WO2022065518A1 - Neurite outgrowth promoter - Google Patents
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- WO2022065518A1 WO2022065518A1 PCT/JP2021/036361 JP2021036361W WO2022065518A1 WO 2022065518 A1 WO2022065518 A1 WO 2022065518A1 JP 2021036361 W JP2021036361 W JP 2021036361W WO 2022065518 A1 WO2022065518 A1 WO 2022065518A1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/18—Sulfonamides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
Definitions
- the present invention relates to a neurite outgrowth promoter and its use. More specifically, the present invention comprises one or more compounds selected from levamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof.
- the present invention relates to agents and their therapeutic uses for autism spectrum disorders.
- Pervasive developmental disorder is a type of neurodevelopmental disorder that is thought to be caused by the subtle abnormalities of the natural brain. Pervasive developmental disorders were subdivided according to symptoms, and were classified into five categories: autism, Asperger's syndrome, childhood disintegrative disorder, unspecified pervasive disorder, and Rett's disorder. However, although there are differences such as intellectual development between Asperger's syndrome and autism, it may be difficult to draw a line because of the common characteristics of "difficulty in communication" and "difficulty in social adaptation due to strong commitment”. Therefore, they are now regarded as different degrees of autism rather than another disorder, and the 2013 revision of the American Psychiatric Association Diagnostic Criteria DSM-5 excludes let disorders. Four were integrated into one diagnostic name as "Autism Spectrum Disorder".
- ASD Autism spectrum disorders
- risperidone (trade name: Risperdal) and aripiprazole (trade name: Abilify) for irritability, which is a peripheral symptom
- drugs that improve core symptoms include rapamycin (trade name: Laparimus) for social interaction disorders, oxytocin nasal spray (trade name: syntocinone) for interpersonal interaction disorders, and peritoxyphyllin for communication disorders.
- rapamycin trade name: Laparimus
- oxytocin nasal spray (trade name: syntocinone) for interpersonal interaction disorders
- peritoxyphyllin for communication disorders.
- Trental tetrahydrobiopterin for social and speech disorders
- Biopten tetrahydrobiopterin for social and speech disorders
- levamipid a dozen or so trace drugs including levamipid (trade name: Mucosta) are added for the purpose of improving immune function.
- Rifabutin an autism ameliorating agent (Patent Document 1), focused on the inhibition of nerve transmission by a neurotoxin produced by Clostridium infection as a cause of autism / ASD, and aimed at eradicating the causative bacteria.
- Autism / ASD treatment / preventive agent containing antibiotics such as (Patent Document 2), and breast cancer resistant proteins such as sotalol in addition to active ingredients for the treatment of neurological conditions including autism.
- BCRP BCRP
- Patent Document 3 A composition (Patent Document 3) that can increase the therapeutic effect by blending an inhibitor to inhibit the excretion of the active ingredient has been proposed.
- rebamipide, rifabutin and sotalol have a neurite outgrowth-promoting effect.
- ASD therapeutic agents or their candidate agents are all found among the drugs already approved as therapeutic agents for other diseases.
- DR drug repositioning
- iPS cells artificial pluripotent stem cells
- Dup15q-iPSC Studies using Dup15q-iPSC have been reported to show phenotypes such as variability in the expression of genes known to be associated with autism and epilepsy, and electrophysiological abnormalities.
- neurite length is shortened in nerve cells derived from Dup15q-iPSC, and it is related to the projection of nerve cells (GO term: Neuron projection development). It is not known that the expression of the gene group is increased.
- the present inventors have so far used disease-specific human iPS cell-derived nervous system cells to treat various neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD).
- ALS amyotrophic lateral sclerosis
- AD Alzheimer's disease
- An object of the present invention is to prepare nerve cells from iPS cells derived from an ASD patient, identify the cells characteristic of the ASD and the molecular phenotype, and use recovery from the phenotype as an index for ASD.
- ASD treatment as a realistic drug using existing drugs that are effective for the phenotype and have been confirmed to be safe for humans, while searching for new drug discovery targets that can exert therapeutic effects. To accelerate the development of agents.
- the present inventors prepared nerve cells (Dup15q nerve cells) from iPS cells established from patients with Dup15q syndrome, many of whom have ASD, and investigated the cells and molecular phenotype. rice field.
- Dup15q neurons showed a cell phenotype of shortening neurite length.
- RNA-seq analysis it was clarified that in Dup15q neurons, the expression of genes (with GO term: Neuron projection development) related to the projection of neurons was significantly changed. Consistent with the cellular phenotype of shortening of neurites.
- the present inventors screened compounds expected to have anti-ASD activity from the compound library of FDA-approved drugs using Dup15q neurons, using the promotion of neurite outgrowth as an index. As a result, it was revealed that rebamipide, sotalol and rifabutin remarkably promote the elongation of neurites of Dup15q neurons.
- levamipide and sotalol reduced the gene expression of nerve protrusion elongation inhibitory molecules such as RTN4, SEMA6A, and DCC, but refabutin had a reducing effect. Did not show.
- rifabutin was found to reduce the expression of the UBE3A gene located within the 15q11-13 region. As a result of further research based on these findings, the present inventors have completed the present invention.
- a neurite outgrowth promoter comprising one or more compounds selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof.
- the agent according to [1] which comprises levamipide or a salt thereof and / or sotalol or a salt thereof.
- the agent according to [1] which comprises rifabutin or a salt thereof.
- Autism spectrum disorders are associated with or non-symptomatic autism selected from the group consisting of Dup15q syndrome, Fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome and Clefstra syndrome.
- the agent according to [4] which is a spectrum disorder.
- the agent according to [4], wherein the autism spectrum disorder is accompanied by Dup15q syndrome.
- a method for promoting nerve projection elongation in a subject which is selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and refabutin or a derivative thereof or a salt thereof.
- a method comprising administering to the subject an effective amount of a compound.
- [7a] One or more compounds selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and refabutin or a derivative thereof or a salt thereof for use in promoting neurite outgrowth.
- [7b] A compound selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and refabutin or a derivative thereof or a salt thereof for the production of a neurite outgrowth promoter. use.
- the method according to [7] wherein the subject has an autism spectrum disorder.
- a screening method for therapeutic agents for autism spectrum disorders (1) A step of contacting a test substance with a nerve cell that has been induced to differentiate from an induced pluripotent stem cell derived from a patient with autism spectrum disorder. (2) One or more genes selected from the group consisting of RNT4, SEMA6A and DCC in the nerve cell, and / or a step of measuring the expression level of the UBE3A gene, and (3) one or more genes in step (2).
- a method comprising the step of selecting a test substance having a reduced expression level as a candidate for a therapeutic agent for autism spectrum disorder.
- a therapeutic agent for autism spectrum disorders which comprises one or more genes selected from the group consisting of RNT4, SEMA6A and DCC, or an agent for suppressing the expression of the UBE3A gene.
- neurite outgrowth can be promoted by administering levamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, or rifabutin or a derivative thereof or a salt thereof to a subject, and ASD. It can improve diseases associated with shortening of neurites such as. Since data on the safety of these drugs have been accumulated, it is expected that the development period for clinical application can be shortened. Further, according to the screening method of the present invention, by using the expression of RTN4, SEMA6A, DCC or UBE3A as an index, candidate substances for ASD therapeutic agents can be efficiently screened.
- iPSCs made from Dup15q patients showed ESC-like morphology and presented pluripotent stem cell markers SSEA4 (red) and NANOG (green). The nuclei were counterstained with DAPI (blue). Scale bar: 100 ⁇ m. The characteristics of iPSC derived from a patient having Dup15q are shown.
- B Probe for D15S10 on 15q11-13 (UBE3A, red), control probe D15Z1 (blue) on the p-arm of chromosome 15, and PML (promyelocytic) on the distal long arm of chromosome 15.
- DNA FISH in iPS cells (Dup15q iPSC) derived from patients with Dup15q using leukemia) (green). Normal chromosome 15 is indicated by a white arrow. Chromosome 15 with duplication is indicated by a yellow arrow. The characteristics of iPSC derived from a patient having Dup15q are shown. (C) Mapping of 15q overlap using array CGH. The characteristics of iPSC derived from a patient having Dup15q are shown. (D) DNA methylation in PWS-ICR and the number of copies of chromosome 15 were assayed by methylation-specific qPCR and genomic qPCR from Dup15q iPSC.
- C Expression of nerve cell markers TUBB3, ELAVL4, MAPT, TBR1, SATB2 and BCL11B. Single cell RNA-seq analysis is shown.
- D Expression of glial cell markers S100B, GFA and GFRA2. Single cell RNA-seq analysis is shown.
- E Expression of neural stem cell markers NES, PAX6 and SOX2 (E). The changes in gene expression in Dup15q neurons are shown.
- A Heat map of genes of Dup15q neurons with increased expression compared to healthy control neurons (P ⁇ 0.05; Wilcoxon test). The changes in gene expression in Dup15q neurons are shown.
- Dup15q Screening of compounds using neurons is shown.
- A Experimental timeline for drug screening for neurite outgrowth inducers. Dup15q Screening of compounds using neurons is shown.
- B High-throughput screen triage strategy. Dup15q Screening of compounds using neurons is shown.
- C 1269 compounds consisting of FDA-approved drugs were screened using Dup15q neurons. Scatter plots show neurite length data treated with each 3 ⁇ M compound. The hit criterion was defined as greater than average + 3SD neurite length (indicated by the red line).
- D Effect of rebamipide, sotalol and rifabutin on neurite length.
- the preparation of iPSC derived from Dup15q patient is shown.
- A G-band analysis of Dup15q and healthy control iPSC. The red asterisk is idic (15). The preparation of iPSC derived from Dup15q patient is shown.
- B Schematic diagram of the overlapping region of chromosome 15 in Dup15q iPSC. BP: Limit point. The neurite length of Dup15q nerve cells is shown. The neurite length was quantified. The neurite lengths from 4 HC strains (HC-3 and HC-4 were derived from the same donor) and 3 Dup15q strains are shown.
- the expression of the differential expression gene is shown. It is a scatter diagram of the differential expression gene. The red and blue dots indicate genes whose expression was significantly increased or decreased in Dup15q neurons as compared to healthy control neurons (Wilcoxson test, P ⁇ 0.05). No change: Black. The x-axis indicates the expression log2 found change, and the y-axis indicates the P value log10. The Gene Ontology of Dup15q neurons is shown.
- A An enriched Gene Ontology term from a gene with increased expression. The data is shown as -log (p-value). The Gene Ontology of Dup15q neurons is shown.
- the data are shown as a fod change of gene expression normalized to a healthy control.
- the Gene Ontology of Dup15q neurons is shown.
- F Expression of the enriched gene in the GO term of "Adherens junction".
- the data are shown as a fod change of gene expression normalized to a healthy control.
- the support data of compound screening is shown.
- the present invention provides a neurite outgrowth promoter (hereinafter, also referred to as "promoter of the present invention”).
- the accelerator contains, as an active ingredient, one or more compounds selected from the group consisting of levamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof.
- neutralrite outgrowth means the elongation of existing neural processes (axons and dendrites) and the growth or expansion of budding of new neuronal processes. Neurite elongation can alter nerve connectivity, resulting in the establishment of new synapses and the reconstruction of existing synapses.
- Rebamipide has a chemical name: (2RS) -2- (4-Chlorobenzoylamino) -3- (2-oxo-1,2-dihydroquinolin-4-yl) pharmaceutical acid and is classified as a gastric mucosa protective factor enhancer. It is an anti-ulcer drug.
- R 1 is a hydrogen atom, lower alkyl, lower alkenyl, lower alkynyl or phenyl lower alkyl
- R 2 is a hydrogen atom, a hydroxyl group or a lower alkoxy
- R 3 is a hydrogen atom or a lower alkyl
- R 4 is a hydrogen atom or group-COR 5
- R 5 is a lower alkyl which may have an amino group or a phenyl lower alkoxycarbonylamino group as a substituent, a cycloalkyl or a halogen atom as a substituent on a phenyl ring, a lower alkyl, (Phenyl group which may have 1 to 3 groups selected from lower alkoxy, nitro and amino).
- the dotted line in is a single bond or a double bond, and the substitution position of this substituent is any of the 3, 4, 5, 6, 7 or 8 positions of the carbostylyl skeleton. Also, the bond between the 3rd and 4th positions of the carbostylyl skeleton indicates a single bond or a double bond. ] Examples thereof include compounds represented by.
- Sotalol is a chemical name: ( ⁇ ) -4 [-(RS) -1-hydroxy-2 (-isotropylamino) ethyl] therapy, and is a sympathetic nerve that contributes to the generation of arrhythmia by ⁇ -receptor blocking (ClassII) action. It has an effect of suppressing an increase in system tension and is used as an arrhythmia therapeutic agent.
- X is a hydrogen atom, hydroxy, amino, lower alkoxy, benzyloxy, halogen atom, lower alkyl or R 2 SO 2 NH-;
- R 1 and R 2 are independently lower alkyl, phenyl, lower alkyl phenyl, halophenyl, lower alkoxyphenyl or benzyloxyphenyl;
- Alk is a C 1-4 alkylene group;
- R4 is a hydrogen atom, lower alkyl or benzyl;
- R 5 is a hydrogen atom or a substituent selected from the group consisting of one or two hydroxyls, carboxyls, aminos, lower alkoxys, benzyloxys, halogen atoms, lower alkyls, methylenedioxy and R2 SO 2 NH.
- sotalol derivatives include the compounds described in Examples of US Pat. No. 3,341,584.
- Rifabutin has the chemical names: (9S, 12E, 14S, 15R, 16S, 17R, 18R, 19R, 20S, 21S, 22E, 24Z) -6,18,20-Trihydroxy-14methoxy-7,9,15, 17,19,21,25-heptamethyl-1'-(2-methylpropyl) -5,10,26-trioxo-3,5,9,10-tetrahydrospiro [9,4- (epoxypentadeca [1,11,13]] trienimino) -2H-furo [2', 3': 7,8] naphtho [1,2-d] imidazole-2,4'-piperidine] -16-yl acetate, which acts on DNA-dependent RNA polymerase.
- Indications include tuberculosis, nontuberculous mycobacteriosis including Mycobacterium avium complex (MAC) disease, and suppression of the onset of disseminated MAC disease in HIV-infected patients.
- MAC Mycobacterium avium complex
- rifabutin examples include rifaximin-based antibiotics such as rifaximin, rifampicin, rifampicin, rifapentine, rifalazil, and bicozamachine.
- Rebamipide or its derivatives, sotalol or its derivatives, and rifabutin or its derivatives shall include not only free forms but also pharmacologically acceptable salts thereof.
- the pharmacologically acceptable salt varies depending on the type of compound, but for example, alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, etc.), aluminum salt, ammonium salt, etc.
- Inorganic base salts such as trimethylamine, triethylamine, pyridine, picolin, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N'-dibenzylethylenediamine and other organic base salts, or hydrochlorides, odors.
- Inorganic acid salts such as hydrobromide, sulfate, hydroiodide, nitrate, phosphate, citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetic acid
- acid addition salts such as salts, maleates, tartrates, methanesulfonates, benzenesulfonates, and organic acid salts such as paratoluenesulfonates.
- Rebamipide or a derivative thereof or a salt thereof hereinafter collectively referred to as “levamipide”
- sotalol or a derivative thereof or a salt thereof hereinafter collectively referred to as “sotalols”
- refabutin or a derivative thereof or If the salt hereinafter collectively referred to as “rebamipides”
- isomers such as optical isomers, steric isomers, positional isomers, and rotational isomers, any one of the isomers may also be used. Mixtures are also included in these agents in the present invention.
- the rebamipides, sotalols and rifabutins may be solvates (eg, hydrates, etc.) or non-solvate (eg, non-hydrates, etc.). It also includes compounds labeled with isotopes (eg, 3 H, 14 C, 35 S, 125 I, etc.) and the like.
- the accelerator of the present invention may contain one compound selected from rebamipides, sotalols and rifabutins, or may contain two or more of them.
- the accelerator of the present invention contains rebamipide or a salt thereof and / or sotalol or a salt thereof.
- Rebamipide can inhibit the expression of neurite outgrowth inhibitory molecules including RNT4, SEMA6A and DCC
- sotalol can inhibit the expression of neurite outgrowth inhibitory molecules including RNT4, SEMA6A.
- the accelerator of the present invention contains rifabutin or a salt thereof.
- Rifabutin is located within the 15q11-13 region and can inhibit the expression of the UBE3A gene, which is upregulated in Dup15q.
- rebamipides, sotalols and rifabutins can be produced by using commercially available products or by methods known per se for each compound.
- rebamipides can be produced, for example, according to the method described in JP-A-59-7168.
- Sotalols can be produced, for example, according to the method described in US Pat. No. 3,341,584.
- Rifabutins can be semi-synthesized from, for example, rifamycin produced by Streptomyces mediaranei by a method known per se.
- the active ingredients of the accelerator of the present invention are nerve cells, preferably subjects having ASD (eg, mammals such as humans, dogs, cats, monkeys, mice, rats, etc., preferably. Can promote neurite outgrowth in nerve cells in humans), so by administration to a subject in which neurite outgrowth is inhibited, preferably a subject with ASD, neurite outgrowth in the subject Diseases involving inhibition can be treated.
