated as a nasal drop, a chelating agent may be added, such as edetic acid, oxalic acid, citric acid, pyrophosphate, hexametaphosphate, gluconic acid, or salts thereof.

[0502] When the pharmaceutical composition is formulated as a nasal drop, a flavoring or refreshing agent may be added. Examples of the flavoring or refreshing agent include peppermint oil, cinnamon oil, clove oil, fennel oil, castor oil, turpentine oil, eucalyptus oil, orange oil, lavender oil, lemon oil, rose oil, lemongrass oil, star anise oil, thymian oil, chenopodium oil, Japanese ginseng oil, touca oil, bergamot oil, citronella oil, camphor oil, rosemary, and sage, and refreshing agents such as l-menthol, camphor, thymol, N-ethyl-p-menthane-carboxamide, p-menthane-3,8-diol, l-isopulegol, and l-menthyl glyceryl ether.

[0503] When the pharmaceutical composition is formulated as a nasal drop, an antioxidant may be added, such as ascorbic acid, propyl gallate, butylhydroxyanisole, dibutylhydroxytoluene, nordihydroguaiaretic acid, tocopherol, or tocopherol acetate.

[0504] When the pharmaceutical composition is formulated as a nasal drop, a preservative may be added, such as thymol, isopropylmethylphenol, benzoic acid and its salts, methyl benzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, benzyl alcohol, benzalkonium chloride, or benzethonium chloride.

[0505] When the pharmaceutical composition is formulated as a nasal drop, an absorption enhancer may be added, such as diisopropyl adipate, lecithin, squalane, squalene, l-menthol, polyethylene glycol, isopropyl myristate, dimethyl sulfoxide, peppermint oil, eucalyptus oil, d-limonene, or dl-limonene.

[0506] When the pharmaceutical composition is used as a nasal drop, a suspending agent may be added. Examples of the suspending agent include celluloses such as methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose, synthetic polymer compounds such as polyvinyl alcohol, polyvinylpyrrolidone, and carboxyvinyl polymer, nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene polypropylene alkyl ethers, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyethylene glycol fatty acid esters, sucrose fatty acid esters, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, and polyoxyethylene polyoxypropylene polymers, amphoteric surfactants such as alkyl betaines, alkylamido betaines, alkyl sulfobetaines, and imidazolines, and anionic surfactants such as saturated higher fatty acid salts, alkyl sulfonates, alkyl ether sulfonates, alkyl ether sulfonates, and polyoxyethylene alkyl ether phosphates.

[0507] The pharmaceutical composition may be in the form of a liquid, which may be filled into a sprayer to prepare a liquid nasal spray formulation. In this case, the sprayer into which the pharmaceutical composition of the present invention is filled is not particularly limited, and for example, a sprayer used for preparing a liquid nasal spray formulation for the treatment of allergic rhinitis may be used. In addition, the specific form of the liquid to be filled into the sprayer is the same as that described above for nasal drops.

[0508] Alternatively, the pharmaceutical composition may be in powder form and filled into a sprayer to prepare a powder nasal spray formulation. In this case, the sprayer into which the pharmaceutical composition of the present invention is filled is not particularly limited, and for example, a sprayer used for powder nasal spray formulations for the treatment of allergic rhinitis can be used. Furthermore, the powder of the pharmaceutical composition to be filled into the sprayer can be produced by freeze-drying, spray-drying, or the like. When the pharmaceutical composition is formulated as a powder nasal spray, optional excipients may be added, such as glucose, sucrose, lactose, and fructose; starch or starch derivatives; oligosaccharides such as dextrin, cyclodextrin, and derivatives thereof; polyvinylpyrrolidone, alginic acid, tylose, silicic acid, cellulose, cellulose derivatives (e.g., cellulose ethers); sugar alcohols such as mannitol, sorbitol, arabinose, ribose, mannose, sucrose, trehalose, maltose, and dextran; calcium carbonate, calcium phosphate, lactose, lactitol, dextrates, dextrose, and maltodextrin.

[0509] When the pharmaceutical composition is in the form of a nasal mucosal injection, which is a preparation for nasal administration, the specific embodiments thereof are the same as those described above for the specific embodiments of the injection. When the pharmaceutical composition is in the form of an ointment, which is a preparation for nasal administration, the specific embodiments thereof are the same as those described above for the eye ointment. When the pharmaceutical composition of the present invention is made into a gel preparation, it can be made into a gel by adding a thickener. For other specific embodiments, the explanation for the specific embodiments of the nasal drops described above is applicable.

[0510] [5] Design method The present invention also relates to a method for designing a pharmaceutical composition, which comprises a step of selecting a pharmaceutical additive to be combined with a substance selected as an active ingredient by the above-described screening method.