- ASD eg, mammals such as humans, dogs, cats, monkeys, mice, rats, etc.
- ASD eg, mammals such as humans, dogs, cats, monkeys, mice, rats, etc.
- ASD eg., mammals such as humans, dogs, cats, monkeys, mice, rats, etc.
- ASD eg.
- ASD eg, mammals such as humans, dogs, cats, monkeys, mice, rats, etc.
- neurite outgrowth in the subject Diseases involving inhibition can be treated.
- treatment includes improvement of symptoms, delay of onset, prevention / delay of recurr
- ASD hereditary syndromes frequently associated with ASD
- ASD hereditary syndromes frequently associated with ASD
- ASD hereditary syndromes frequently associated with ASD
- Cornelia de Lange Syndrome Catless Syndrome, Angelman Syndrome, Down Syndrome, Charge Syndrome, Prader Willy Syndrome, Kleinfelder Syndrome, Phelan McDermid Syndrome, etc. (Parkinson syndrome, etc.)
- spinal cord injury epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.
- mental system diseases eg, psychiatric division disease, depression, etc.
- It is preferably ASD, more preferably ASD with hereditary syndrome preferably Dup15q syndrome, fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome, Clefstra syndrome, more preferably Dup15q syndrome).
- the accelerator of the present invention is an appropriate dosage form obtained by mixing the active ingredients rebamipides, sotalols, rifabutins as they are, or by mixing them with a pharmacologically acceptable carrier, excipient, diluent, etc. It can be administered orally or parenterally as a pharmaceutical composition.
- compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups. Agents, emulsions, suspending agents and the like can be mentioned.
- the composition for parenteral administration for example, injections, suppositories and the like are used, and the injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like.
- the dosage form may be included.
- excipients eg, sugar derivatives such as lactose, sucrose, grape sugar, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Rubber arabic; dextran; organic excipients such as purulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonic acid Carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate), lubricants (eg, metal stearate such as stearic acid, calcium stearate, magnesium stearate; talc; Colloidal silica; bead wax, waxes such as gay wax; boric acid; adipic acid; stearic acid such as sodium sulfate;
- disintegrants eg, cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, internally cross-linked sodium carboxymethyl cellulose; carboxymethyl starch, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone.
- emulsifiers eg, colloidal clays such as bentonite, bee gum; metal hydroxides such as magnesium hydroxide, aluminum hydroxide; sodium lauryl sulfate, calcium stearate.
- Anionic surfactants such as; cationic surfactants such as benzalconium chloride; and nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters.
- Stabilizers paraoxybenzoic acid esters such as methylparaben, propylparaben; alcohols such as chlorobutanol, benzyl alcohol, phenylethyl alcohol; benzalconium chloride; phenol, cresol With additives such as phenols; thimerosal; dehydroacetic acid; and sorbic acid
- flavoring agents eg, commonly used sweeteners, acidulants, flavors, etc.
- diluents and the like Manufactured by a well-known method.
- the doses of rebamipides, sotalols, and rifabutins which are the active ingredients of the accelerator of the present invention, may vary depending on various conditions such as the type of compound, the symptom of the subject to be administered, age, body weight, drug acceptability, and the like.
- the lower limit is 0.1 mg (preferably 0.5 mg) and the upper limit is 1000 mg (preferably 500 mg), and in the case of parenteral administration, the lower limit is 0.01 mg (preferably 500 mg).
- a suitable amount of 0.05 mg) and an upper limit of 100 mg (preferably 50 mg) can be administered to an adult 1 to 6 times a day.
- the dose may be increased or decreased depending on the symptoms.
- the active ingredient has already been put on the market as a drug for diseases other than the above-mentioned diseases, the dose of each compound can be appropriately selected as long as the safety has been confirmed.
- the accelerator of the present invention may be used in combination with other agents such as risperidone, aripiprazole, rapamycin, oxytocin, pentoxifylline, tetrahydrobiopterin and the like.
- the accelerator of the present invention and other agents thereof can be administered simultaneously, sequentially or separately.
- the present invention also provides a screening method for a therapeutic agent for ASD (hereinafter, also referred to as “screening method of the present invention”).
- the screening method of the present invention (1) A step of contacting a test substance with nerve cells induced to differentiate from iPS cells derived from an ASD patient. (2) One or more genes selected from the group consisting of RNT4, SEMA6A and DCC in the nerve cell, and / or a step of measuring the expression level of the UBE3A gene, and (3) one or more genes in step (2). A step of selecting a test substance having a reduced expression level of ASD as a candidate for a therapeutic agent for ASD is included.
- the ASD patient from which the iPS cells used in step (1) are derived is not particularly limited, but is preferably hereditary syndrome (eg, Dup15q syndrome, fragile X syndrome, Let syndrome, nodular sclerosis, Costello syndrome, Clefstra syndrome). , Cornelia de Lange Syndrome, Catless Syndrome, Angelman Syndrome, Down Syndrome, Charge Syndrome, Prader-Willi Syndrome, Kleinfelder Syndrome, Phelan McDermid Syndrome, etc., preferably Dup15q Syndrome, Vulnerable X Syndrome, Let Syndrome, Nodular ASD patients with sclerosis, Costello syndrome, Clefstra syndrome, more preferably Dup15q syndrome).
- hereditary syndrome eg, Dup15q syndrome, fragile X syndrome, Let syndrome, nodular sclerosis, Costello syndrome, Clefstra syndrome.
- tissue stem cells such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells
- tissue precursor cells (2) tissue precursor cells
- Lymphocytes eg, peripheral blood mononuclear cells
- epithelial cells e.g, epithelial cells
- endothelial cells e.g., endothelial cells
- muscle cells e.g., hematopoietic stem cells
- fibroblasts skin cells, etc.
- hair cells hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic cells) Exocrine cells, etc.
- brain cells lung cells, renal cells, fat cells, and other differentiated cells are exemplified.
- iPS cells can be made by introducing specific reprogramming factors into somatic cells in the form of DNA or protein, with properties similar to ES cells, such as pluripotency and self-renewal proliferation.
- the reprogramming factor is a gene specifically expressed in ES cells, its gene product or non-coding RNA, or a gene that plays an important role in maintaining undifferentiated ES cells, its gene product or non-coding RNA, Alternatively, it may be composed of a low molecular weight compound.
- Genes contained in the reprogramming factor include, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, Eras, ECAT15.
- initialization factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 /.
- the method for inducing differentiation of nerve cells from iPS cells is not particularly limited, but is a differentiation induction method by high-density culture on a fibroblast feeder layer (Japanese Patent Laid-Open No. 2008-201792) and a differentiation induction method by co-culture with stromal cells. (SDIA method) (for example, WO2001 / 088100, WO / 2003/042384), differentiation induction method by suspension culture (SFEB method) (WO2005 / 123902), adhered to a culture dish coated after formation of neurosphere, and adhered to the culture dish.
- SDIA method for example, WO2001 / 088100, WO / 2003/042384
- SFEB method suspension culture
- a modified SFEBq method (WO2013 / 108926) in which the additives are appropriately changed and cultured in an arbitrary medium, and a method based on a combination thereof can be used. More specifically, for example, the method described in Examples described later can be used.
- test substance used in the screening method of the present invention is not particularly limited, and examples thereof include proteins, peptides, nucleic acids, inorganic compounds, and organic compounds prepared naturally or synthetically.
- specific examples of the test substance include a peptide library of 3 to 50 amino acid residues, preferably 5 to 20 residues, and a molecular weight of 100 to 2000 prepared using a combinatorial chemistry technique known to those skilled in the art.
- Preferred are 200-800 small molecule organic compound libraries.
- a library of existing pharmaceutical compounds eg, Presswick Chemical Library, which is a compound library of FDA-approved drugs
- the concentration of the test substance to be brought into contact with the nerve cells is not particularly limited, and may be usually about 0.01 ⁇ M to about 100 ⁇ M, preferably 0.1 ⁇ M to 50 ⁇ M.
- the time for contacting the cells with the test substance is not particularly limited and is set in a timely manner, but is, for example, about 5 minutes to 5 days, preferably about 10 minutes to 3 days.
- the test substance can be appropriately dissolved or suspended in a solvent such as water, a buffer such as a phosphate buffer or a Tris buffer, ethanol, acetone, dimethyl sulfoxide (DMSO) or a mixture thereof.
- the gene expression level in step (2) is determined by, for example, designing and synthesizing an appropriate primer set, probe, etc. from the nucleotide sequence information of each gene, and using RNA extracted from nerve cells (eg, total RNA) as a template. It can be measured by using a known RNA quantification method, preferably a quantitative RT-PCR method or the like.
- the expression level of one or more genes selected from the group consisting of RNT4, SEMA6A and DCC in the cells supplemented with the test substance and / or the UBE3A gene was compared with the expression level in the control cells not supplemented with the test substance. If it is statistically significantly reduced, the test substance can be selected as a candidate for an ASD therapeutic agent.
- the hit compound is preferably used as an hereditary syndrome (preferably Dup15q syndrome, Fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome, Clefstra syndrome, etc.). More preferably, it can be selected as a therapeutic drug candidate for ASD with Dup15q syndrome).
- the hit compound can be preferably selected as a therapeutic drug candidate for ASD with Dup15q syndrome.
- the present invention also provides an ASD therapeutic agent comprising one or more genes selected from the group consisting of RNT4, SEMA6A and DCC, or a UBE3A gene expression inhibitor.
- the RNT4, SEMA6A and DCC genes are known to be neurite outgrowth inhibitory molecules, and their expression is increased in neurons derived from iPS cells derived from patients with Dup15q syndrome, as described in Examples below. It is in good agreement with the phenotype of reduced neurite length in the nerve cells. Therefore, agents that suppress the expression of these genes can exert a therapeutic effect on ASD by promoting neurite outgrowth.
- the type of ASD to be treated by the therapeutic agent is not particularly limited, but is preferably hereditary syndrome (preferably Dup15q syndrome, fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome, Clefstra syndrome, and more preferably. Can be ASD with Dup15q syndrome).
- the UBE3A gene is located in the 15q11-13 region and its expression is increased in Dup15q syndrome. Therefore, a drug that suppresses the expression of the gene can restore the etiology associated with Dup15q, promote neurite outgrowth, and exert a therapeutic effect on ASD.
- the type of ASD to be treated by the therapeutic agent is not particularly limited, but is preferably hereditary syndrome (preferably Dup15q syndrome, fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome, Clefstra syndrome, and more preferably. Can be ASD with Dup15q syndrome).
- Examples of the expression inhibitor of one or more genes selected from the group consisting of RNT4, SEMA6A and DCC include the above-mentioned rebamipides or sotalols.
- examples of the UBE3A gene expression inhibitor include the above-mentioned rifabutins.
- examples of the expression-suppressing agent for these genes include antisense nucleic acids for each gene, and inhibitory nucleic acids such as siRNA, shRNA, miRNA, and ribozyme.
- inhibitory nucleic acids can be appropriately designed based on the nucleotide sequence information of the gene encoding each gene, for example, using design software known per se, and easily synthesized using a DNA / RNA automatic synthesizer. can.
- some of these inhibitory nucleic acids are commercially available, and they can also be used.
- a neutralizing antibody against a translation product of each gene can be mentioned.
- These antibodies can be appropriately produced by using a method known per se, or commercially available antibodies can also be used.
- test substance selected by the above-mentioned screening method of the present invention can be mentioned as an expression inhibitor for these genes.
- the RNT4, SEMA6A, DCC or UBE3A gene expression inhibitor can be used alone or mixed with a pharmacologically acceptable carrier, excipient, diluent and the like in the same manner as the above-mentioned accelerator of the present invention.
- a pharmacologically acceptable carrier excipient, diluent and the like in the same manner as the above-mentioned accelerator of the present invention.
- Dup15q-1 GM20562A
- Dup15q-2 GM22571
- int dup 15) lymphoblastoma cell line (LCL)
- Dup15q iPSCs were made from LCL using episome vectors for Oct3 / 4, Sox2, Klf4, L-Myc, Lin28, and dominant negative p53 as previously reported.
- healthy control iPSCs (HC-1, HC-2, HC-3 and HC-4) are prepared from peripheral blood mononuclear cells of healthy individuals, and using StemFit (AK02N or AK03N, Ajinomoto, Tokyo, Japan). The cells were cultured under feeder-free conditions on iMatrix-511 (Nippi, Tokyo, Japan) coated plates. HC-3 and HC-4 were derived from the same donor. Dup15q-3 (NMI005_1-13) iPSCs were made from patients with Dup15q using episome vectors for Oct3 / 4, Sox4, Klf4, L-Myc, Lin28, EBNA1, and dominant negative p53. The background information of iPS cell clones is shown in Table 1.
- PWS-ICR Prader-Willi Syndrome Imprinting Control Region Methylation Analysis and Genomic Copy Count Analysis of Chromosome 15 iPSC Genomic DNA PureLink genomic DNA kit (Invitrogen, Thermo Fisher Scientific, Somerset, NJUS) Purified using according to the vendor's protocol. Genomic DNA was subjected to bisulfite conversion using Methyl Easy Xceed Rapid DNA Bisulfite Modification Kit (Takara, Otsu, Shiga, Japan). The converted DNA was then used as a template for PCR reactions using primers for maternal PWS-ICR and paternal PWS-ICR. Maternal PWS-ICR was standardized for paternal PWS-ICR.
- Purified genomic DNA is Taqman quantitative real-time PCR using primers (Thermo FISHer Scientific, Waltham, MA, USA) for SNORD116-2 on chromosome 15 and the RNase P RNA component H1 gene (RPPH1) on chromosome 14. Used as a template for. The number of copies of chromosome 15 was normalized to the RNase P internal control. The primer list is shown in Table 2.
- iPSCs were dissociated into single cells using Accumax (Innovative Cell Technologies, Inc., San Diego, CA, USA) and V-bottomed 96-well plates for suspension culture (Sumitomo Bakelite). Co., Ltd. Tokyo, Japan) was rapidly reaggregated.
- DFK 5% containing 2 ⁇ M Dolsomorphin (Chemdea LLC, Ridgewood, NJ, USA) and 10 ⁇ M SB431542 (Cayman Chemical, Ann Arbor, MI, USA) in the embryonic body (EB) during the nerve induction phase (day 0-8).
- KSR KnockOut Serum Replacement
- NEAA MEM Non-Essential Amino Acids Solution
- DMEM / Ham's F12 Gib's F12
- 2-mercaptoethanol Sigma-Aldrich
- EB was transferred onto a Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) coated 6-well culture plate and in the patterning phase (8-24 days) 1x N2 supplement (Invitrogen), 2 ⁇ M dolsomorphin, And DFK 10% supplemented with 10 ⁇ M SB431542 (DMEM / Ham's F12 supplemented with 10% KSR, NEAA, L-glutamine, 0.1% 2-mercaptoethanol).
- Neuroprogenitor cells were isolated from the bottom of the plate using Accumax, and during neuromaturation (25-28 days), 1x B27 without Vitamin A (Invitrogen), 1x Glutamax (Invitrogen), 10 ng / ml BDNF, 10 ng / Cultures were performed on Matrigel-coated 96-well or 384-well culture plates in Neurobasal Medium (Invitrogen) supplemented with ml GDNF and 10 ng / ml NT-3.
- Immunostained cells were fixed in PBS in 4% paraformaldehyde for 30 minutes at room temperature, washed with PBS, and in PBS containing 0.1% Triton X-100 and 20% Block Ace (Yukijirushi, Tokyo, Japan). Permeation treatment / blocking at room temperature for 60 minutes. After incubation with the primary antibody in PBS containing 0.1% Triton X100 overnight at 4 ° C., cells were washed 3 times with PBS and incubated with the appropriate secondary antibody for 1 hour at room temperature. BIOREVO BZ-9000 (Keyence, Osaka, Japan), Opera phenix (PerkinElmer, Waltham, MA, USA) or IN Cell Analyzer 6000 (GE Healthcare, Chicago, Illinois) acquired cells, Illinois. The antibodies are listed in Table 3.
- Quantification of neurites iPSCs were differentiated into cortical neurons as described above.
- cells were dissociated into single cells using Accumax and then seeded in a Matrigel coated plate.
- the culture medium used was Neurobasal Medium supplemented with 1x B27 without Vitamin A, 1x Glutamax, 10 ng / ml BDNF, 10 ng / ml GDNF and 10 ng / ml NT-3.
- cells were fixed with 4% PFA and stained with anti- ⁇ III tubulin and DAPI. Stained cells were imaged using Opera Phenix or IN CELL 6000 and neurite length was measured by Harmony High Content Imaging and Analysis Software (PerkinElmer) or IN Cell Developer Tole.
- RNA-seq Three healthy control iPSCs and three Dup15q iPSCs were used. Cortical neurons were differentiated from these iPSCs based on the method described above. On day 28, cells were harvested and single-cell RNA-seq was performed. Single cell transcriptome analysis was performed using the 10X genomics chromium plateform. Cell suspensions were loaded onto a 10X Chromium System (10x Genomics, Pleasanton, CA, USA) according to the manufacturer's instructions for single cell isolation. Single cell transcriptome analysis was performed using the 10X genomics chromium plateform. RNA sequencing libraries were prepared according to the 10X genomics Single Cell 3'v2 protocol.
- the quantification and quality control of the obtained cDNA were performed using the Bioanalyzer High Sensitivity DNA Assay (Aglient).
- a single-cell cDNA library was sequenced using GENEWIZ JAPAN (Saitama, Japan) on the Illumina HiSeq2500 platform. The analysis was performed by Amelieff Co., Inc. (Tokyo, Japan). Leads were processed and aligned to human reference GRCh38 using 10X Genomics Cell Ranger software (v 3.0.1) and visualized by Loope Cell browser (v 3.0.1). Single cell data of neuronal clusters were analyzed using scatter (v1.10.1) and Seurat (v 2.3.0) for gene expression variation analysis.
- Cytotoxicity test Cortical neurons were treated with compound (concentration, 3 ⁇ M or 10 ⁇ M) on day 26 and fixed with 4% PFA on day 28. Cells stained with ⁇ III tubulin antibody and DAPI were imaged using Cell Analyzer 6000, and the number of neurons was quantified by IN Cell Developer toolbox software 1.9 (GE Healthcare) to assess cytotoxicity. ..
- Array comparative genomic hybridization was performed using iPSC genomic DNA, and Dup15q-1 had four duplications of 15q11.2q13.2 (2010254-30562489) and three of 15q11.2q13.2. Overlapping, three duplications of 15q11.2q13.2 (23208842-28535051) for Dup15q-2 and three duplications of 15q11.2q13.2. (22765628-28535051) for Dup15q-3 were confirmed (FIGS. 1C and 6B).
- the methylation status of ICR was analyzed.
- the Dup15q iPSC had about 2 or 3 copies of maternal 15q 11.2-13 and 3 or 4 copies of 15q 11.2-13 (FIG. 1D).
- 1E and Table 5 show the genetic information of chromosome 15 structure and Dup15q iPSC, respectively. These results indicate that the increased copy number on chromosome 15 and DNA methylation in PWS-ICR are consistent with the CNV predicted for iPSC from Dup15q patients.
- Dup15q neurons showed a significant reduction in neurite length Cortical dysfunction is thought to cause central symptoms in ASD. It has also been reported that nerve cells of autistic patients exhibit changes in nerve differentiation ability and nerve cell morphology (for example, neurite length).
- FIG. 2A cortical neurons from iPSC strains using a modified SFEBq (serum-free culture of embryoid body-like aggregates with rapid aggregation) differentiation method (FIG. 2A) and ⁇ III tubulin immunostaining.
- the neuronal differentiation potential and morphology of Dup15q neurons were analyzed using the image of (Fig. 2B). When the differentiation efficiency of the healthy control and the Dup15q neuron was compared, no significant difference was observed (Fig. 2C). It was confirmed that the Dup15q neurons had a significantly shorter neurite length as compared with the healthy control neurons (FIGS. 2D and 7).
- scRNA-seq Single-cell RNA sequencing
- t-SNE t-distributed established neighborhood embedding
- cluster 4 is a neuron cluster having neural cell-specific gene expression
- cluster 8 is a glial cell cluster containing glial cell-specific gene expression
- the other clusters are neural stem cell-specific gene expression. It was found to be a neural stem cell cluster with (FIGS. 3C to E).
- cluster 4 contained TUBB3, ELAVL4, MAPT and TBR1
- cluster 8 contained S100B and GFRA2
- other clusters contained PAX6 and SOX2, respectively.
- Results include UBE3A, HERC2 (HECT and RLD domain-containing E3 ubiquitin protein ligase) and NIPA2 (NIPA magnesium transporter 2) located within chromosome 15q11-13 in Dup15q neurons compared to healthy control neurons. 114 genes with increased expression and 13 genes with decreased expression were found (FIGS. 4A, B and 8). In addition, Gene Ontology (GO) analysis showed that genes with increased expression were enriched in the GO term and other developmental related terms of the Neuron projection development (FIGS. 4C, 9A, and 6). ). The expression level of the gene contained in the GO of Neuron projection development is shown in FIG. 4D.
- RTN4 Reticulon 4
- SEMA6A Semaphorin 6A
- DCC Deleted in colorectal canceler
- Revamipids, sotalols and rifabutins promote neurite outgrowth in nerve cells, especially those in ASD patients, and are therefore useful in the treatment of ASD, especially in the treatment of Dup15q syndrome.
- rebamipide, sotalol, and rifabutin which have already been marketed as drugs for other diseases, have accumulated clinical and non-clinical data such as safety, and develop drugs that can treat ASD at low cost and quickly. There is a possibility that it can be done.
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Abstract
The present invention provides a neurite outgrowth promoter containing at least one compound selected from the group consisting of: rebamipide or a derivative or salt thereof; sotalol or a derivative or salt thereof; and rifabutin or a derivative or salt thereof.
Description
本発明は、神経突起伸長促進剤及びその用途に関する。より詳細には、本発明は、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩から選ばれる1以上の化合物を含有してなる、神経突起伸長促進剤、並びにその自閉症スペクトラム障害の治療用途等に関する。
The present invention relates to a neurite outgrowth promoter and its use. More specifically, the present invention comprises one or more compounds selected from levamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof. The present invention relates to agents and their therapeutic uses for autism spectrum disorders.
(発明の背景)
広汎性発達障害は、生まれつきの脳の微細な異常が原因と考えられている神経発達障害の一種である。広汎性発達障害には症状によって細かな分類があり、自閉症、アスペルガー症候群、小児期崩壊性障害、特定不能の広汎性障害、レット障害の5つに区分されていた。しかし、アスペルガー症候群や自閉症では知能の発達などの違いがあるものの、「コミュニケーションの困難さ」と「強いこだわりによる社会適応の困難さ」という共通の特徴を示し、その線引きが難しい場合があるので、それらを別の障害ではなく、連続した自閉障害の中での程度の差として捉えるようになり、2013年に改訂されたアメリカ精神医学会の診断基準DSM−5では、レット障害を除く4つが「自閉症スペクトラム障害」として1つの診断名に統合された。 (Background of invention)
Pervasive developmental disorder is a type of neurodevelopmental disorder that is thought to be caused by the subtle abnormalities of the natural brain. Pervasive developmental disorders were subdivided according to symptoms, and were classified into five categories: autism, Asperger's syndrome, childhood disintegrative disorder, unspecified pervasive disorder, and Rett's disorder. However, although there are differences such as intellectual development between Asperger's syndrome and autism, it may be difficult to draw a line because of the common characteristics of "difficulty in communication" and "difficulty in social adaptation due to strong commitment". Therefore, they are now regarded as different degrees of autism rather than another disorder, and the 2013 revision of the American Psychiatric Association Diagnostic Criteria DSM-5 excludes let disorders. Four were integrated into one diagnostic name as "Autism Spectrum Disorder".
広汎性発達障害は、生まれつきの脳の微細な異常が原因と考えられている神経発達障害の一種である。広汎性発達障害には症状によって細かな分類があり、自閉症、アスペルガー症候群、小児期崩壊性障害、特定不能の広汎性障害、レット障害の5つに区分されていた。しかし、アスペルガー症候群や自閉症では知能の発達などの違いがあるものの、「コミュニケーションの困難さ」と「強いこだわりによる社会適応の困難さ」という共通の特徴を示し、その線引きが難しい場合があるので、それらを別の障害ではなく、連続した自閉障害の中での程度の差として捉えるようになり、2013年に改訂されたアメリカ精神医学会の診断基準DSM−5では、レット障害を除く4つが「自閉症スペクトラム障害」として1つの診断名に統合された。 (Background of invention)
Pervasive developmental disorder is a type of neurodevelopmental disorder that is thought to be caused by the subtle abnormalities of the natural brain. Pervasive developmental disorders were subdivided according to symptoms, and were classified into five categories: autism, Asperger's syndrome, childhood disintegrative disorder, unspecified pervasive disorder, and Rett's disorder. However, although there are differences such as intellectual development between Asperger's syndrome and autism, it may be difficult to draw a line because of the common characteristics of "difficulty in communication" and "difficulty in social adaptation due to strong commitment". Therefore, they are now regarded as different degrees of autism rather than another disorder, and the 2013 revision of the American Psychiatric Association Diagnostic Criteria DSM-5 excludes let disorders. Four were integrated into one diagnostic name as "Autism Spectrum Disorder".
自閉症スペクトラム障害(以下、「ASD」ともいう。)は、(1)社会的コミュニケーション及び対人相互反応における持続的な欠陥、並びに(2)行動・興味・活動の限定された反復的な様式を中核症状とする複雑な神経発達障害である。最近の疫学調査によると、ASDの有病率は1%を超えると考えられ、近年ますます増加傾向にあり、社会的な問題としても議論されている。ASDは遺伝率が高いことから、発症の原因としてゲノム変異が示唆されているが、原因遺伝子や原因染色体領域の候補は多数存在し、それらの機能的な多様性からも、ASD発症の分子メカニズムを解明することは未だに困難である。
Autism spectrum disorders (hereinafter also referred to as "ASD") are (1) persistent defects in social communication and interpersonal interactions, and (2) limited repetitive modes of behavior, interests, and activities. It is a complicated neurodevelopmental disorder with the core symptom. According to a recent epidemiological survey, the prevalence of ASD is thought to exceed 1%, and has been increasing in recent years, and is being discussed as a social problem. The high heritability of ASD suggests genomic mutations as the cause of the onset, but there are many candidates for the causative gene and the causative chromosomal region, and their functional diversity also suggests the molecular mechanism of the onset of ASD. Is still difficult to elucidate.
現在、ASD治療薬として承認されているのは、周辺症状である易刺激性に対するリスペリドン(商品名:リスパダール)とアリピプラゾール(商品名:エビリファイ)のみである。一方、中核症状を改善する薬剤としては、社会的交流障害に対するラパマイシン(商品名:ラパリムス)、対人場面での相互作用の障害に対するオキシトシン点鼻スプレー(商品名:シントシノン)、コミュニケーション障害に対するペリトキシフィリン(商品名:トレンタール)、社会性と言語障害に対するテトラヒドロビオプテリン(商品名:ビオプテン)等が、臨床もしくは非臨床試験段階にあるが、未だ承認には至っていない。
Currently, only risperidone (trade name: Risperdal) and aripiprazole (trade name: Abilify) for irritability, which is a peripheral symptom, are approved as ASD therapeutic agents. On the other hand, drugs that improve core symptoms include rapamycin (trade name: Laparimus) for social interaction disorders, oxytocin nasal spray (trade name: syntocinone) for interpersonal interaction disorders, and peritoxyphyllin for communication disorders. (Product name: Trental), tetrahydrobiopterin for social and speech disorders (trade name: Biopten), etc. are in the clinical or non-clinical trial stage, but have not yet been approved.
上記以外に、自閉症と免疫機能異常や自己免疫疾患との関連性に着目し、免疫機能の改善を目的とした、レバミピド(商品名:ムコスタ)を含む十数種の微量の薬剤を配合した自閉症改善剤(特許文献1)、自閉症/ASDの一因としてのクロストリジウム感染により産生される神経毒による神経伝達阻害に着目し、該原因菌の除菌を目的とした、リファブチン等の抗生物質を含む自閉症/ASD治療・予防剤(特許文献2)、さらに、自閉症を含む神経学的状態の治療のために、活性成分に加えて、ソタロール等の乳がん耐性タンパク質(BCRP)阻害剤を配合して活性成分の排出を阻害することにより治療効果を増大させ得る組成物(特許文献3)などが提唱されている。しかしながら、レバミピド、リファブチン及びソタロールが神経突起の伸長促進作用を有することは全く知られていない。
In addition to the above, focusing on the relationship between autism and immune dysfunction and autoimmune diseases, a dozen or so trace drugs including levamipid (trade name: Mucosta) are added for the purpose of improving immune function. Rifabutin, an autism ameliorating agent (Patent Document 1), focused on the inhibition of nerve transmission by a neurotoxin produced by Clostridium infection as a cause of autism / ASD, and aimed at eradicating the causative bacteria. Autism / ASD treatment / preventive agent containing antibiotics such as (Patent Document 2), and breast cancer resistant proteins such as sotalol in addition to active ingredients for the treatment of neurological conditions including autism. (BCRP) A composition (Patent Document 3) that can increase the therapeutic effect by blending an inhibitor to inhibit the excretion of the active ingredient has been proposed. However, it is not known at all that rebamipide, rifabutin and sotalol have a neurite outgrowth-promoting effect.
上記のASD治療薬又はその候補薬剤は、いずれも他の疾患の治療薬として既に承認された医薬品の中から見出されたものである。昨今見られる新薬開発研究の行き詰まりを打開する手段の1つとして、ヒトでの安全性と体内動態が実績によって既に確認されている既存薬の中から、新たな薬効を有する薬剤を見つけ出し、実用化につなげていこうというドラッグ・リポジショニング(DR)の研究が盛んに行われている。多くの既存のデータが使用できるので、開発コストを低く抑えることができ、蓄積されたノウハウと材料(周辺化合物など)が存在するなどの更なる利点もある。
The above-mentioned ASD therapeutic agents or their candidate agents are all found among the drugs already approved as therapeutic agents for other diseases. As one of the means to break the deadlock of new drug development research that has been seen in recent years, we have found a drug with new efficacy from existing drugs whose safety and pharmacokinetics in humans have already been confirmed by actual results, and put them into practical use. Research on drug repositioning (DR) is being actively conducted to connect it to. Since a lot of existing data can be used, the development cost can be kept low, and there are further advantages such as the existence of accumulated know-how and materials (peripheral compounds, etc.).
一方、初期化技術を用いて患者由来の細胞より人工多能性幹細胞(iPS細胞)を樹立し、このiPS細胞から病因となる細胞へと分化誘導することで、in vitroで病態の再現ができると考えられている。ASDを合併する種々の遺伝的障害の患者からiPS細胞を樹立し、神経細胞に分化誘導した例は多数報告されている。15番染色体長腕の15q11−13領域の重複を原因とし、知的障害、てんかん、低血圧、統合失調症及び自閉等の症状により特徴づけられる15q重複(Dup15q)症候群患者由来のiPS細胞(Dup15q−iPSC)を用いた研究では、自閉症やてんかんに関連することが既知の遺伝子の発現変動や、電気生理学的異常などの表現型を示すことが報告されている。しかし、健常人由来iPSCから誘導した神経細胞との比較において、Dup15q−iPSCから誘導した神経細胞において神経突起長が短縮することや、神経細胞の投射に関連する(GOターム:Neuron projection developmentを付与された)遺伝子群の発現が増加することは何ら知られていない。
On the other hand, by establishing artificial pluripotent stem cells (iPS cells) from patient-derived cells using reprogramming technology and inducing differentiation from these iPS cells into cells that cause the pathogenesis, the pathological condition can be reproduced in vitro. It is believed that. Many cases have been reported in which iPS cells were established from patients with various genetic disorders associated with ASD and induced to differentiate into nerve cells. IDS cells from patients with 15q duplication (Dup15q) syndrome characterized by symptoms such as intellectual disability, epilepsy, hypotension, schizophrenia and autism due to duplication of the 15q11-13 region of the long arm of chromosome 15 (Dup15q) Studies using Dup15q-iPSC) have been reported to show phenotypes such as variability in the expression of genes known to be associated with autism and epilepsy, and electrophysiological abnormalities. However, in comparison with nerve cells derived from healthy human-derived iPSC, neurite length is shortened in nerve cells derived from Dup15q-iPSC, and it is related to the projection of nerve cells (GO term: Neuron projection development). It is not known that the expression of the gene group is increased.
本発明者らはこれまで、疾患特異的ヒトiPS細胞由来の神経系細胞を用いて、筋萎縮性側索硬化症(ALS)やアルツハイマー病(AD)等の種々の神経変性疾患の治療薬のスクリーニング・薬効評価系を開発し、既存薬の中から有望な候補物質(治療薬シーズ)を見出してきた。
本発明の目的は、ASD患者由来のiPS細胞から神経細胞を作製して、そのASDに特徴的な細胞及び分子的な表現型を同定し、当該表現型からの回復を指標として、ASDに対して治療効果を発揮し得る新規な創薬標的を探索するとともに、当該表現型に対して有効で、かつヒトへの安全性が確認されている既存薬を用い、現実的な医薬品としてのASD治療剤の開発を加速させることである。 The present inventors have so far used disease-specific human iPS cell-derived nervous system cells to treat various neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). We have developed a screening / drug efficacy evaluation system and have found promising candidate substances (therapeutic drug seeds) from existing drugs.
An object of the present invention is to prepare nerve cells from iPS cells derived from an ASD patient, identify the cells characteristic of the ASD and the molecular phenotype, and use recovery from the phenotype as an index for ASD. ASD treatment as a realistic drug using existing drugs that are effective for the phenotype and have been confirmed to be safe for humans, while searching for new drug discovery targets that can exert therapeutic effects. To accelerate the development of agents.
本発明の目的は、ASD患者由来のiPS細胞から神経細胞を作製して、そのASDに特徴的な細胞及び分子的な表現型を同定し、当該表現型からの回復を指標として、ASDに対して治療効果を発揮し得る新規な創薬標的を探索するとともに、当該表現型に対して有効で、かつヒトへの安全性が確認されている既存薬を用い、現実的な医薬品としてのASD治療剤の開発を加速させることである。 The present inventors have so far used disease-specific human iPS cell-derived nervous system cells to treat various neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). We have developed a screening / drug efficacy evaluation system and have found promising candidate substances (therapeutic drug seeds) from existing drugs.