[0511] The practitioner of the design method of the present invention may implement the above-described screening method himself or herself, or may implement the invention of the design method based on the results of another person implementing the above-described screening method. That is, one embodiment of the present invention includes a screening step of screening for an active ingredient by the above-mentioned screening method. The embodiment of the screening step is as described above. In one embodiment of the present invention, the design method invention is carried out using a substance that has already been determined to be an effective ingredient by the above-mentioned screening method.

[0512] According to the present invention, regenerative medicines and / or anticancer drugs can be designed. In a preferred embodiment of the present invention, the method includes a step of selecting an indication for the pharmaceutical composition, such as a disease, disorder, or illness of the nervous system, heart, or pancreas, or a symptom thereof, or cancer, the specific details of which are as detailed above.

[0513] A preferred embodiment of the present invention includes a step of selecting a dosage form of the pharmaceutical composition. In a preferred embodiment of the present invention, a liquid is selected as the dosage form, and an aqueous medium to be mixed with the active ingredient is selected. In the present invention, an injection can be selected as the dosage form. In the present invention, eye drops can be selected as the dosage form. In the present invention, a lyophilized formulation can be selected as the dosage form. In the present invention, an ointment can be selected as the dosage form, and a base to be mixed with the active ingredient can be selected. An eye ointment can also be selected as the ointment. In the present invention, a nasal administration formulation can be selected as the dosage form. In the present invention, an oral administration formulation can be selected as the dosage form.

[0514] The explanation of the manufacturing method of the present invention described above can be applied as is to the pharmaceutical additives to be combined with the substance selected as the active ingredient in the screening, the dosage form of the pharmaceutical composition, and the like.

[0515] [6] Clinical trial methods The present invention also relates to a method comprising designing a pharmaceutical composition by the above-described method, preparing a substance selected as an active ingredient by the above-described screening method, producing a pharmaceutical composition by the above-described production method, and statistically processing a data set obtained from people to whom the pharmaceutical composition has been administered in a clinical trial. Implementation of the method of the present invention is essential for pharmaceuticals containing active ingredients screened by the above-mentioned screening method to be approved by regulatory authorities and commercialized.

[0516] In one embodiment of the present invention, data sets obtained from individuals administered the pharmaceutical composition (test group) and individuals administered a control drug (control group) in a clinical trial are compared and statistically processed. The data sets obtained from the test group and the control group are then statistically processed to determine whether there is a statistically significant difference between the two groups.

[0517] In one embodiment of the present invention, the present invention relates to a method comprising statistically processing a data set obtained from healthy subjects to whom the pharmaceutical composition has been administered. This embodiment relates to a clinical trial called a "Phase 1 clinical trial" in Japanese pharmaceutical practice. The acquired data sets include data sets such as the dosage, number of administrations, administration time, administration period, blood concentration of the active ingredient after administration or its time course, and concentration of the active ingredient or its metabolite in urine.

[0518] In one embodiment of the present invention, the present invention also relates to a method comprising statistically processing a data set obtained from patients having an indication for the pharmaceutical composition to which the pharmaceutical composition has been administered. This embodiment relates to a "Phase 2 clinical trial" or a "Phase 3 clinical trial" in Japanese pharmaceutical practice. The data sets to be acquired include the dosage, number of doses, time of administration, duration of administration, blood concentration of the active ingredient after administration or its time course, concentration of the active ingredient or its metabolite in urine, or physiological data regarding safety, side effects, and efficacy for the indicated conditions.

[0519] The statistical processing is preferably performed by computer, and the specific method of statistical processing can be appropriately selected based on the test plan of the clinical trial.

[0520] [7] Pharmaceutical composition The present invention also relates to a pharmaceutical composition containing an active ingredient screened by the above-mentioned screening method, a pharmaceutical composition produced by the above-mentioned production method, and a pharmaceutical composition designed by the above-mentioned design method. The pharmaceutical composition of the present invention will be described in detail below.

[0521] The target animal of the pharmaceutical composition of the present invention is not particularly limited, but vertebrates are preferred. Specifically, the present invention can be applied to any species of mammals, birds, reptiles, amphibians, and fish. More specifically, the target of the present invention can be, for example, humans or non-human animals other than humans. Examples of non-human animals include fish such as zebrafish, and non-human mammals such as mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, and guinea pigs.

[0522] The pharmaceutical composition of the present invention has the effect of promoting the self-replication of undifferentiated cells, and therefore can be used for the self-replication of undifferentiated cells. The pharmaceutical composition of the present invention is used for the self-renewal of adult stem cells. The pharmaceutical composition of the present invention is used for the self-renewal of adult stem cells in one or more tissues selected from the nervous system, heart, and pancreas.