An object of the present invention is to prepare nerve cells from iPS cells derived from an ASD patient, identify the cells characteristic of the ASD and the molecular phenotype, and use recovery from the phenotype as an index for ASD. ASD treatment as a realistic drug using existing drugs that are effective for the phenotype and have been confirmed to be safe for humans, while searching for new drug discovery targets that can exert therapeutic effects. To accelerate the development of agents.
上記の目的を達成すべく、本発明者らは、その多くがASDを有するDup15q症候群患者から樹立したiPS細胞から神経細胞(Dup15q神経細胞)を作製し、その細胞及び分子的な表現型を調べた。その結果、Dup15q神経細胞は、神経突起長の短縮という細胞表現型を示した。シングルセルRNA−seq解析の結果、Dup15q神経細胞では、神経細胞の投射に関連する(GOターム:Neuron projection developmentを付与された)遺伝子群の発現が有意に変動していることが明らかとなり、神経突起の短縮という細胞表現型と一致した。
In order to achieve the above object, the present inventors prepared nerve cells (Dup15q nerve cells) from iPS cells established from patients with Dup15q syndrome, many of whom have ASD, and investigated the cells and molecular phenotype. rice field. As a result, Dup15q neurons showed a cell phenotype of shortening neurite length. As a result of single-cell RNA-seq analysis, it was clarified that in Dup15q neurons, the expression of genes (with GO term: Neuron projection development) related to the projection of neurons was significantly changed. Consistent with the cellular phenotype of shortening of neurites.
さらに、本発明者らは、Dup15q神経細胞を用い、FDA承認薬の化合物ライブラリーの中から、神経突起の伸長促進を指標として、抗ASD活性を有することが期待される化合物をスクリーニングした。その結果、レバミピド、ソタロール及びリファブチンが、Dup15q神経細胞の神経突起の伸長を顕著に促進することが明らかとなった。これらの化合物の神経突起伸長阻害分子の遺伝子発現に及ぼす効果を調べたところ、レバミピドとソタロールは、RTN4、SEMA6A、DCC等の神経突起伸長阻害分子の遺伝子発現を低減させたが、リファブチンは低減効果を示さなかった。一方で、リファブチンは、15q11−13領域内に位置するUBE3A遺伝子の発現を低減させることが分かった。
本発明者らは、これらの知見に基づいてさらに研究を重ねた結果、本発明を完成するに至った。 Furthermore, the present inventors screened compounds expected to have anti-ASD activity from the compound library of FDA-approved drugs using Dup15q neurons, using the promotion of neurite outgrowth as an index. As a result, it was revealed that rebamipide, sotalol and rifabutin remarkably promote the elongation of neurites of Dup15q neurons. When the effects of these compounds on the gene expression of nerve protrusion elongation inhibitory molecules were investigated, levamipide and sotalol reduced the gene expression of nerve protrusion elongation inhibitory molecules such as RTN4, SEMA6A, and DCC, but refabutin had a reducing effect. Did not show. On the other hand, rifabutin was found to reduce the expression of the UBE3A gene located within the 15q11-13 region.
As a result of further research based on these findings, the present inventors have completed the present invention.
本発明者らは、これらの知見に基づいてさらに研究を重ねた結果、本発明を完成するに至った。 Furthermore, the present inventors screened compounds expected to have anti-ASD activity from the compound library of FDA-approved drugs using Dup15q neurons, using the promotion of neurite outgrowth as an index. As a result, it was revealed that rebamipide, sotalol and rifabutin remarkably promote the elongation of neurites of Dup15q neurons. When the effects of these compounds on the gene expression of nerve protrusion elongation inhibitory molecules were investigated, levamipide and sotalol reduced the gene expression of nerve protrusion elongation inhibitory molecules such as RTN4, SEMA6A, and DCC, but refabutin had a reducing effect. Did not show. On the other hand, rifabutin was found to reduce the expression of the UBE3A gene located within the 15q11-13 region.
As a result of further research based on these findings, the present inventors have completed the present invention.
すなわち、本発明は以下の通りのものである。
[1] レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物を含有してなる、神経突起伸長促進剤。
[2] レバミピドもしくはその塩、及び/又はソタロールもしくはその塩を含有してなる、[1]に記載の剤。
[3] リファブチン又はその塩を含有してなる、[1]に記載の剤。
[4] 自閉症スペクトラム障害の治療のための、[1]~[3]のいずれかに記載の剤。
[5] 自閉症スペクトラム障害が、Dup15q症候群、脆弱X症候群、レット症候群、結節性硬化症、コステロ症候群及びクレーフストラ症候群からなる群より選択される疾患を伴うか、あるいは非症候性の自閉症スペクトラム障害である、[4]に記載の剤。
[6] 自閉症スペクトラム障害がDup15q症候群を伴う、[4]に記載の剤。
[7] 対象における神経突起伸長を促進する方法であって、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物の有効量を、該対象に投与することを含む、方法。
[7a]神経突起伸長の促進における使用のための、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物。
[7b]神経突起伸長促進剤の製造のための、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物の使用。
[8] 対象が自閉症スペクトラム障害を有する、[7]に記載の方法。
[8a] 神経突起伸長の促進が自閉症スペクトラム障害の治療のためである、[7a]に記載の化合物。
[8b] 神経突起伸長促進剤が自閉症スペクトラム障害の治療用である、[7b]に記載の化合物。
[9] 自閉症スペクトラム障害の治療薬のスクリーニング方法であって、
(1)自閉症スペクトラム障害患者由来の人工多能性幹細胞から分化誘導した神経細胞に被検物質を接触させる工程、
(2)該神経細胞におけるRNT4、SEMA6A及びDCCからなる群より選択される1以上の遺伝子、並びに/或いはUBE3A遺伝子の発現レベルを測定する工程、及び
(3)工程(2)において1以上の遺伝子の発現レベルを低下させた被検物質を、自閉症スペクトラム障害の治療薬の候補として選択する工程
を含む方法。
[10] RNT4、SEMA6A及びDCCからなる群より選択される1以上の遺伝子、又はUBE3A遺伝子の発現抑制薬を含有してなる、自閉症スペクトラム障害治療剤。 That is, the present invention is as follows.
[1] A neurite outgrowth promoter comprising one or more compounds selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof.
[2] The agent according to [1], which comprises levamipide or a salt thereof and / or sotalol or a salt thereof.
[3] The agent according to [1], which comprises rifabutin or a salt thereof.
[4] The agent according to any one of [1] to [3] for the treatment of autism spectrum disorder.
[5] Autism spectrum disorders are associated with or non-symptomatic autism selected from the group consisting of Dup15q syndrome, Fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome and Clefstra syndrome. The agent according to [4], which is a spectrum disorder.
[6] The agent according to [4], wherein the autism spectrum disorder is accompanied by Dup15q syndrome.
[7] A method for promoting nerve projection elongation in a subject, which is selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and refabutin or a derivative thereof or a salt thereof. A method comprising administering to the subject an effective amount of a compound.
[7a] One or more compounds selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and refabutin or a derivative thereof or a salt thereof for use in promoting neurite outgrowth.
[7b] A compound selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and refabutin or a derivative thereof or a salt thereof for the production of a neurite outgrowth promoter. use.
[8] The method according to [7], wherein the subject has an autism spectrum disorder.
[8a] The compound according to [7a], wherein the promotion of neurite outgrowth is for the treatment of autism spectrum disorders.
[8b] The compound according to [7b], wherein the neurite outgrowth promoter is for the treatment of autism spectrum disorders.
[9] A screening method for therapeutic agents for autism spectrum disorders.
(1) A step of contacting a test substance with a nerve cell that has been induced to differentiate from an induced pluripotent stem cell derived from a patient with autism spectrum disorder.
(2) One or more genes selected from the group consisting of RNT4, SEMA6A and DCC in the nerve cell, and / or a step of measuring the expression level of the UBE3A gene, and (3) one or more genes in step (2). A method comprising the step of selecting a test substance having a reduced expression level as a candidate for a therapeutic agent for autism spectrum disorder.
[10] A therapeutic agent for autism spectrum disorders, which comprises one or more genes selected from the group consisting of RNT4, SEMA6A and DCC, or an agent for suppressing the expression of the UBE3A gene.
[1] レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物を含有してなる、神経突起伸長促進剤。
[2] レバミピドもしくはその塩、及び/又はソタロールもしくはその塩を含有してなる、[1]に記載の剤。
[3] リファブチン又はその塩を含有してなる、[1]に記載の剤。
[4] 自閉症スペクトラム障害の治療のための、[1]~[3]のいずれかに記載の剤。
[5] 自閉症スペクトラム障害が、Dup15q症候群、脆弱X症候群、レット症候群、結節性硬化症、コステロ症候群及びクレーフストラ症候群からなる群より選択される疾患を伴うか、あるいは非症候性の自閉症スペクトラム障害である、[4]に記載の剤。
[6] 自閉症スペクトラム障害がDup15q症候群を伴う、[4]に記載の剤。
[7] 対象における神経突起伸長を促進する方法であって、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物の有効量を、該対象に投与することを含む、方法。
[7a]神経突起伸長の促進における使用のための、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物。
[7b]神経突起伸長促進剤の製造のための、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物の使用。
[8] 対象が自閉症スペクトラム障害を有する、[7]に記載の方法。
[8a] 神経突起伸長の促進が自閉症スペクトラム障害の治療のためである、[7a]に記載の化合物。
[8b] 神経突起伸長促進剤が自閉症スペクトラム障害の治療用である、[7b]に記載の化合物。
[9] 自閉症スペクトラム障害の治療薬のスクリーニング方法であって、
(1)自閉症スペクトラム障害患者由来の人工多能性幹細胞から分化誘導した神経細胞に被検物質を接触させる工程、
(2)該神経細胞におけるRNT4、SEMA6A及びDCCからなる群より選択される1以上の遺伝子、並びに/或いはUBE3A遺伝子の発現レベルを測定する工程、及び
(3)工程(2)において1以上の遺伝子の発現レベルを低下させた被検物質を、自閉症スペクトラム障害の治療薬の候補として選択する工程
を含む方法。
[10] RNT4、SEMA6A及びDCCからなる群より選択される1以上の遺伝子、又はUBE3A遺伝子の発現抑制薬を含有してなる、自閉症スペクトラム障害治療剤。 That is, the present invention is as follows.
[1] A neurite outgrowth promoter comprising one or more compounds selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof.
[2] The agent according to [1], which comprises levamipide or a salt thereof and / or sotalol or a salt thereof.
[3] The agent according to [1], which comprises rifabutin or a salt thereof.
[4] The agent according to any one of [1] to [3] for the treatment of autism spectrum disorder.
[5] Autism spectrum disorders are associated with or non-symptomatic autism selected from the group consisting of Dup15q syndrome, Fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome and Clefstra syndrome. The agent according to [4], which is a spectrum disorder.
[6] The agent according to [4], wherein the autism spectrum disorder is accompanied by Dup15q syndrome.
[7] A method for promoting nerve projection elongation in a subject, which is selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and refabutin or a derivative thereof or a salt thereof. A method comprising administering to the subject an effective amount of a compound.
[7a] One or more compounds selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and refabutin or a derivative thereof or a salt thereof for use in promoting neurite outgrowth.
[7b] A compound selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and refabutin or a derivative thereof or a salt thereof for the production of a neurite outgrowth promoter. use.
[8] The method according to [7], wherein the subject has an autism spectrum disorder.
[8a] The compound according to [7a], wherein the promotion of neurite outgrowth is for the treatment of autism spectrum disorders.
[8b] The compound according to [7b], wherein the neurite outgrowth promoter is for the treatment of autism spectrum disorders.
[9] A screening method for therapeutic agents for autism spectrum disorders.
(1) A step of contacting a test substance with a nerve cell that has been induced to differentiate from an induced pluripotent stem cell derived from a patient with autism spectrum disorder.
(2) One or more genes selected from the group consisting of RNT4, SEMA6A and DCC in the nerve cell, and / or a step of measuring the expression level of the UBE3A gene, and (3) one or more genes in step (2). A method comprising the step of selecting a test substance having a reduced expression level as a candidate for a therapeutic agent for autism spectrum disorder.
[10] A therapeutic agent for autism spectrum disorders, which comprises one or more genes selected from the group consisting of RNT4, SEMA6A and DCC, or an agent for suppressing the expression of the UBE3A gene.
本発明によれば、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、あるいはリファブチンもしくはその誘導体又はその塩を対象に投与することで、神経突起の伸長を促進することができ、ASD等の神経突起の短縮が関与する疾患を改善し得る。これらの薬剤は、安全性に関するデータが蓄積されているので、臨床応用に向けた開発期間を短縮できることが期待される。また、本発明のスクリーニング法によれば、RTN4、SEMA6A、DCC又はUBE3Aの発現を指標とすることで、ASD治療薬の候補物質を効率よくスクリーニングすることができる。
According to the present invention, neurite outgrowth can be promoted by administering levamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, or rifabutin or a derivative thereof or a salt thereof to a subject, and ASD. It can improve diseases associated with shortening of neurites such as. Since data on the safety of these drugs have been accumulated, it is expected that the development period for clinical application can be shortened. Further, according to the screening method of the present invention, by using the expression of RTN4, SEMA6A, DCC or UBE3A as an index, candidate substances for ASD therapeutic agents can be efficiently screened.
(発明の詳細な説明)
本発明は神経突起伸長促進剤(以下、「本発明の促進剤」ともいう。)を提供する。該促進剤は、有効成分として、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物を含有する。 (Detailed description of the invention)
The present invention provides a neurite outgrowth promoter (hereinafter, also referred to as "promoter of the present invention"). The accelerator contains, as an active ingredient, one or more compounds selected from the group consisting of levamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof.
本発明は神経突起伸長促進剤(以下、「本発明の促進剤」ともいう。)を提供する。該促進剤は、有効成分として、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物を含有する。 (Detailed description of the invention)
The present invention provides a neurite outgrowth promoter (hereinafter, also referred to as "promoter of the present invention"). The accelerator contains, as an active ingredient, one or more compounds selected from the group consisting of levamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof.
本明細書において、「神経突起伸長」とは、既存の神経プロセス(軸索及び樹状突起)の伸長及び新たな神経細胞プロセスの成長又は出芽の拡張を意味する。神経突起伸長は、神経の接続性を変化させ得、新たなシナプスの確立及び既存のシナプスの再構築を生じ得る。
As used herein, "neurite outgrowth" means the elongation of existing neural processes (axons and dendrites) and the growth or expansion of budding of new neuronal processes. Neurite elongation can alter nerve connectivity, resulting in the establishment of new synapses and the reconstruction of existing synapses.
レバミピドは、化学名:(2RS)−2−(4−Chlorobenzoylamino)−3−(2−oxo−1,2−dihydroquinolin−4−yl)propanoic acidであり、胃粘膜防御因子増強薬に分類される抗潰瘍薬である。
Rebamipide has a chemical name: (2RS) -2- (4-Chlorobenzoylamino) -3- (2-oxo-1,2-dihydroquinolin-4-yl) pharmaceutical acid and is classified as a gastric mucosa protective factor enhancer. It is an anti-ulcer drug.
レバミピドの誘導体としては、下記式:
As a derivative of rebamipide, the following formula:
[式中:
R1は水素原子、低級アルキル、低級アルケニル、低級アルキニル又はフェニル低級アルキル;
R2は水素原子、水酸基又は低級アルコキシ;
R3は水素原子又は低級アルキル;
R4は水素原子又は基−COR5(R5はアミノ基又はフェニル低級アルコキシカルボニルアミノ基を置換基として有することのある低級アルキル、シクロアルキル又はフェニル環上に置換基としてハロゲン原子、低級アルキル、低級アルコキシ、ニトロ及びアミノから選ばれる基の1~3個を有することのあるフェニル基)を示し、
置換基の式: [During the ceremony:
R 1 is a hydrogen atom, lower alkyl, lower alkenyl, lower alkynyl or phenyl lower alkyl;
R 2 is a hydrogen atom, a hydroxyl group or a lower alkoxy;
R 3 is a hydrogen atom or a lower alkyl;
R 4 is a hydrogen atom or group-COR 5 (R 5 is a lower alkyl which may have an amino group or a phenyl lower alkoxycarbonylamino group as a substituent, a cycloalkyl or a halogen atom as a substituent on a phenyl ring, a lower alkyl, (Phenyl group which may have 1 to 3 groups selected from lower alkoxy, nitro and amino).