[0523] Furthermore, the pharmaceutical composition of the present invention has the effect of inducing differentiation of undifferentiated cells into various cells, and therefore can be used for inducing differentiation of undifferentiated cells. Furthermore, the pharmaceutical composition of the present invention can be used to induce differentiation of adult stem cells. Furthermore, the pharmaceutical composition of the present invention can be used to induce differentiation of adult stem cells in one or more tissues selected from the nervous system, heart, and pancreas.

[0524] The pharmaceutical composition of the present invention is used for either or both of two purposes: "self-replication of undifferentiated cells" and "induction of differentiation of undifferentiated cells." In a preferred embodiment of the present invention, the present invention is used for both "self-replication of undifferentiated cells" and "induction of differentiation of undifferentiated cells."

[0525] In conventional regenerative medicine processes, the self-renewal process and the differentiation induction process are carried out in vitro (in a petri dish or in a test tube). The pharmaceutical composition of the present invention can achieve these two steps in vivo, i.e., the present invention can be used to induce self-renewal and differentiation of undifferentiated cells in vivo.

[0526] In one embodiment of the present invention, the drug is a regenerative medicine and / or an anti-cancer drug. One embodiment of the present invention is a regenerative medicine that induces self-replication and differentiation of undifferentiated cells. In one embodiment of the present invention, the pharmaceutical composition is used for inducing differentiation of cancer cells into normal cells. In a more preferred embodiment, the pharmaceutical composition is for cancer treatment by inducing differentiation of cancer cells into normal cells.

[0527] The pharmaceutical composition of the present invention can promote the self-renewal of undifferentiated neuronal cells and subsequently promote the differentiation of the expanded undifferentiated neuronal cells. In other words, the present invention can realize the regeneration or enhancement of the nervous system, which has been considered impossible or extremely difficult to regenerate until now.

[0528] As described above, the pharmaceutical composition of the present invention can induce differentiation into various cells that constitute the nervous system. That is, the pharmaceutical composition of the present invention can induce differentiation of undifferentiated neural cells into neurons and glial cells.

[0529] The pharmaceutical composition of the present invention can be used to induce differentiation of undifferentiated neural cells, thereby enabling proliferation of mature neural cells such as glutaminergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, and cholinergic neurons. Furthermore, the pharmaceutical composition of the present invention can be used for the proliferation of retinal ganglion cells and Purkinje cells.

[0530] The pharmaceutical composition of the present invention can be used to induce differentiation of undifferentiated neural cells, thereby promoting the proliferation of glial cells such as astrocytes (star-shaped cells), oligodendrocytes (oligodendrocytes), ependymal cells, Schwann cells (theca cells), and satellite cells.

[0531] The pharmaceutical composition of the present invention may be used in either the central nervous system or the peripheral nervous system, and in either nervous system, the pharmaceutical composition of the present invention exerts the effect of inducing self-renewal and / or differentiation of undifferentiated neural cells. Once damaged, the central nervous system does not regenerate or is extremely difficult to regenerate. Therefore, the pharmaceutical composition of the present invention is preferably used for inducing self-renewal and / or differentiation of undifferentiated neural cells in the central nervous system.

[0532] The central nervous system to which the present invention can be suitably applied includes the brain, spinal cord, optic nerve, and olfactory nerve. In particular, since damage to the brain, spinal cord, or optic nerve can lead to serious diseases that significantly reduce the patient's quality of life, it is preferable to use the pharmaceutical composition of the present invention for inducing self-renewal and / or differentiation of undifferentiated neural cells in the brain, spinal cord, or optic nerve.

[0533] As described above, the pharmaceutical composition of the present invention exerts the effect of inducing self-renewal and / or differentiation of undifferentiated neural cells, thereby enabling the regeneration of the nervous system, which is said to be impossible or extremely difficult to achieve. Due to this action, the present invention exerts a therapeutic or preventive effect when applied to nervous system disorders, disorders, or diseases, or symptoms thereof. That is, the pharmaceutical composition of the present invention is preferably used for treating or preventing nervous system disorders, disorders, or diseases, or symptoms thereof.

[0534] The present invention is effective for treating both central and peripheral nervous system disorders. Diseases of the central nervous system include diseases of the brain, spinal cord, optic nerve and / or olfactory nerve. Diseases of the peripheral nervous system include diseases of the somatic nervous system and autonomic nervous system.

[0535] Diseases of the central nervous system include Parkinson's disease, Alzheimer's disease, Creutzfeldt-Jakob disease, corticobasal degeneration, amyotrophic lateral sclerosis, multiple sclerosis, progressive motor weakness, immune-mediated neuropathies, central nervous system injuries such as brain injury, spinal cord injury, optic nerve injury, and olfactory nerve injury, Alzheimer's disease with parkinsonism, bradykinesia, akinesia, movement disorders that impair fine motor control and manual dexterity, dysphonia, monotonous speech, rigidity, dystonia, and inflammation associated with Parkinson's disease. ; tremors of the face, jaw, tongue, or posture; parkinsonian gait; shuffling gait; shuffling gait; accelerated gait; mood, cognitive, sensory, or sleep disorders; dementia; depression; drug-induced parkinsonism; vascular parkinsonism; multiple system atrophy; progressive supranuclear palsy; disorders with primary tau pathology; corticobasal ganglionic degeneration; parkinsonism with dementia; hyperkinetic disorder; chorea; Huntington's disease; dystonia; Wilson's disease; Tourette's syndrome; essential tremor; myoclonus; tardive movement disorder; schizophrenia; bipolar disorder, and autism spectrum disorder.