Substituent formula:
R1は水素原子、低級アルキル、低級アルケニル、低級アルキニル又はフェニル低級アルキル;
R2は水素原子、水酸基又は低級アルコキシ;
R3は水素原子又は低級アルキル;
R4は水素原子又は基−COR5(R5はアミノ基又はフェニル低級アルコキシカルボニルアミノ基を置換基として有することのある低級アルキル、シクロアルキル又はフェニル環上に置換基としてハロゲン原子、低級アルキル、低級アルコキシ、ニトロ及びアミノから選ばれる基の1~3個を有することのあるフェニル基)を示し、
置換基の式: [During the ceremony:
R 1 is a hydrogen atom, lower alkyl, lower alkenyl, lower alkynyl or phenyl lower alkyl;
R 2 is a hydrogen atom, a hydroxyl group or a lower alkoxy;
R 3 is a hydrogen atom or a lower alkyl;
R 4 is a hydrogen atom or group-COR 5 (R 5 is a lower alkyl which may have an amino group or a phenyl lower alkoxycarbonylamino group as a substituent, a cycloalkyl or a halogen atom as a substituent on a phenyl ring, a lower alkyl, (Phenyl group which may have 1 to 3 groups selected from lower alkoxy, nitro and amino).
Substituent formula:
における点線は一重結合又は二重結合であることを意味し、かつこの置換基の置換位置はカルボスチリル骨格の3、4、5、6、7又は8位のいずれかである。また、カルボスチリル骨格の3位と4位の間の結合は一重結合又は二重結合を示す。]
で表される化合物が挙げられる。 The dotted line in is a single bond or a double bond, and the substitution position of this substituent is any of the 3, 4, 5, 6, 7 or 8 positions of the carbostylyl skeleton. Also, the bond between the 3rd and 4th positions of the carbostylyl skeleton indicates a single bond or a double bond. ]
Examples thereof include compounds represented by.
で表される化合物が挙げられる。 The dotted line in is a single bond or a double bond, and the substitution position of this substituent is any of the 3, 4, 5, 6, 7 or 8 positions of the carbostylyl skeleton. Also, the bond between the 3rd and 4th positions of the carbostylyl skeleton indicates a single bond or a double bond. ]
Examples thereof include compounds represented by.
上記式の各基の説明において上位概念として記載される各用語(例えば、「低級アルキル」、「低級アルケニル」等)やその他の用語は、特開昭59−7168号公報における定義に従う。
Each term (for example, "lower alkyl", "lower alkenyl", etc.) described as a superordinate concept in the description of each group of the above formula and other terms follow the definitions in JP-A-59-7168.
レバミピドの誘導体の具体的な例としては、特開昭59−7168号公報の実施例に記載される化合物が挙げられる。
Specific examples of the derivative of rebamipide include the compounds described in Examples of JP-A-59-7168.
ソタロールは、化学名:(±)−4[−(RS)−1−hydroxy−2(−isopropylamino)ethyl]methanesulfonanilideであり、β受容体遮断(ClassII)作用により不整脈発生の一因である交感神経系の緊張増加を抑制する等の作用を有し、不整脈治療薬として使用されている。
Sotalol is a chemical name: (±) -4 [-(RS) -1-hydroxy-2 (-isotropylamino) ethyl] therapy, and is a sympathetic nerve that contributes to the generation of arrhythmia by β-receptor blocking (ClassII) action. It has an effect of suppressing an increase in system tension and is used as an arrhythmia therapeutic agent.
ソタロールの誘導体としては、下記式:
As a derivative of sotalol, the following formula:
[式中:
Xは水素原子、ヒドロキシ、アミノ、低級アルコキシ、ベンジルオキシ、ハロゲン原子、低級アルキル又はR2SO2NH−であり;
R1及びR2は、それぞれ独立して、低級アルキル、フェニル、低級アルキルフェニル、ハロフェニル、低級アルコキシフェニル又はベンジルオキシフェニルであり;
Zは>C=0又は>CHOHであり;
AlkはC1−4アルキレン基であり;
R4は水素原子、低級アルキル又はベンジルであり;
R5は水素原子、あるいは、1もしくは2個の、ヒドロキシル、カルボキシル、アミノ、低級アルコキシ、ベンジルオキシ、ハロゲン原子、低級アルキル、メチレンジオキシ及びR2SO2NHからなる群より選択される置換基を有してもよい、炭素数10以下のアルキル、アルケニル、シクロアルキル、シクロアルケニル、シクロアルキルアルキル、シクロアルケニルアルキル、ビシクロアルキル、トリシクロアルキル、ビシクロアルケニル、ビシクロアルキルアルキル、ビシクロアルケニルアルキル、アリール、フェニルアルキル、フェニルアルケニル、フェノキシアルキル、ヘテロモノシクリック、ヘテロモノシクロアルキル又はヘテロビシクリックである。]
で表される化合物が挙げられる。 [During the ceremony:
X is a hydrogen atom, hydroxy, amino, lower alkoxy, benzyloxy, halogen atom, lower alkyl or R 2 SO 2 NH-;
R 1 and R 2 are independently lower alkyl, phenyl, lower alkyl phenyl, halophenyl, lower alkoxyphenyl or benzyloxyphenyl;
Z is> C = 0 or>CHOH;
Alk is a C 1-4 alkylene group;
R4 is a hydrogen atom, lower alkyl or benzyl;
R 5 is a hydrogen atom or a substituent selected from the group consisting of one or two hydroxyls, carboxyls, aminos, lower alkoxys, benzyloxys, halogen atoms, lower alkyls, methylenedioxy and R2 SO 2 NH. Alkoxy, alkoxy, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxyalkyl, bicycloalkyl, tricycloalkyl, bicycloalkenyl, bicycloalkylalkyl, bicycloalkenylalkyl, aryl, which may have 10 or less carbon atoms. Phenylalkyl, phenylalkoxy, phenoxyalkyl, heteromonocyclic, heteromonocycloalkyl or heterobicyclic. ]
Examples thereof include compounds represented by.
Xは水素原子、ヒドロキシ、アミノ、低級アルコキシ、ベンジルオキシ、ハロゲン原子、低級アルキル又はR2SO2NH−であり;
R1及びR2は、それぞれ独立して、低級アルキル、フェニル、低級アルキルフェニル、ハロフェニル、低級アルコキシフェニル又はベンジルオキシフェニルであり;
Zは>C=0又は>CHOHであり;
AlkはC1−4アルキレン基であり;
R4は水素原子、低級アルキル又はベンジルであり;
R5は水素原子、あるいは、1もしくは2個の、ヒドロキシル、カルボキシル、アミノ、低級アルコキシ、ベンジルオキシ、ハロゲン原子、低級アルキル、メチレンジオキシ及びR2SO2NHからなる群より選択される置換基を有してもよい、炭素数10以下のアルキル、アルケニル、シクロアルキル、シクロアルケニル、シクロアルキルアルキル、シクロアルケニルアルキル、ビシクロアルキル、トリシクロアルキル、ビシクロアルケニル、ビシクロアルキルアルキル、ビシクロアルケニルアルキル、アリール、フェニルアルキル、フェニルアルケニル、フェノキシアルキル、ヘテロモノシクリック、ヘテロモノシクロアルキル又はヘテロビシクリックである。]
で表される化合物が挙げられる。 [During the ceremony:
X is a hydrogen atom, hydroxy, amino, lower alkoxy, benzyloxy, halogen atom, lower alkyl or R 2 SO 2 NH-;
R 1 and R 2 are independently lower alkyl, phenyl, lower alkyl phenyl, halophenyl, lower alkoxyphenyl or benzyloxyphenyl;
Z is> C = 0 or>CHOH;
Alk is a C 1-4 alkylene group;
R4 is a hydrogen atom, lower alkyl or benzyl;
R 5 is a hydrogen atom or a substituent selected from the group consisting of one or two hydroxyls, carboxyls, aminos, lower alkoxys, benzyloxys, halogen atoms, lower alkyls, methylenedioxy and R2 SO 2 NH. Alkoxy, alkoxy, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxyalkyl, bicycloalkyl, tricycloalkyl, bicycloalkenyl, bicycloalkylalkyl, bicycloalkenylalkyl, aryl, which may have 10 or less carbon atoms. Phenylalkyl, phenylalkoxy, phenoxyalkyl, heteromonocyclic, heteromonocycloalkyl or heterobicyclic. ]
Examples thereof include compounds represented by.
上記式の各基の説明において上位概念として記載される各用語(例えば、「低級アルキル」、「低級アルケニル」等)やその他の用語は、米国特許第3,341,584号明細書における定義に従う。
Each term (eg, "lower alkyl", "lower alkenyl", etc.) and other terms described as superordinate concepts in the description of each group of the above formulas are as defined in US Pat. No. 3,341,584. ..
ソタロールの誘導体の具体的な例としては、米国特許第3,341,584号明細書の実施例に記載される化合物が挙げられる。
Specific examples of sotalol derivatives include the compounds described in Examples of US Pat. No. 3,341,584.
リファブチンは、化学名:(9S,12E,14S,15R,16S,17R,18R,19R,20S,21S,22E,24Z)−6,18,20−Trihydroxy−14−methoxy−7,9,15,17,19,21,25−heptamethyl−1’−(2−methylpropyl)−5,10,26−trioxo−3,5,9,10−tetrahydrospiro[9,4−(epoxypentadeca[1,11,13]trienimino)−2H−furo[2’,3’:7,8]naphtho[1,2−d]imidazole−2,4’−piperidine]−16−yl acetateであり、DNA依存性RNAポリメラーゼに作用してRNA合成を阻害する抗酸菌症治療薬である。結核症、マイコバクテリウム・アビウムコンプレックス(MAC)症を含む非結核性抗酸菌症、HIV感染患者における播種性MAC症の発症抑制を適応症とする。
Rifabutin has the chemical names: (9S, 12E, 14S, 15R, 16S, 17R, 18R, 19R, 20S, 21S, 22E, 24Z) -6,18,20-Trihydroxy-14methoxy-7,9,15, 17,19,21,25-heptamethyl-1'-(2-methylpropyl) -5,10,26-trioxo-3,5,9,10-tetrahydrospiro [9,4- (epoxypentadeca [1,11,13]] trienimino) -2H-furo [2', 3': 7,8] naphtho [1,2-d] imidazole-2,4'-piperidine] -16-yl acetate, which acts on DNA-dependent RNA polymerase. It is a therapeutic agent for acidobacterial disease that inhibits RNA synthesis. Indications include tuberculosis, nontuberculous mycobacteriosis including Mycobacterium avium complex (MAC) disease, and suppression of the onset of disseminated MAC disease in HIV-infected patients.
リファブチンの誘導体としては、リファマイシン系の抗生物質、例えば、リファキシミン、リファマイシン、リファンピシン、リファペンチン、リファラジル、ビコザマシン等が挙げられる。
Examples of the derivative of rifabutin include rifaximin-based antibiotics such as rifaximin, rifampicin, rifampicin, rifapentine, rifalazil, and bicozamachine.
レバミピドもしくはその誘導体、ソタロールもしくはその誘導体、及びリファブチンもしくはその誘導体は、フリー体だけでなく、その薬理学的に許容される塩も包含されるものとする。薬理学的に許容される塩は化合物の種類によって異なるが、例えば、アルカリ金属塩(ナトリウム塩、カリウム塩等)、アルカリ土類金属塩(カルシウム塩、マグネシウム塩等)、アルミニウム塩、アンモニウム塩等の無機塩基塩、並びにトリメチルアミン、トリエチルアミン、ピリジン、ピコリン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、ジシクロヘキシルアミン、N,N’−ジベンジルエチレンジアミン等の有機塩基塩などの塩基付加塩、あるいは塩酸塩、臭化水素酸塩、硫酸塩、ヨウ化水素酸塩、硝酸塩、リン酸塩等の無機酸塩、クエン酸塩、シュウ酸塩、酢酸塩、ギ酸塩、プロピオン酸塩、安息香酸塩、トリフルオロ酢酸塩、マレイン酸塩、酒石酸塩、メタンスルホン酸塩、ベンゼンスルホン酸塩、パラトルエンスルホン酸塩等の有機酸塩などの酸付加塩が挙げられる。
Rebamipide or its derivatives, sotalol or its derivatives, and rifabutin or its derivatives shall include not only free forms but also pharmacologically acceptable salts thereof. The pharmacologically acceptable salt varies depending on the type of compound, but for example, alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, etc.), aluminum salt, ammonium salt, etc. Inorganic base salts such as trimethylamine, triethylamine, pyridine, picolin, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N'-dibenzylethylenediamine and other organic base salts, or hydrochlorides, odors. Inorganic acid salts such as hydrobromide, sulfate, hydroiodide, nitrate, phosphate, citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetic acid Examples thereof include acid addition salts such as salts, maleates, tartrates, methanesulfonates, benzenesulfonates, and organic acid salts such as paratoluenesulfonates.
レバミピドもしくはその誘導体又はその塩(以下、包括して「レバミピド類」ともいう。)、ソタロールもしくはその誘導体又はその塩(以下、包括して「ソタロール類」ともいう。)、及びリファブチンもしくはその誘導体又はその塩(以下、包括して「リファブチン類」ともいう。)が、光学異性体、立体異性体、位置異性体、回転異性体等の異性体を有する場合には、いずれか一方の異性体も混合物も本発明におけるこれらの薬剤に包含される。
これらの異性体は、自体公知の合成手法、分離手法(例、濃縮、溶媒抽出、カラムクロマトグラフィー、再結晶等)、光学分割手法(例、分別再結晶法、キラルカラム法、ジアステレオマー法等)等によりそれぞれを単品として得ることができる。
レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩は、結晶であってもよく、結晶形が単一であっても結晶形混合物であっても本発明の化合物に包含される。結晶は、自体公知の結晶化法を適用して、結晶化することによって製造することができる。
レバミピド類、ソタロール類及びリファブチン類は、溶媒和物(例、水和物等)であっても、無溶媒和物(例、非水和物等)であってもよい。
また、同位元素(例、3H,14C,35S,125I等)等で標識された化合物も包含される。 Rebamipide or a derivative thereof or a salt thereof (hereinafter collectively referred to as "levamipide"), sotalol or a derivative thereof or a salt thereof (hereinafter collectively referred to as "sotalols"), and refabutin or a derivative thereof or If the salt (hereinafter collectively referred to as "rebamipides") has isomers such as optical isomers, steric isomers, positional isomers, and rotational isomers, any one of the isomers may also be used. Mixtures are also included in these agents in the present invention.
These isomers can be used in synthetic methods known per se, separation methods (eg, concentration, solvent extraction, column chromatography, recrystallization, etc.), optical resolution methods (eg, fractional recrystallization method, chiral column method, diastereomer method, etc.). ) Etc., each can be obtained as a single item.
Revamipid or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof may be crystalline, and may be a single crystalline form or a crystalline mixed form. Included in the compounds of the invention. Crystals can be produced by crystallization by applying a crystallization method known per se.
The rebamipides, sotalols and rifabutins may be solvates (eg, hydrates, etc.) or non-solvate (eg, non-hydrates, etc.).
It also includes compounds labeled with isotopes (eg, 3 H, 14 C, 35 S, 125 I, etc.) and the like.
これらの異性体は、自体公知の合成手法、分離手法(例、濃縮、溶媒抽出、カラムクロマトグラフィー、再結晶等)、光学分割手法(例、分別再結晶法、キラルカラム法、ジアステレオマー法等)等によりそれぞれを単品として得ることができる。
レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩は、結晶であってもよく、結晶形が単一であっても結晶形混合物であっても本発明の化合物に包含される。結晶は、自体公知の結晶化法を適用して、結晶化することによって製造することができる。
レバミピド類、ソタロール類及びリファブチン類は、溶媒和物(例、水和物等)であっても、無溶媒和物(例、非水和物等)であってもよい。
また、同位元素(例、3H,14C,35S,125I等)等で標識された化合物も包含される。 Rebamipide or a derivative thereof or a salt thereof (hereinafter collectively referred to as "levamipide"), sotalol or a derivative thereof or a salt thereof (hereinafter collectively referred to as "sotalols"), and refabutin or a derivative thereof or If the salt (hereinafter collectively referred to as "rebamipides") has isomers such as optical isomers, steric isomers, positional isomers, and rotational isomers, any one of the isomers may also be used. Mixtures are also included in these agents in the present invention.
These isomers can be used in synthetic methods known per se, separation methods (eg, concentration, solvent extraction, column chromatography, recrystallization, etc.), optical resolution methods (eg, fractional recrystallization method, chiral column method, diastereomer method, etc.). ) Etc., each can be obtained as a single item.
Revamipid or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof may be crystalline, and may be a single crystalline form or a crystalline mixed form. Included in the compounds of the invention. Crystals can be produced by crystallization by applying a crystallization method known per se.
The rebamipides, sotalols and rifabutins may be solvates (eg, hydrates, etc.) or non-solvate (eg, non-hydrates, etc.).
It also includes compounds labeled with isotopes (eg, 3 H, 14 C, 35 S, 125 I, etc.) and the like.
本発明の促進剤は、レバミピド類、ソタロール類及びリファブチン類から選ばれる1種の化合物を含有してもよいし、2種以上を含有してもよい。
The accelerator of the present invention may contain one compound selected from rebamipides, sotalols and rifabutins, or may contain two or more of them.
好ましい一実施態様において、本発明の促進剤は、レバミピドもしくはその塩、及び/又はソタロールもしくはその塩を含有する。レバミピドは、RNT4、SEMA6A及びDCCを含む神経突起伸長阻害分子の発現を阻害することができ、ソタロールは、RNT4及びSEMA6Aを含む神経突起伸長阻害分子の発現を阻害することができる。
In one preferred embodiment, the accelerator of the present invention contains rebamipide or a salt thereof and / or sotalol or a salt thereof. Rebamipide can inhibit the expression of neurite outgrowth inhibitory molecules including RNT4, SEMA6A and DCC, and sotalol can inhibit the expression of neurite outgrowth inhibitory molecules including RNT4, SEMA6A.
別の好ましい一実施態様において、本発明の促進剤は、リファブチンもしくはその塩を含有する。リファブチンは、15q11−13領域内に位置し、Dup15qにおいて発現が上昇しているUBE3A遺伝子の発現を阻害することができる。
In another preferred embodiment, the accelerator of the present invention contains rifabutin or a salt thereof. Rifabutin is located within the 15q11-13 region and can inhibit the expression of the UBE3A gene, which is upregulated in Dup15q.
上記のレバミピド類、ソタロール類及びリファブチン類は、市販品を用いるか、あるいは各化合物についてそれぞれ自体公知の方法により製造することができる。
例えば、レバミピド類は、例えば、特開昭59−7168号公報等に記載される方法に従って製造することができる。
ソタロール類は、例えば、米国特許第3,341,584号明細書等に記載される方法に従って製造することができる。
リファブチン類は、例えば、Streptomyces mediterraneiが生産するリファマイシンから、自体公知の方法により半合成することができる。 The above-mentioned rebamipides, sotalols and rifabutins can be produced by using commercially available products or by methods known per se for each compound.
For example, rebamipides can be produced, for example, according to the method described in JP-A-59-7168.
Sotalols can be produced, for example, according to the method described in US Pat. No. 3,341,584.
Rifabutins can be semi-synthesized from, for example, rifamycin produced by Streptomyces mediaranei by a method known per se.
例えば、レバミピド類は、例えば、特開昭59−7168号公報等に記載される方法に従って製造することができる。
ソタロール類は、例えば、米国特許第3,341,584号明細書等に記載される方法に従って製造することができる。
リファブチン類は、例えば、Streptomyces mediterraneiが生産するリファマイシンから、自体公知の方法により半合成することができる。 The above-mentioned rebamipides, sotalols and rifabutins can be produced by using commercially available products or by methods known per se for each compound.
For example, rebamipides can be produced, for example, according to the method described in JP-A-59-7168.
Sotalols can be produced, for example, according to the method described in US Pat. No. 3,341,584.
Rifabutins can be semi-synthesized from, for example, rifamycin produced by Streptomyces mediaranei by a method known per se.
本発明の促進剤の有効成分であるレバミピド類、ソタロール類及びリファブチン類は、神経細胞、好ましくは、ASDを有する対象(例、ヒト、イヌ、ネコ、サル、マウス、ラット等の哺乳動物、好ましくはヒト)中の神経細胞において、神経突起の伸長を促進することができるので、神経突起伸長が阻害されている対象、好ましくはASDを有する対象に投与することにより、該対象における神経突起伸長の阻害が関与する疾患を治療することができる。ここで「治療」とは、症状の改善、発症の遅延、再発の防止・遅延等を包含する。
The active ingredients of the accelerator of the present invention, levamipids, sotalols and rifabutins, are nerve cells, preferably subjects having ASD (eg, mammals such as humans, dogs, cats, monkeys, mice, rats, etc., preferably. Can promote neurite outgrowth in nerve cells in humans), so by administration to a subject in which neurite outgrowth is inhibited, preferably a subject with ASD, neurite outgrowth in the subject Diseases involving inhibition can be treated. Here, "treatment" includes improvement of symptoms, delay of onset, prevention / delay of recurrence, and the like.
神経突起伸長の阻害が関与する疾患としては、例えば、ASD、ASDに高頻度に併発する遺伝性症候群(例、Dup15q症候群、脆弱X症候群、レット症候群、結節性硬化症、コステロ症候群、クレーフストラ症候群、コルネリア・デ・ランゲ症候群、ネコなき症候群、Angelman症候群、ダウン症候群、チャージ症候群、プラダー・ウィリー症候群、クラインフェルター症候群、Phelan McDermid症候群等)、神経系疾患〔例、神経変性疾患(例、アルツハイマー病、パーキンソン症候群など)、脊髄損傷、てんかん、脳虚血性障害、脳血管性痴呆など〕、精神系疾患(例、精神分裂病、うつ病など)等が挙げられるが、それらに限定されない。好ましくはASDであり、より好ましくは、遺伝性症候群(好ましくは、Dup15q症候群、脆弱X症候群、レット症候群、結節性硬化症、コステロ症候群、クレーフストラ症候群、さらに好ましくはDup15q症候群)を伴うASDである。
Diseases associated with inhibition of neurite elongation include, for example, ASD, hereditary syndromes frequently associated with ASD (eg, Dup15q syndrome, fragile X syndrome, Let syndrome, nodular sclerosis, Costello syndrome, Clefstra syndrome, etc.) Cornelia de Lange Syndrome, Catless Syndrome, Angelman Syndrome, Down Syndrome, Charge Syndrome, Prader Willy Syndrome, Kleinfelder Syndrome, Phelan McDermid Syndrome, etc. (Parkinson syndrome, etc.), spinal cord injury, epilepsy, cerebral ischemic disorder, cerebrovascular dementia, etc.], mental system diseases (eg, psychiatric division disease, depression, etc.), but are not limited thereto. It is preferably ASD, more preferably ASD with hereditary syndrome (preferably Dup15q syndrome, fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome, Clefstra syndrome, more preferably Dup15q syndrome).
本発明の促進剤は、有効成分であるレバミピド類、ソタロール類、リファブチン類をそのまま単独で、又は薬理学的に許容される担体、賦形剤、希釈剤等と混合し、適当な剤型の医薬組成物として経口的又は非経口的に投与することができる。
The accelerator of the present invention is an appropriate dosage form obtained by mixing the active ingredients rebamipides, sotalols, rifabutins as they are, or by mixing them with a pharmacologically acceptable carrier, excipient, diluent, etc. It can be administered orally or parenterally as a pharmaceutical composition.
経口投与のための組成物としては、固体又は液体の剤形、具体的には錠剤(糖衣錠、フィルムコーティング錠を含む)、丸剤、顆粒剤、散剤、カプセル剤(ソフトカプセル剤を含む)、シロップ剤、乳剤、懸濁剤等が挙げられる。一方、非経口投与のための組成物としては、例えば、注射剤、坐剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。これらの製剤は、賦形剤(例えば、乳糖、白糖、葡萄糖、マンニトール、ソルビトールのような糖誘導体;トウモロコシデンプン、バレイショデンプン、α澱粉、デキストリンのような澱粉誘導体;結晶セルロースのようなセルロース誘導体;アラビアゴム;デキストラン;プルランのような有機系賦形剤;及び、軽質無水珪酸、合成珪酸アルミニウム、珪酸カルシウム、メタ珪酸アルミン酸マグネシウムのような珪酸塩誘導体;燐酸水素カルシウムのような燐酸塩;炭酸カルシウムのような炭酸塩;硫酸カルシウムのような硫酸塩等の無機系賦形剤である)、滑沢剤(例えば、ステアリン酸、ステアリン酸カルシウム、ステアリン酸マグネシウムのようなステアリン酸金属塩;タルク;コロイドシリカ;ビーズワックス、ゲイ蝋のようなワックス類;硼酸;アジピン酸;硫酸ナトリウムのような硫酸塩;グリコール;フマル酸;安息香酸ナトリウム;DLロイシン;ラウリル硫酸ナトリウム、ラウリル硫酸マグネシウムのようなラウリル硫酸塩;無水珪酸、珪酸水和物のような珪酸類;及び、上記澱粉誘導体である)、結合剤(例えば、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、マクロゴール、及び、前記賦形剤と同様の化合物である)、崩壊剤(例えば、低置換度ヒドロキシプロピルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、内部架橋カルボキシメチルセルロースナトリウムのようなセルロース誘導体;カルボキシメチルスターチ、カルボキシメチルスターチナトリウム、架橋ポリビニルピロリドンのような化学修飾されたデンプン・セルロース類である)、乳化剤(例えば、ベントナイト、ビーガムのようなコロイド性粘土;水酸化マグネシウム、水酸化アルミニウムのような金属水酸化物;ラウリル硫酸ナトリウム、ステアリン酸カルシウムのような陰イオン界面活性剤;塩化ベンザルコニウムのような陽イオン界面活性剤;及び、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンソルビタン脂肪酸エステル、ショ糖脂肪酸エステルのような非イオン界面活性剤である)、安定剤(メチルパラベン、プロピルパラベンのようなパラオキシ安息香酸エステル類;クロロブタノール、ベンジルアルコール、フェニルエチルアルコールのようなアルコール類;塩化ベンザルコニウム;フェノール、クレゾールのようなフェノール類;チメロサール;デヒドロ酢酸;及び、ソルビン酸である)、矯味矯臭剤(例えば、通常使用される、甘味料、酸味料、香料等である)、希釈剤等の添加剤を用いて周知の方法で製造される。
Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups. Agents, emulsions, suspending agents and the like can be mentioned. On the other hand, as the composition for parenteral administration, for example, injections, suppositories and the like are used, and the injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. The dosage form may be included. These formulations include excipients (eg, sugar derivatives such as lactose, sucrose, grape sugar, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Rubber arabic; dextran; organic excipients such as purulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonic acid Carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate), lubricants (eg, metal stearate such as stearic acid, calcium stearate, magnesium stearate; talc; Colloidal silica; bead wax, waxes such as gay wax; boric acid; adipic acid; stearic acid such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL leucine; sodium lauryl sulfate, lauryl such as magnesium lauryl sulfate Sulfates; stearic acids such as stearic acid anhydride, silicate hydrate; and the starch derivatives mentioned above), binders (eg, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, macrogol, and the excipients. (Similar compounds), disintegrants (eg, cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, internally cross-linked sodium carboxymethyl cellulose; carboxymethyl starch, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone. Chemically modified starches and celluloses such as), emulsifiers (eg, colloidal clays such as bentonite, bee gum; metal hydroxides such as magnesium hydroxide, aluminum hydroxide; sodium lauryl sulfate, calcium stearate. Anionic surfactants such as; cationic surfactants such as benzalconium chloride; and nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters. Stabilizers (paraoxybenzoic acid esters such as methylparaben, propylparaben; alcohols such as chlorobutanol, benzyl alcohol, phenylethyl alcohol; benzalconium chloride; phenol, cresol With additives such as phenols; thimerosal; dehydroacetic acid; and sorbic acid), flavoring agents (eg, commonly used sweeteners, acidulants, flavors, etc.), diluents and the like. Manufactured by a well-known method.
本発明の促進剤の有効成分であるレバミピド類、ソタロール類、リファブチン類の投与量は、化合物の種類、投与対象の症状、齢、体重、薬物受容性等の種々の条件により変化し得るが、経口投与の場合には、1回当たり下限0.1mg(好適には0.5mg)、上限1000mg(好適には500mg)を、非経口的投与の場合には、1回当たり下限0.01mg(好適には0.05mg)、上限100mg(好適には50mg)を、成人に対して1日当たり1乃至6回投与することができる。症状に応じて増量もしくは減量してもよい。特に、有効成分が上記疾患以外の疾患に対する医薬品として、既に上市されている場合には、各化合物について、安全性が確認されている範囲で、適宜投与量を選択することができる。
The doses of rebamipides, sotalols, and rifabutins, which are the active ingredients of the accelerator of the present invention, may vary depending on various conditions such as the type of compound, the symptom of the subject to be administered, age, body weight, drug acceptability, and the like. In the case of oral administration, the lower limit is 0.1 mg (preferably 0.5 mg) and the upper limit is 1000 mg (preferably 500 mg), and in the case of parenteral administration, the lower limit is 0.01 mg (preferably 500 mg). A suitable amount of 0.05 mg) and an upper limit of 100 mg (preferably 50 mg) can be administered to an adult 1 to 6 times a day. The dose may be increased or decreased depending on the symptoms. In particular, when the active ingredient has already been put on the market as a drug for diseases other than the above-mentioned diseases, the dose of each compound can be appropriately selected as long as the safety has been confirmed.
さらに、本発明の促進剤は、他の薬剤、例えば、リスペリドン、アリピプラゾール、ラパマイシン、オキシトシン、ペントキシフィリン、テトラヒドロビオプテリンなどと併用してもよい。本発明の促進剤及びこれらの他の薬剤は、同時に、順次又は別個に投与することができる。
Furthermore, the accelerator of the present invention may be used in combination with other agents such as risperidone, aripiprazole, rapamycin, oxytocin, pentoxifylline, tetrahydrobiopterin and the like. The accelerator of the present invention and other agents thereof can be administered simultaneously, sequentially or separately.
本発明はまた、ASDの治療薬のスクリーニング方法(以下、「本発明のスクリーニング方法」ともいう。)を提供する。
The present invention also provides a screening method for a therapeutic agent for ASD (hereinafter, also referred to as "screening method of the present invention").
本発明のスクリーニング方法は、
(1)ASD患者由来のiPS細胞から分化誘導した神経細胞に被検物質を接触させる工程、
(2)該神経細胞におけるRNT4、SEMA6A及びDCCからなる群より選択される1以上の遺伝子、並びに/或いはUBE3A遺伝子の発現レベルを測定する工程、及び
(3)工程(2)において1以上の遺伝子の発現レベルを低下させた被検物質を、ASDの治療薬の候補として選択する工程
を含む。 The screening method of the present invention
(1) A step of contacting a test substance with nerve cells induced to differentiate from iPS cells derived from an ASD patient.
(2) One or more genes selected from the group consisting of RNT4, SEMA6A and DCC in the nerve cell, and / or a step of measuring the expression level of the UBE3A gene, and (3) one or more genes in step (2). A step of selecting a test substance having a reduced expression level of ASD as a candidate for a therapeutic agent for ASD is included.
(1)ASD患者由来のiPS細胞から分化誘導した神経細胞に被検物質を接触させる工程、
(2)該神経細胞におけるRNT4、SEMA6A及びDCCからなる群より選択される1以上の遺伝子、並びに/或いはUBE3A遺伝子の発現レベルを測定する工程、及び
(3)工程(2)において1以上の遺伝子の発現レベルを低下させた被検物質を、ASDの治療薬の候補として選択する工程
を含む。 The screening method of the present invention
(1) A step of contacting a test substance with nerve cells induced to differentiate from iPS cells derived from an ASD patient.
(2) One or more genes selected from the group consisting of RNT4, SEMA6A and DCC in the nerve cell, and / or a step of measuring the expression level of the UBE3A gene, and (3) one or more genes in step (2). A step of selecting a test substance having a reduced expression level of ASD as a candidate for a therapeutic agent for ASD is included.
工程(1)に用いられるiPS細胞の由来となるASD患者は特に制限されないが、好ましくは、遺伝性症候群(例、Dup15q症候群、脆弱X症候群、レット症候群、結節性硬化症、コステロ症候群、クレーフストラ症候群、コルネリア・デ・ランゲ症候群、ネコなき症候群、Angelman症候群、ダウン症候群、チャージ症候群、プラダー・ウィリー症候群、クラインフェルター症候群、Phelan McDermid症候群等、好ましくは、Dup15q症候群、脆弱X症候群、レット症候群、結節性硬化症、コステロ症候群、クレーフストラ症候群、さらに好ましくはDup15q症候群)を伴うASD患者である。
The ASD patient from which the iPS cells used in step (1) are derived is not particularly limited, but is preferably hereditary syndrome (eg, Dup15q syndrome, fragile X syndrome, Let syndrome, nodular sclerosis, Costello syndrome, Clefstra syndrome). , Cornelia de Lange Syndrome, Catless Syndrome, Angelman Syndrome, Down Syndrome, Charge Syndrome, Prader-Willi Syndrome, Kleinfelder Syndrome, Phelan McDermid Syndrome, etc., preferably Dup15q Syndrome, Vulnerable X Syndrome, Let Syndrome, Nodular ASD patients with sclerosis, Costello syndrome, Clefstra syndrome, more preferably Dup15q syndrome).