[0536] Diseases of the peripheral nervous system include motor disorders, which are characterized by symptoms such as "weakness in the hands and feet," "frequent dropping of things," "trouble walking or running," "trouble standing up," and "trending of the toes, making one prone to tripping"; sensory disorders, which are characterized by symptoms such as "tingling and numbness," "tingling pain," and "loss of feeling" in the hands and feet; and autonomic nervous system disorders, which are characterized by symptoms such as "cold skin in the hands and feet" and "lack of sweating in the lower body."

[0537] The pharmaceutical composition of the present invention has the effect of inducing regeneration of the nervous system by promoting the self-renewal and / or differentiation induction of undifferentiated neural cells, and is therefore effective for diseases caused by damage or functional impairment of the nervous system.

[0538] Furthermore, the pharmaceutical composition of the present invention is effective for diseases caused by damage or functional decline of nerve cells or glial cells. Furthermore, the pharmaceutical composition of the present invention is effective for diseases caused by damage or functional impairment of cell bodies, myelin sheaths, axons, and neuromuscular junctions. The pharmaceutical composition of the present invention is effective for diseases caused by damage or functional decline of mature neurons such as glutaminergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, and cholinergic neurons. Furthermore, the pharmaceutical composition of the present invention is effective for diseases caused by damage or decreased function of retinal ganglion cells or Purkinje cells. The pharmaceutical composition of the present invention is effective for diseases caused by damage or impaired function of glial cells such as astrocytes (star-shaped glial cells), oligodendrocytes (oligodendrocytes), ependymal cells, Schwann cells (theca cells), and satellite cells. The term "decreased function" as used herein includes not only a state in which the function of the cells themselves is reduced, but also a case in which the function of the entire tissue is reduced due to a decrease in the number of cells.

[0539] Glaucoma and spinal cord injury, among the indications of the present invention, will be described in more detail below.

[0540] The pharmaceutical composition of the present invention is effective for treating or preventing glaucoma. The present invention is effective for both high-tension glaucoma and normal-tension glaucoma.

[0541] High-tension glaucoma is a disease in which high intraocular pressure gradually causes the retinal optic nerve to atrophy, swell, and become damaged, leading to the death of retinal ganglion cells, a type of nerve cell present in the retina, through apoptosis. This then causes the optic nerve head to collapse, gradually narrowing the field of view and ultimately leading to loss of vision. Normal-tension glaucoma is a disease that clinically exhibits specific optic nerve lesions and is characterized by the comprehensive presence of all five clinical findings: (1) intraocular pressure within the normal range of 10 to 21 mmHg, (2) peripheral narrowing and depression of the optic disc, (3) retinal optic nerve fiber layer defects, (4) optic nerve deformation and posterior deviation at the lamina cribrosa (orbital lamina), and (5) a decrease in optic ganglion cells and glial cells. The pharmaceutical composition of the present invention exerts a therapeutic effect by regenerating damaged or reduced optic nerves (retinal ganglion cells, glial cells) observed in both high-tension and normal-tension glaucoma.

[0542] The spinal cord does not regenerate spontaneously, so once the spinal cord is damaged, it is impossible to completely repair it. The pharmaceutical composition of the present invention has a regenerative effect on the nervous system. Therefore, the pharmaceutical composition of the present invention is highly suitable for treating spinal cord injury. The pharmaceutical composition of the present invention brings about nerve regeneration at the site of spinal cord injury, and can improve or completely cure motor disorders and the like associated with spinal cord injury.

[0543] Spinal cord injuries to which the pharmaceutical composition of the present invention can be applied include those caused by trauma due to accidents such as traffic accidents, sports accidents, and falls, as well as those caused by diseases such as spinal cord tumors and hernias.

[0544] The pharmaceutical composition of the present invention is preferably administered to or near the site where undifferentiated neural cells are present in a living body, and such administration allows the pharmaceutical composition of the present invention to efficiently induce self-replication and differentiation of undifferentiated neural cells. The term "vicinity" refers to the range within which the active ingredient contained in the pharmaceutical composition can reach the location of undifferentiated neural cells by diffusion or dispersion through body fluids.