ASD患者から採取する体細胞は特に限定されず、例えば(1)神経幹細胞、造血幹細胞、間葉系幹細胞、歯髄幹細胞等の組織幹細胞(体性幹細胞)、(2)組織前駆細胞、(3)リンパ球(例、末梢血単核球)、上皮細胞、内皮細胞、筋肉細胞、線維芽細胞(皮膚細胞等)、毛細胞、肝細胞、胃粘膜細胞、腸細胞、脾細胞、膵細胞(膵外分泌細胞等)、脳細胞、肺細胞、腎細胞及び脂肪細胞等の分化した細胞などが例示される。
The somatic cells collected from ASD patients are not particularly limited, and are, for example, (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, (3). Lymphocytes (eg, peripheral blood mononuclear cells), epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic cells) Exocrine cells, etc.), brain cells, lung cells, renal cells, fat cells, and other differentiated cells are exemplified.
iPS細胞は、特定の初期化因子を、DNA又はタンパク質の形態で体細胞に導入することによって作製することができる、ES細胞とほぼ同等の特性、例えば分化多能性と自己複製による増殖能、を有する体細胞由来の人工の幹細胞である(K.Takahashi and S.Yamanaka(2006)Cell,126:663−676;K.Takahashi et al.(2007),Cell,131:861−872;J.Yu et al.(2007),Science,318:1917−1920;Nakagawa,M.ら,Nat.Biotechnol.26:101−106(2008);WO 2007/069666)。初期化因子は、ES細胞に特異的に発現している遺伝子、その遺伝子産物もしくはnon−coding RNA、又はES細胞の未分化維持に重要な役割を果たす遺伝子、その遺伝子産物もしくはnon−coding RNA、あるいは低分子化合物によって構成されてもよい。初期化因子に含まれる遺伝子として、例えば、Oct3/4、Sox2、Sox1、Sox3、Sox15、Sox17、Klf4、Klf2、c−Myc、N−Myc、L−Myc、Nanog、Lin28、Fbx15、ERas、ECAT15−2、Tcl1、beta−catenin、Lin28b、Sall1、Sall4、Esrrb、Nr5a2、Tbx3又はGlis1等が例示され、これらの初期化因子は、単独で用いても良く、組み合わせて用いても良い。初期化因子の組み合わせとしては、WO2007/069666、WO2008/118820、WO2009/007852、WO2009/032194、WO2009/058413、WO2009/057831、WO2009/075119、WO2009/079007、WO2009/091659、WO2009/101084、WO2009/101407、WO2009/102983、WO2009/114949、WO2009/117439、WO2009/126250、WO2009/126251、WO2009/126655、WO2009/157593、WO2010/009015、WO2010/033906、WO2010/033920、WO2010/042800、WO2010/050626、WO2010/056831、WO2010/068955、WO2010/098419、WO2010/102267、WO 2010/111409、WO 2010/111422、WO2010/115050、WO2010/124290、WO2010/147395、WO2010/147612、Huangfu D,et al.(2008),Nat.Biotechnol.,26:795−797、Shi Y,et al.(2008),Cell Stem Cell,2:525−528、Eminli S,et al.(2008),Stem Cells.26:2467−2474、Huangfu D,et al.(2008),Nat Biotechnol.26:1269−1275、Shi Y,et al.(2008),Cell Stem Cell,3,568−574、Zhao Y,et al.(2008),Cell Stem Cell,3:475−479、Marson A,(2008),Cell Stem Cell,3,132−135、Feng B,et al.(2009),Nat Cell Biol.11:197−203、R.L.Judson et al.,(2009),Nat.Biotech.,27:459−461、Lyssiotis CA,et al.(2009),Proc Natl Acad Sci U S A.106:8912−8917、Kim JB,et al.(2009),Nature.461:649−643、Ichida JK,et al.(2009),Cell Stem Cell.5:491−503、Heng JC,et al.(2010),Cell Stem Cell.6:167−74、Han J,et al.(2010),Nature.463:1096−100、Mali P,et al.(2010),Stem Cells.28:713−720、Maekawa M,et al.(2011),Nature.474:225−9.に記載の組み合わせが例示される。
iPS cells can be made by introducing specific reprogramming factors into somatic cells in the form of DNA or protein, with properties similar to ES cells, such as pluripotency and self-renewal proliferation. (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. et al. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); WO 2007/069666). The reprogramming factor is a gene specifically expressed in ES cells, its gene product or non-coding RNA, or a gene that plays an important role in maintaining undifferentiated ES cells, its gene product or non-coding RNA, Alternatively, it may be composed of a low molecular weight compound. Genes contained in the reprogramming factor include, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, Eras, ECAT15. -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc. are exemplified, and these initialization factors may be used alone or in combination. The combinations of initialization factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 /. 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 11550, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Hungfu D, et. (2008), Nat. Biotechnol. , 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467-2474, Hungfu D, et al. (2008), Nat Biotechnology. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3,568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3,132-135, Feng B, et al. (2009), Nature Cell Biology. 11: 197-203, R.M. L. Judson et al. , (2009), Nat. Biotechnology. , 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106: 8912-8917, Kim JB, et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5: 491-503, Heng JC, et al. (2010), Cell Stem Cell. 6: 167-74, Han J, et al. (2010), Nature. 463: 1096-100, Mari P, et al. (2010), Stem Cells. 28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-9. The combinations described in are exemplified.
iPS細胞から神経細胞を分化誘導する方法として、特に限定されないが、線維芽細胞フィーダー層上での高密度培養による分化誘導法(特開2008−201792)、ストローマ細胞との共培養による分化誘導法(SDIA法)(例えば、WO2001/088100、WO/2003/042384)、浮遊培養による分化誘導法(SFEB法)(WO2005/123902)、ニューロスフェアの形成後コーティング処理された培養皿に接着させて、任意の培地中で適宜添加物を変えて培養する改変SFEBq法(WO2013/108926)及びそれらの組み合わせによる方法を利用することができる。より具体的には、例えば、後述の実施例に記載の方法を用いることができる。
The method for inducing differentiation of nerve cells from iPS cells is not particularly limited, but is a differentiation induction method by high-density culture on a fibroblast feeder layer (Japanese Patent Laid-Open No. 2008-201792) and a differentiation induction method by co-culture with stromal cells. (SDIA method) (for example, WO2001 / 088100, WO / 2003/042384), differentiation induction method by suspension culture (SFEB method) (WO2005 / 123902), adhered to a culture dish coated after formation of neurosphere, and adhered to the culture dish. A modified SFEBq method (WO2013 / 108926) in which the additives are appropriately changed and cultured in an arbitrary medium, and a method based on a combination thereof can be used. More specifically, for example, the method described in Examples described later can be used.
本発明のスクリーニング方法において用いられる被検物質に特に限定は無く、タンパク質、ペプチド、核酸、無機化合物、天然もしくは合成化学的に調製された有機化合物等が挙げられる。被検物質として、具体的には、アミノ酸3~50残基、好ましくは5~20残基のペプチドライブラリーや、当業者に公知のコンビナトリアルケミストリーの技術を用いて調製された分子量100~2000、好ましくは200~800の低分子有機化合物ライブラリーを挙げることができる。別の好ましい態様においては、既存の医薬品化合物のライブラリー(例、FDA承認薬の化合物ライブラリーであるPrestwick Chemical Library)等を用いることもできる。
The test substance used in the screening method of the present invention is not particularly limited, and examples thereof include proteins, peptides, nucleic acids, inorganic compounds, and organic compounds prepared naturally or synthetically. Specific examples of the test substance include a peptide library of 3 to 50 amino acid residues, preferably 5 to 20 residues, and a molecular weight of 100 to 2000 prepared using a combinatorial chemistry technique known to those skilled in the art. Preferred are 200-800 small molecule organic compound libraries. In another preferred embodiment, a library of existing pharmaceutical compounds (eg, Presswick Chemical Library, which is a compound library of FDA-approved drugs) and the like can also be used.
神経細胞と接触させる被検物質の濃度としては、特に限定は無く、通常約0.01μM~約100μMであればよく、好ましくは0.1μM~50μMであればよい。細胞と被検物質とを接触させる時間は、特に限定されるものでなく適時設定するものであるが、例えば5分間~5日間程度あり、好ましくは10分間~3日間程度である。被検物質は適宜、水、リン酸バッファーもしくはトリスバッファー等のバッファー、エタノール、アセトン、ジメチルスルホキシド(DMSO)もしくはこれらの混合物などの溶媒に溶解又は懸濁して用いることができる。
The concentration of the test substance to be brought into contact with the nerve cells is not particularly limited, and may be usually about 0.01 μM to about 100 μM, preferably 0.1 μM to 50 μM. The time for contacting the cells with the test substance is not particularly limited and is set in a timely manner, but is, for example, about 5 minutes to 5 days, preferably about 10 minutes to 3 days. The test substance can be appropriately dissolved or suspended in a solvent such as water, a buffer such as a phosphate buffer or a Tris buffer, ethanol, acetone, dimethyl sulfoxide (DMSO) or a mixture thereof.
工程(2)における遺伝子の発現レベルは、例えば、各遺伝子のヌクレオチド配列情報から適当なプライマーセット、プローブ等を設計・合成し、神経細胞から抽出したRNA(例、全RNA)を鋳型として、自体公知のRNA定量法、好ましくは定量的RT−PCR法等を用いて測定することができる。
The gene expression level in step (2) is determined by, for example, designing and synthesizing an appropriate primer set, probe, etc. from the nucleotide sequence information of each gene, and using RNA extracted from nerve cells (eg, total RNA) as a template. It can be measured by using a known RNA quantification method, preferably a quantitative RT-PCR method or the like.
被検物質を添加した細胞におけるRNT4、SEMA6A及びDCCからなる群より選択される1以上の遺伝子、並びに/或いはUBE3A遺伝子の発現レベルが、被検物質を添加しない対照細胞での発現レベルと比較して統計学的に有意に低下していれば、該被検物質をASD治療薬の候補として選択することができる。RNT4、SEMA6A又はDCC遺伝子の発現レベルを指標とする場合、好ましくは、ヒット化合物を、遺伝性症候群(好ましくは、Dup15q症候群、脆弱X症候群、レット症候群、結節性硬化症、コステロ症候群、クレーフストラ症候群、さらに好ましくはDup15q症候群)を伴うASDの治療薬候補として選択することができる。また、UBE3A遺伝子の発現レベルを指標とする場合、好ましくは、ヒット化合物を、Dup15q症候群を伴うASDの治療薬候補として選択することができる。
The expression level of one or more genes selected from the group consisting of RNT4, SEMA6A and DCC in the cells supplemented with the test substance and / or the UBE3A gene was compared with the expression level in the control cells not supplemented with the test substance. If it is statistically significantly reduced, the test substance can be selected as a candidate for an ASD therapeutic agent. When the expression level of the RNT4, SEMA6A or DCC gene is used as an index, the hit compound is preferably used as an hereditary syndrome (preferably Dup15q syndrome, Fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome, Clefstra syndrome, etc.). More preferably, it can be selected as a therapeutic drug candidate for ASD with Dup15q syndrome). In addition, when the expression level of the UBE3A gene is used as an index, the hit compound can be preferably selected as a therapeutic drug candidate for ASD with Dup15q syndrome.
本発明はまた、RNT4、SEMA6A及びDCCからなる群より選択される1以上の遺伝子、又はUBE3A遺伝子の発現抑制薬を含有してなる、ASD治療剤を提供する。RNT4、SEMA6A及びDCC遺伝子は、神経突起伸長阻害分子であることが知られており、後述の実施例に記載されるとおり、Dup15q症候群患者由来のiPS細胞から誘導した神経細胞で発現が上昇しており、該神経細胞における神経突起長の減少という表現型とよく一致する。従って、これらの遺伝子発現を抑制する薬剤は、神経突起伸長を促進することにより、ASDの治療効果を奏し得る。該治療剤の治療対象となるASDの種類は特に制限されないが、好ましくは、遺伝性症候群(好ましくは、Dup15q症候群、脆弱X症候群、レット症候群、結節性硬化症、コステロ症候群、クレーフストラ症候群、さらに好ましくはDup15q症候群)を伴うASDであり得る。
The present invention also provides an ASD therapeutic agent comprising one or more genes selected from the group consisting of RNT4, SEMA6A and DCC, or a UBE3A gene expression inhibitor. The RNT4, SEMA6A and DCC genes are known to be neurite outgrowth inhibitory molecules, and their expression is increased in neurons derived from iPS cells derived from patients with Dup15q syndrome, as described in Examples below. It is in good agreement with the phenotype of reduced neurite length in the nerve cells. Therefore, agents that suppress the expression of these genes can exert a therapeutic effect on ASD by promoting neurite outgrowth. The type of ASD to be treated by the therapeutic agent is not particularly limited, but is preferably hereditary syndrome (preferably Dup15q syndrome, fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome, Clefstra syndrome, and more preferably. Can be ASD with Dup15q syndrome).
UBE3A遺伝子は、15q11−13領域内に位置し、Dup15q症候群において発現が上昇している。従って、該遺伝子の発現を抑制する薬剤は、Dup15qに伴う病因を回復させ、神経突起伸長を促進して、ASDの治療効果を奏し得る。該治療剤の治療対象となるASDの種類は特に制限されないが、好ましくは、遺伝性症候群(好ましくは、Dup15q症候群、脆弱X症候群、レット症候群、結節性硬化症、コステロ症候群、クレーフストラ症候群、さらに好ましくはDup15q症候群)を伴うASDであり得る。
The UBE3A gene is located in the 15q11-13 region and its expression is increased in Dup15q syndrome. Therefore, a drug that suppresses the expression of the gene can restore the etiology associated with Dup15q, promote neurite outgrowth, and exert a therapeutic effect on ASD. The type of ASD to be treated by the therapeutic agent is not particularly limited, but is preferably hereditary syndrome (preferably Dup15q syndrome, fragile X syndrome, Rett syndrome, tuberous sclerosis, Costello syndrome, Clefstra syndrome, and more preferably. Can be ASD with Dup15q syndrome).
RNT4、SEMA6A及びDCCからなる群より選択される1以上の遺伝子の発現抑制薬として、例えば、上記のレバミピド類又はソタロール類を挙げることができる。また、UBE3A遺伝子の発現抑制薬として、例えば、上記のリファブチン類を挙げることができる。
Examples of the expression inhibitor of one or more genes selected from the group consisting of RNT4, SEMA6A and DCC include the above-mentioned rebamipides or sotalols. In addition, examples of the UBE3A gene expression inhibitor include the above-mentioned rifabutins.
別の実施態様において、これらの遺伝子の発現抑制薬として、各遺伝子に対するアンチセンス核酸、siRNA、shRNA、miRNA、リボザイムなどの抑制性核酸が挙げられる。これらの抑制性核酸は、各遺伝子をコードする遺伝子のヌクレオチド配列情報に基づいて、例えば、自体公知の設計ソフトを用いて適宜設計し、DNA/RNA自動合成機を用いて容易に合成することができる。また、これらの抑制性核酸の一部は市販されており、それを用いることもできる。
In another embodiment, examples of the expression-suppressing agent for these genes include antisense nucleic acids for each gene, and inhibitory nucleic acids such as siRNA, shRNA, miRNA, and ribozyme. These inhibitory nucleic acids can be appropriately designed based on the nucleotide sequence information of the gene encoding each gene, for example, using design software known per se, and easily synthesized using a DNA / RNA automatic synthesizer. can. In addition, some of these inhibitory nucleic acids are commercially available, and they can also be used.
また別の実施態様において、これらの遺伝子の発現(機能)抑制薬として、各遺伝子の翻訳産物に対する中和抗体が挙げられる。これらの抗体は、自体公知の手法を用いて、適宜製造することができ、また、市販の抗体を用いることもできる。
In another embodiment, as an expression (function) inhibitor of these genes, a neutralizing antibody against a translation product of each gene can be mentioned. These antibodies can be appropriately produced by using a method known per se, or commercially available antibodies can also be used.
さらに別の実施態様において、これらの遺伝子の発現抑制薬として、上記の本発明のスクリーニング方法により選択された被検物質を挙げることができる。
In still another embodiment, the test substance selected by the above-mentioned screening method of the present invention can be mentioned as an expression inhibitor for these genes.
RNT4、SEMA6A、DCC又はUBE3A遺伝子の発現抑制薬は、上記の本発明の促進剤と同様にして、そのまま単独で、又は薬理学的に許容される担体、賦形剤、希釈剤等と混合し、適当な剤型の医薬組成物として経口的又は非経口的に投与することができる。
The RNT4, SEMA6A, DCC or UBE3A gene expression inhibitor can be used alone or mixed with a pharmacologically acceptable carrier, excipient, diluent and the like in the same manner as the above-mentioned accelerator of the present invention. , Can be administered orally or parenterally as a pharmaceutical composition of a suitable dosage form.
以下に実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに何ら限定されるものではない。
The present invention will be described in more detail with reference to examples below, but the present invention is not limited thereto.
材料及び方法
倫理陳述
ヒトiPSCの作製及び使用は、Department of Medicine and Graduate School of Medicine,Kyoto Universityの倫理委員会、Department of Psychiatry,Nagoya University Graduate School of Medicine及び武田薬品工業株式会社によって承認されている。全ての方法を、承認されたガイドラインに従って実施した。正式なインフォームドコンセントを全ての対象から得た。 Materials and Methods Ethics Statements The production and use of human iPSCs is approved by the Department of Medicine and Graduate School of Medicine, Kyoto University's Institutional Review Board, Department of Medicine, and Nagoya University. .. All methods were performed according to approved guidelines. Formal informed consent was obtained from all subjects.