[0545] The pharmaceutical composition of the present invention is preferably administered to or near the site where undifferentiated neural cells of the central nervous system are present in a living body. By using such an administration form, the effect of the pharmaceutical composition of the present invention is to efficiently induce self-replication and differentiation of undifferentiated neural cells of the central nervous system.

[0546] The pharmaceutical composition of the present invention is preferably administered to the brain, spinal cord, or eyeball, and such an administration form can achieve regeneration or neogenesis of the brain, spinal cord, or optic nerve.

[0547] The stem cells responsible for neurogenesis in the adult brain are called adult neural stem cells, and are known to exist in at least two locations: the subventricular zone surrounding the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus. Therefore, the pharmaceutical composition of the present invention may be administered to the lateral ventricle and / or hippocampus where adult neural stem cells are present. More specifically, the pharmaceutical composition of the present invention may be administered to the subventricular zone surrounding the lateral ventricles and / or the subgranular zone of the dentate gyrus of the hippocampus.

[0548] The pharmaceutical composition of the present invention is preferably administered to a site of nervous system damage or functional decline, thereby enabling efficient regeneration of the nervous system at the site of damage or functional decline, and efficient treatment of diseases, etc.

[0549] Furthermore, the pharmaceutical composition of the present invention is preferably administered to a site where damage or functional decline of nerve cells or glial cells is observed, thereby enabling efficient regeneration of nerve cells or glial cells at the site and efficient treatment of diseases, etc.

[0550] Furthermore, the pharmaceutical composition of the present invention is preferably administered to a site where damage or functional impairment of a cell body, myelin sheath, axon, or neuromuscular junction is observed, thereby enabling efficient regeneration of the damaged or functionally impaired cell body, myelin sheath, axon, or neuromuscular junction, and efficient treatment of diseases, etc.

[0551] The pharmaceutical composition of the present invention is preferably administered to a site where damaged or impaired function of mature neurons such as glutaminergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, and cholinergic neurons is observed, thereby enabling efficient regeneration of damaged or impaired mature neurons and efficient treatment of diseases, etc.

[0552] Furthermore, the pharmaceutical composition of the present invention is preferably administered to a site where damage or decreased function of retinal ganglion cells or Purkinje cells is observed, thereby enabling efficient regeneration of damaged or decreased function of retinal ganglion cells or Purkinje cells and efficient treatment of diseases, etc.

[0553] The pharmaceutical composition of the present invention is preferably administered to a site where damage or impaired function of glial cells such as astrocytes, oligodendrocytes, ependymal cells, Schwann cells (theca cells), and satellite cells is observed, thereby enabling efficient regeneration of damaged or impaired glial cells and efficient treatment of diseases, etc.

[0554] Furthermore, the pharmaceutical composition of the present invention can be used to treat diseases caused by damage or functional decline of cardiomyocytes. The pharmaceutical composition of the present invention acts on cardiac stem cells / cardiac progenitor cells present in the heart, inducing their self-renewal and differentiation, thereby enabling the regeneration of damaged or functionally declined cardiomyocytes. The pharmaceutical composition of the present invention can be used to treat heart diseases such as angina pectoris, myocardial infarction, valvular disease, cardiomyopathy, atrial septal defect, cardiac tumor, heart failure, and arrhythmia. When the pharmaceutical composition of the present invention is used to treat heart diseases and the like, it can be administered in the form of local administration to the heart, oral administration, injection into a blood vessel, intravenous injection, or the like.

[0555] Furthermore, the pharmaceutical composition of the present invention can be used to treat diseases caused by damage or functional impairment of pancreatic cells. Examples of pancreatic cells include insulin-secreting β cells. The pharmaceutical composition of the present invention acts on pancreatic stem cells / progenitor cells present in the pancreatic duct, inducing their self-renewal and differentiation, thereby enabling the regeneration of damaged or functionally impaired pancreatic cells. The pharmaceutical composition of the present invention can be used to treat pancreatic diseases such as type 1 diabetes, type 2 diabetes, acute pancreatitis, chronic pancreatitis, pancreatic cancer, and mucus-producing tumors. When the pharmaceutical composition of the present invention is used to treat heart disease or the like, it can be administered by local administration to the pancreas, oral administration, injection into a blood vessel, intravenous injection, or the like.

[0556] The pharmaceutical composition of the present invention can be used to treat blood cancers, including leukemias such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia; Hodgkin lymphomas such as classical Hodgkin lymphoma and nodular lymphocyte-predominant Hodgkin lymphoma; B-cell lymphoblastic leukemia / lymphoma; T-cell lymphoblastic leukemia / lymphoma; chronic lymphocytic leukemia / small lymphocytic lymphoma; follicular lymphoma; MALT lymphoma; lymphoplasmacytic lymphoma; Malignant lymphomas such as idiopathic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, and lymphoma of the skin (mycosis fungoides, Sézary syndrome); multiple myelomas such as symptomatic multiple myeloma, benign monoclonal hypergammaglobulinemia, asymptomatic myeloma, non-secretory myeloma, and solitary plasmacytoma of bone. The pharmaceutical composition of the present invention can be used to induce the differentiation of cancer cells present in the blood into vascular endothelial cells. In one embodiment of the present invention, the present invention can be used to treat blood cancer by inducing the differentiation of cancer cells present in the blood into vascular endothelial cells.

[0557] The dosage is preferably 0.0001 mg or more, more preferably 0.001 mg or more, more preferably 0.01 mg or more, more preferably 0.1 mg or more per kg of body weight per administration, and preferably 10,000 mg or less, more preferably 1,000 mg or less, more preferably 100 mg or less per kg of body weight per administration.

[0558] The dosage, in terms of the weight of the active ingredient, per patient is preferably 0.01 mg or more, more preferably 0.1 mg or more, more preferably 1 mg or more, more preferably 10 mg or more, and preferably 100,000 mg or less, more preferably 10,000 mg or less, more preferably 1,000 mg or less, more preferably 100 mg or less.

[0559] The administration schedule can be adjusted, for example, between 3 times / day and 1 time / month, for example, 1 time / day to 1 time / week, while observing the condition and trends in blood test values.

[0560] Hereinafter, the use of the pharmaceutical composition of the present invention in the treatment or prevention of diseases of the cranial nervous system, the treatment or prevention of glaucoma, and the treatment of spinal cord injury will be described in more detail.

[0561] When treating or preventing diseases of the central nervous system, the pharmaceutical composition of the present invention is preferably administered by intracerebral injection, more specifically, intraventricular injection. In this case, it is preferable to use an intracerebral administration system comprising an injection device equipped with an injection pump and a syringe filled with an injection solution containing the pharmaceutical composition of the present invention, and a ventricular access device connected to the injection device via an extension line. The ventricular access device is inserted into the brain by drilling the skull, and the syringe is inserted to the injection target site in the brain. The infusion pump is then started, and the injection solution is infused at a rate of preferably 0.1 mL / hour to 10 mL / hour, more preferably 1 mL / hour to 5 mL / hour. The intracerebral injection can be carried out preferably once a day to once a month, more preferably once every three days to once every three weeks.

[0562] It is also known that there is a route in the nasal cavity whereby administered substances can be transferred directly to the cerebrospinal fluid or brain via the nasal mucous membrane layer, without passing through the blood. Therefore, in order to allow the active ingredient of the medicament of the present invention to reach the brain, a preferred example is intranasal administration. In this case, the medicament of the present invention can be administered by spraying it into the nasal cavity in the form of a powder nasal spray or a liquid nasal spray. The medicament of the present invention can also be administered in the form of nasal drops by dropping them into the nasal cavity. The medicament of the present invention can also be administered intranasally via a tube or catheter in the form of a gel preparation or ointment. Furthermore, the pharmaceutical agent of the present invention can be administered intranasally in the form of an injection by submucosal injection into the nasal mucosa. The administration rate can be preferably once a day to once a week, more preferably twice a day to once every three days.

[0563] When treating or preventing glaucoma, the pharmaceutical composition of the present invention is preferably in the form of eye drops or eye ointment, which is dropped onto or applied to the eyeball. The administration rate can be preferably once a day to once a week, more preferably twice a day to once every three days.

[0564] When treating spinal cord injury, it is preferable to inject the pharmaceutical composition of the present invention in the form of an injection directly into the site of spinal cord injury. Although the injection can be performed manually intermittently, it is preferable to administer the drug continuously to the injured area using a mini-pump and a catheter connected thereto. Administration is preferably initiated as soon as possible after injury, and administration is preferably continued for at least one week, more preferably at least two weeks, after injury.

[0565] When treating heart disease or the like, the pharmaceutical composition of the present invention may be administered directly to the heart in the form of an injection. Alternatively, the pharmaceutical composition of the present invention may be administered intravenously in the form of an injection. Alternatively, the pharmaceutical composition of the present invention may be administered orally in the form of an oral administration agent.

[0566] When treating pancreatic diseases, the pharmaceutical composition of the present invention may be administered directly to the pancreas in the form of an injection. Alternatively, the pharmaceutical composition of the present invention may be administered intravenously in the form of an injection. Alternatively, the pharmaceutical composition of the present invention may be administered orally in the form of an oral administration agent.

[0567] When the pharmaceutical composition of the present invention is used as a therapeutic agent for blood cancer, it may be administered intravenously as an injection, or orally as an oral administration agent.

[0568] The present invention also relates to pharmaceutical compositions containing, as an active ingredient, a compound encompassed by general formula (I), general formula (II), or general formula (III), or a salt thereof, or a hydrate thereof. Specific embodiments thereof are as described above, and therefore further description will be omitted here. In one embodiment of the present invention, the active ingredient of the present invention is a compound selected from the above-mentioned compounds 1 to 7, or a salt thereof, or a hydrate thereof. Specific embodiments thereof are as described above, and therefore further explanation is omitted here. [Example]

[0569] [Test Example 1] Neural stem cell proliferation test <Animals used> Transgenic zebrafish embryos expressing green fluorescent protein (GFP) specifically in midbrain neural stem cells

[0570] <Test compound> Compound 1: (3R)-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile phosphate Compound 2: 4-[3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoline Compound 3: 2-amino-5-(ethylamino)-5-oxopentanoic acid Compound 4:N 1 -hydroxy-N 8 -phenyloctanediamino Compound 5: 4-[(5-bromopyridin-2-yl)amino]-4-oxobutyric acid Compound 6: 5-chloro-2-N-{4-[4-(dimethylamino)piperidin-1-yl]-2-methoxyphenyl}-4-N-[2-(dimethylphosphoryl)phenyl]pyrimidine-2,4-diamine Compound 7: 2-[[5-chloro-2-[2-methoxy-4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]amino]-N-methylbenzenesulfonamide (Compounds 1 to 7 were used separately in the experiments described below.)

[0571] <Experimental Method> A test compound was dissolved in E3 Ringer (zebrafish physiological saline) to prepare a solution (100 μM). This solution was injected into the ventricle of embryos 24 hours after fertilization under a microscope, and the embryos were cultured at 28.5°C for up to 72 hours after fertilization. In addition, 0.2 mM phenylthiourea (PTU) was added to the culture medium to inhibit the synthesis of melanin pigment, which interferes with observation by fluorescence microscope. For microinjection, embryos were anesthetized with 0.02% MS-222 (tricaine) / E3 ringer, held head-down on a coffin (a plastic plate with a V-shaped groove), and a glass capillary was inserted near the pineal gland. Nitrogen gas pressure (15 picosiemens) was applied for 15–45 ms before injection into the ventricles. At this time, 0.025% (final concentration) of phenol red was mixed as a tracer into the injection solution. The amount of injection was adjusted by applying pulsed gas pressure until the anterior ventricle, diencephalon, and middle ventricle turned a uniform pale red color. As a control experiment, the same amount of E3 Ringer alone was administered intracerebroventricularly. The embryos were cultured for 72 hours after fertilization and then placed on a slide glass in a 3% methylcellulose / E3 Ringer / 0.02% tricaine solution. GFP was then emitted using 488 nm excitation light, and images were taken with a CCD camera (Figures 6-8). The number of samples was 80 in each experiment using each test compound (N=80).

[0572] Brains from individual zebrafish treated with each treatment were dissected, washed with sterile distilled water, and then disrupted in 500 μl of Sepasol-RNA I Super G (Nacalai Tesque) by pipetting or by aspirating with a 1 ml syringe with an 18G–24G needle. Protein was removed once with an equal volume of phenol-chloroform and once with chloroform. The supernatant was purified using an RNA purification column (RNeasy Plus Mini Kit, Qiagen). Elution was reverse transcribed (ReverTra Ace qPCR Kit, TOYOBO) using a mixed primer set of random hexamers and oligo-dT at 37°C for 15 minutes and 98°C for 5 minutes. Quantitative PCR was performed to measure the expression of neural stem cell markers sox1α, sox2, sox3, and her6. Quantitative PCR was performed using SYBER Green Realtime PCR Master Mix (TOYOBO). PCR reactions were performed using an ABI PRISM 7700 or a Roche Diagnostics LightCycler. Quantification was performed in three wells per sample (n=3). The obtained data were compared and quantified using the ΔΔCt method with a normalizer (β-actin, GAPDH, 18S, etc.). To confirm the reliability of the quantitative reaction, the amplification product was confirmed to form a band by agarose electrophoresis.

[0573] <Result> As shown in Figures 6 to 8, in the zebrafish embryos injected with the test compound, the area in which GFP fluorescence, which indicates midbrain neural stem cells, was observed was significantly larger than in the control. This result indicates that the test compounds, Compounds 1 to 7, have the effect of significantly promoting the self-renewal of neural stem cells.

[0574] Furthermore, no tumors were observed in any of the samples, demonstrating that the self-renewal of nerve cells can be promoted without causing cancer, which is one of the risks associated with regenerative medicine.

[0575] Furthermore, quantitative PCR revealed that the expression levels of neural stem cell markers sox1α, sox2, sox3, and her6 were significantly elevated in the brains of zebrafish individuals in which proliferation of undifferentiated neural cells was observed. Figures 9 and 10 show the results of quantitative PCR in individuals administered with compounds 2 and 7.

[0576] [Test Example 2] Proliferation test of differentiated neural cells <Experimental Method> In Test Example 1, the individual to which the test compound was intracerebroventricularly administered was cultured for 40 hours until it became an embryo, and then fixed in 4% paraformaldehyde / phosphate buffered saline (PBS) at room temperature for 90 minutes. The embryos were then immunofluorescently labeled with anti-acetylated tubulin antibody (monoclonal, diluted 1 / 3000), a marker for differentiated neurons, and Alexa 488 anti-mouse IgG secondary antibody, and observed with an excitation light of 488 nm (Figures 11 to 13). The number of samples was 10 in each experiment using each test compound (N=10).

[0577] <Result> The areas where green fluorescence is observed in Figures 11 to 13 are differentiated neurons, more specifically, neuronal cell body clusters (telencephalon and diencephalon) and the axon bundles connecting them. As shown in Figures 11 to 13, in zebrafish embryos injected with the test compound, the range and intensity of the tendency to show differentiated neuronal cell body clusters were significantly greater than in the control. Furthermore, no tumors were observed in any of the samples.

[0578] Considering the results of Test Examples 1 and 2 together, it can be said that the test compounds, Compounds 1 to 7, promote the self-replication of undifferentiated neural cells, thereby inducing differentiation of the proliferated undifferentiated neural cells, and have the effect of proliferating differentiated neural cells.

[0579] Conventional wisdom in regenerative medicine has been that self-renewal and differentiation of undifferentiated cells must be induced by separate factors. However, the results of Test Examples 1 and 2 demonstrate the surprising fact that a single agent can achieve the two processes of self-renewal and differentiation induction of undifferentiated cells (and without causing canceration).

[0580] [Test Example 3] Screening using zebrafish embryos A library of compounds with completely different structures was prepared and screened according to Test Examples 1 and 2. As a result, an increase in undifferentiated neural cells and differentiated neural cells was observed for 37 compounds. Table 1 summarizes the primary effects of compounds confirmed to increase undifferentiated neural cells and differentiated neural cells.

[0581] [Table 1]

[0582] As shown in Table 1, the compounds determined to be active ingredients through screening are concentrated in those that act on signaling pathways involved in stem cell self-renewal, maintenance of totipotency, or differentiation / development induction (Notch signaling pathway, PI3K / AKT / mTOR signaling pathway, JAK / STAT signaling pathway, MAPK signaling pathway, TGFβ / SMAD signaling pathway, and Wnt signaling pathway). These signaling pathways are involved in the control of stem cell self-renewal and differentiation induction.

[0583] Interestingly, previous in vitro studies have shown that compounds believed to maintain or promote stem cell self-renewal and totipotency, and compounds believed to promote differentiation and development, can increase the number of undifferentiated neural cells and differentiated neural cells, respectively, when used alone. These results indicate that in vitro tests only reveal one aspect of stem cell property control, i.e., that in vitro test systems can only observe one side of the mechanism: either "maintenance of stem cell self-renewal and pluripotency" or "induction of differentiation." On the other hand, the in vivo screening system of the present invention using living organisms can provide extremely effective results that reflect the complex signal transduction between cells and tissues.

[0584] [Test Example 4] Regeneration...

Claims

1. A method for screening an active ingredient of a pharmaceutical, comprising: local administration of a candidate substance to or near a region in an animal (excluding human) embryo that develops into a specific tissue; a step of observing the specific tissue or differentiated cells of the specific tissue in the animal after the local administration; selecting, as the active ingredient, a candidate substance that has been observed to improve the function of the specific tissue or the differentiated cells compared to when the candidate substance is not administered; A screening method comprising:

2. The screening method according to claim 1 , wherein the observing step includes observing the motor function of the specific tissue or the amount of secretion of a substance secreted by the specific tissue or the differentiated cell.

3. 3. The screening method according to claim 1 or 2, which is a screening method for an active ingredient for treating or preventing a disease, disorder, or illness of the nervous system, heart, or pancreas, or symptoms thereof.

4. The screening method according to any one of claims 1 to 3, wherein the specific tissue is the nervous system, the heart, or the pancreas.

5. The screening method according to claim 3 or 4, wherein the nervous system is the central nervous system.

6. The method of claim 5, wherein the central nervous system is the brain, spinal cord, or optic nerve.

7. the step of locally administering comprises locally administering the candidate substance to or near a region that will develop into a heart in an embryo of the animal; the observing step includes observing the motor function of the heart. The screening method according to claim 1 or 2.

8. the step of locally administering comprises locally administering the candidate substance to or near a region that will develop into a pancreas in an embryo of the animal; the observing step includes observing the amount of insulin produced or secreted by β cells. The screening method according to claim 1 or 2.

9. The screening method according to any one of claims 1 to 8, which is a screening method for active ingredients of regenerative medicine.

10. The screening method according to any one of claims 1 to 9, wherein the animal is a fish.

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