倫理陳述
ヒトiPSCの作製及び使用は、Department of Medicine and Graduate School of Medicine,Kyoto Universityの倫理委員会、Department of Psychiatry,Nagoya University Graduate School of Medicine及び武田薬品工業株式会社によって承認されている。全ての方法を、承認されたガイドラインに従って実施した。正式なインフォームドコンセントを全ての対象から得た。 Materials and Methods Ethics Statements The production and use of human iPSCs is approved by the Department of Medicine and Graduate School of Medicine, Kyoto University's Institutional Review Board, Department of Medicine, and Nagoya University. .. All methods were performed according to approved guidelines. Formal informed consent was obtained from all subjects.
iPSの作製及び細胞培養
idic(15)を有するDup15q−1(GM20562A)及びint dup(15)を有するDup15q−2(GM22571)のリンパ芽球腫細胞株(LCL)を、Coriell Institute(Camden,NJ,USA)から購入した。以前の報告のようにOct3/4、Sox2、Klf4、L−Myc、Lin28、及びドミナントネガティブp53用のエピソームベクターを使用して、Dup15q iPSC(Dup15q−1、Dup15q−2)をLCLから作製し、そして健常対照iPSC(HC−1、HC−2、HC−3及びHC−4)を健常個体の末梢血単核細胞から作製し、そしてStemFit(AK02N又はAK03N,Ajinomoto,Tokyo,Japan)を用いてiMatrix−511(Nippi,Tokyo,Japan)コートプレート上でフィーダーフリー条件下で培養した。HC−3及びHC−4は同じドナーに由来した。Dup15q−3(NMI005_1−13)iPSCをDup15q患者からOct3/4、Sox4、Klf4、L−Myc、Lin28、EBNA1、及びドミナントネガティブp53用のエピソームベクターを使用して作製した。iPS細胞クローンのバックグラウンド情報を表1に示す。 Preparation of iPS and Cell Culture Dup15q-1 (GM20562A) with idic (15) and Dup15q-2 (GM22571) with int dup (15) lymphoblastoma cell line (LCL) were used in Coriell Institute (Camden, NJ). , USA). Dup15q iPSCs (Dup15q-1, Dup15q-2) were made from LCL using episome vectors for Oct3 / 4, Sox2, Klf4, L-Myc, Lin28, and dominant negative p53 as previously reported. Then, healthy control iPSCs (HC-1, HC-2, HC-3 and HC-4) are prepared from peripheral blood mononuclear cells of healthy individuals, and using StemFit (AK02N or AK03N, Ajinomoto, Tokyo, Japan). The cells were cultured under feeder-free conditions on iMatrix-511 (Nippi, Tokyo, Japan) coated plates. HC-3 and HC-4 were derived from the same donor. Dup15q-3 (NMI005_1-13) iPSCs were made from patients with Dup15q using episome vectors for Oct3 / 4, Sox4, Klf4, L-Myc, Lin28, EBNA1, and dominant negative p53. The background information of iPS cell clones is shown in Table 1.
idic(15)を有するDup15q−1(GM20562A)及びint dup(15)を有するDup15q−2(GM22571)のリンパ芽球腫細胞株(LCL)を、Coriell Institute(Camden,NJ,USA)から購入した。以前の報告のようにOct3/4、Sox2、Klf4、L−Myc、Lin28、及びドミナントネガティブp53用のエピソームベクターを使用して、Dup15q iPSC(Dup15q−1、Dup15q−2)をLCLから作製し、そして健常対照iPSC(HC−1、HC−2、HC−3及びHC−4)を健常個体の末梢血単核細胞から作製し、そしてStemFit(AK02N又はAK03N,Ajinomoto,Tokyo,Japan)を用いてiMatrix−511(Nippi,Tokyo,Japan)コートプレート上でフィーダーフリー条件下で培養した。HC−3及びHC−4は同じドナーに由来した。Dup15q−3(NMI005_1−13)iPSCをDup15q患者からOct3/4、Sox4、Klf4、L−Myc、Lin28、EBNA1、及びドミナントネガティブp53用のエピソームベクターを使用して作製した。iPS細胞クローンのバックグラウンド情報を表1に示す。 Preparation of iPS and Cell Culture Dup15q-1 (GM20562A) with idic (15) and Dup15q-2 (GM22571) with int dup (15) lymphoblastoma cell line (LCL) were used in Coriell Institute (Camden, NJ). , USA). Dup15q iPSCs (Dup15q-1, Dup15q-2) were made from LCL using episome vectors for Oct3 / 4, Sox2, Klf4, L-Myc, Lin28, and dominant negative p53 as previously reported. Then, healthy control iPSCs (HC-1, HC-2, HC-3 and HC-4) are prepared from peripheral blood mononuclear cells of healthy individuals, and using StemFit (AK02N or AK03N, Ajinomoto, Tokyo, Japan). The cells were cultured under feeder-free conditions on iMatrix-511 (Nippi, Tokyo, Japan) coated plates. HC-3 and HC-4 were derived from the same donor. Dup15q-3 (NMI005_1-13) iPSCs were made from patients with Dup15q using episome vectors for Oct3 / 4, Sox4, Klf4, L-Myc, Lin28, EBNA1, and dominant negative p53. The background information of iPS cell clones is shown in Table 1.
核型分析、FISH分析及びアレイCGH分析
核型分析及び蛍光インサイチュハイブシダイゼーション(FISH)分析はそれぞれLSI Medience Corp.(Tokyo,Japan)及びSRL Corp.(Tokyo,Japan)によって行われた。アレイ比較ゲノムハイブリダイゼーション(CGH)分析はMacrogen Japan(Kyoto,Japan)によってSurePrint G3 Human Gene CGH Microarray(Agilent,Santa Clara,CA,USA)を使用して行われた。コピー数変動の座標はhgアセンブリーに基づく。 Karyotype analysis, FISH analysis and array CGH analysis Karyotype analysis and fluorescence in situ hybridization (FISH) analysis are described in LSI Medience Corp., respectively. (Tokyo, Japan) and SRL Corp. (Tokyo, Japan). Array comparative genomic hybridization (CGH) analysis was performed by Macrogen Japan (Kyoto, Japan) using the SurePrint G3 Human Gene CGH Microarray (Agilent, Santa Clara, CA, USA). The coordinates of the copy number variation are based on the hg assembly.
核型分析及び蛍光インサイチュハイブシダイゼーション(FISH)分析はそれぞれLSI Medience Corp.(Tokyo,Japan)及びSRL Corp.(Tokyo,Japan)によって行われた。アレイ比較ゲノムハイブリダイゼーション(CGH)分析はMacrogen Japan(Kyoto,Japan)によってSurePrint G3 Human Gene CGH Microarray(Agilent,Santa Clara,CA,USA)を使用して行われた。コピー数変動の座標はhgアセンブリーに基づく。 Karyotype analysis, FISH analysis and array CGH analysis Karyotype analysis and fluorescence in situ hybridization (FISH) analysis are described in LSI Medience Corp., respectively. (Tokyo, Japan) and SRL Corp. (Tokyo, Japan). Array comparative genomic hybridization (CGH) analysis was performed by Macrogen Japan (Kyoto, Japan) using the SurePrint G3 Human Gene CGH Microarray (Agilent, Santa Clara, CA, USA). The coordinates of the copy number variation are based on the hg assembly.
プラダー・ウィリー症候群インプリンティング対照領域(PWS−ICR)におけるメチル化分析及び15番染色体のゲノムコピー数分析
iPSCのゲノムDNAをPureLink genomic DNA kit(Invitrogen,Thermo FISHer Scientific,Somerset,NJ,USA)を製造業者のプロトコルに従って使用して精製した。ゲノムDNAを、Methyl Easy Xceed Rapid DNA Bisuphite Modification Kit(Takara,Otsu,Shiga,Japan)を使用してバイサルファイト変換に供した。次いで、変換されたDNAを母性PWS−ICR及び父性PWS−ICR用のプライマーを使用するPCR反応の鋳型として使用した。母性PWS−ICRを父性PWS−ICRに対して規準化した。精製ゲノムDNAを、15番染色体上のSNORD116−2及び14番染色体上のRNase P RNA component H1遺伝子(RPPH1)用のプライマー(Thermo FISHer Scientific,Waltham,MA,USA)を使用するTaqman定量的リアルタイムPCRの鋳型として使用した。15番染色体のコピー数を、RNase P内部対照に対して規準化した。プライマーリストを表2に示す。 Prader-Willi Syndrome Imprinting Control Region (PWS-ICR) Methylation Analysis and Genomic Copy Count Analysis ofChromosome 15 iPSC Genomic DNA PureLink genomic DNA kit (Invitrogen, Thermo Fisher Scientific, Somerset, NJUS) Purified using according to the vendor's protocol. Genomic DNA was subjected to bisulfite conversion using Methyl Easy Xceed Rapid DNA Bisulfite Modification Kit (Takara, Otsu, Shiga, Japan). The converted DNA was then used as a template for PCR reactions using primers for maternal PWS-ICR and paternal PWS-ICR. Maternal PWS-ICR was standardized for paternal PWS-ICR. Purified genomic DNA is Taqman quantitative real-time PCR using primers (Thermo FISHer Scientific, Waltham, MA, USA) for SNORD116-2 on chromosome 15 and the RNase P RNA component H1 gene (RPPH1) on chromosome 14. Used as a template for. The number of copies of chromosome 15 was normalized to the RNase P internal control. The primer list is shown in Table 2.
iPSCのゲノムDNAをPureLink genomic DNA kit(Invitrogen,Thermo FISHer Scientific,Somerset,NJ,USA)を製造業者のプロトコルに従って使用して精製した。ゲノムDNAを、Methyl Easy Xceed Rapid DNA Bisuphite Modification Kit(Takara,Otsu,Shiga,Japan)を使用してバイサルファイト変換に供した。次いで、変換されたDNAを母性PWS−ICR及び父性PWS−ICR用のプライマーを使用するPCR反応の鋳型として使用した。母性PWS−ICRを父性PWS−ICRに対して規準化した。精製ゲノムDNAを、15番染色体上のSNORD116−2及び14番染色体上のRNase P RNA component H1遺伝子(RPPH1)用のプライマー(Thermo FISHer Scientific,Waltham,MA,USA)を使用するTaqman定量的リアルタイムPCRの鋳型として使用した。15番染色体のコピー数を、RNase P内部対照に対して規準化した。プライマーリストを表2に示す。 Prader-Willi Syndrome Imprinting Control Region (PWS-ICR) Methylation Analysis and Genomic Copy Count Analysis of
iPSCからの皮質神経細胞の作製
iPSCをAccumax(Innovative Cell Technologies,Inc.,San Diego,CA,USA)を使用して単一細胞に解離し、そして懸濁培養用V底96ウェルプレート(Sumitomo Bakelite Co.,Ltd.Tokyo,Japan)中で迅速に再凝集させた。胚様体(EB)を神経誘導期(0~8日目)において、2μMドルソモルフィン(Chemdea LLC,Ridgewood,NJ,USA)及び10μM SB431542(Cayman Chemical,Ann Arbor,MI,USA)を含むDFK5%培地(5%KnockOut Serum Replacement(KSR)(Gibco)、MEM Non−Essential Amino Acids Solution(NEAA)(Invitrogen,Thermo FISHer Scientific)、L−グルタミン(Sigma−Aldrich,St.Louis,MO,USA)、0.1%2−メルカプトエタノール(Sigma−Aldrich)を補充したDMEM/Ham’s F12(Gibco,Thermo FISHer Scientific))中で培養した。神経誘導後、EBをMatrigel(Becton Dickinson,Franklin Lakes,NJ,USA)コート6ウェル培養プレート上に移し、そしてパターン形成期(8~24日目)において1x N2サプリメント(Invitrogen)、2μMドルソモルフィン、及び10μM SB431542を補充したDFK10%(10%KSR、NEAA、L−グルタミン、0.1%2−メルカプトエタノールを補充したDMEM/Ham’s F12)中で培養した。神経前駆細胞をAccumaxを使用してプレート底から分離し、そして神経成熟期(25~28日目)において、1x B27 without Vitamin A(Invitrogen)、1x Glutamax(Invitrogen)、10ng/ml BDNF、10ng/ml GDNF及び10ng/ml NT−3を補充したNeurobasal Medium(Invitrogen)中Matrigelコート96ウェル培養プレート又は384ウェル培養プレート上で培養した。 Preparation of Cortical Neurons from iPSCs iPSCs were dissociated into single cells using Accumax (Innovative Cell Technologies, Inc., San Diego, CA, USA) and V-bottomed 96-well plates for suspension culture (Sumitomo Bakelite). Co., Ltd. Tokyo, Japan) was rapidly reaggregated.DFK 5% containing 2 μM Dolsomorphin (Chemdea LLC, Ridgewood, NJ, USA) and 10 μM SB431542 (Cayman Chemical, Ann Arbor, MI, USA) in the embryonic body (EB) during the nerve induction phase (day 0-8). Medium (5% KnockOut Serum Replacement (KSR) (Gibco), MEM Non-Essential Amino Acids Solution (NEAA) (Invitrogen, Thermo Fisher Scientific, L-Glutamine), L-Glutamine, L-Glutamine. .Cultivated in DMEM / Ham's F12 (Gibco, Thermo Fisher Scientific) supplemented with 1% 2-mercaptoethanol (Sigma-Aldrich). After nerve induction, EB was transferred onto a Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) coated 6-well culture plate and in the patterning phase (8-24 days) 1x N2 supplement (Invitrogen), 2 μM dolsomorphin, And DFK 10% supplemented with 10 μM SB431542 (DMEM / Ham's F12 supplemented with 10% KSR, NEAA, L-glutamine, 0.1% 2-mercaptoethanol). Neuroprogenitor cells were isolated from the bottom of the plate using Accumax, and during neuromaturation (25-28 days), 1x B27 without Vitamin A (Invitrogen), 1x Glutamax (Invitrogen), 10 ng / ml BDNF, 10 ng / Cultures were performed on Matrigel-coated 96-well or 384-well culture plates in Neurobasal Medium (Invitrogen) supplemented with ml GDNF and 10 ng / ml NT-3.
iPSCをAccumax(Innovative Cell Technologies,Inc.,San Diego,CA,USA)を使用して単一細胞に解離し、そして懸濁培養用V底96ウェルプレート(Sumitomo Bakelite Co.,Ltd.Tokyo,Japan)中で迅速に再凝集させた。胚様体(EB)を神経誘導期(0~8日目)において、2μMドルソモルフィン(Chemdea LLC,Ridgewood,NJ,USA)及び10μM SB431542(Cayman Chemical,Ann Arbor,MI,USA)を含むDFK5%培地(5%KnockOut Serum Replacement(KSR)(Gibco)、MEM Non−Essential Amino Acids Solution(NEAA)(Invitrogen,Thermo FISHer Scientific)、L−グルタミン(Sigma−Aldrich,St.Louis,MO,USA)、0.1%2−メルカプトエタノール(Sigma−Aldrich)を補充したDMEM/Ham’s F12(Gibco,Thermo FISHer Scientific))中で培養した。神経誘導後、EBをMatrigel(Becton Dickinson,Franklin Lakes,NJ,USA)コート6ウェル培養プレート上に移し、そしてパターン形成期(8~24日目)において1x N2サプリメント(Invitrogen)、2μMドルソモルフィン、及び10μM SB431542を補充したDFK10%(10%KSR、NEAA、L−グルタミン、0.1%2−メルカプトエタノールを補充したDMEM/Ham’s F12)中で培養した。神経前駆細胞をAccumaxを使用してプレート底から分離し、そして神経成熟期(25~28日目)において、1x B27 without Vitamin A(Invitrogen)、1x Glutamax(Invitrogen)、10ng/ml BDNF、10ng/ml GDNF及び10ng/ml NT−3を補充したNeurobasal Medium(Invitrogen)中Matrigelコート96ウェル培養プレート又は384ウェル培養プレート上で培養した。 Preparation of Cortical Neurons from iPSCs iPSCs were dissociated into single cells using Accumax (Innovative Cell Technologies, Inc., San Diego, CA, USA) and V-bottomed 96-well plates for suspension culture (Sumitomo Bakelite). Co., Ltd. Tokyo, Japan) was rapidly reaggregated.
免疫染色
細胞をPBS中4%パラホルムアルデヒド中で30分間室温で固定し、PBSで洗浄し、そして0.1%Triton X−100及び20%Block Ace(Yukijirushi,Tokyo,Japan)を含有するPBS中で60分間室温で透過処理/ブロックした。0.1%Triton X100を含有するPBS中一晩4℃での一次抗体とのインキュベーションの後、細胞をPBSで3回洗浄し、そして適切な二次抗体と1時間室温でインキュベートした。BIOREVO BZ−9000(Keyence,Osaka,Japan)、Opera phenix(PerkinElmer,Waltham,MA,USA)又はIN Cell Analyzer 6000(GE Healthcare,Chicago,Illinois,USA)を用いて細胞イメージを獲得した。抗体を表3に列挙する。 Immunostained cells were fixed in PBS in 4% paraformaldehyde for 30 minutes at room temperature, washed with PBS, and in PBS containing 0.1% Triton X-100 and 20% Block Ace (Yukijirushi, Tokyo, Japan). Permeation treatment / blocking at room temperature for 60 minutes. After incubation with the primary antibody in PBS containing 0.1% Triton X100 overnight at 4 ° C., cells were washed 3 times with PBS and incubated with the appropriate secondary antibody for 1 hour at room temperature. BIOREVO BZ-9000 (Keyence, Osaka, Japan), Opera phenix (PerkinElmer, Waltham, MA, USA) or IN Cell Analyzer 6000 (GE Healthcare, Chicago, Illinois) acquired cells, Illinois. The antibodies are listed in Table 3.
細胞をPBS中4%パラホルムアルデヒド中で30分間室温で固定し、PBSで洗浄し、そして0.1%Triton X−100及び20%Block Ace(Yukijirushi,Tokyo,Japan)を含有するPBS中で60分間室温で透過処理/ブロックした。0.1%Triton X100を含有するPBS中一晩4℃での一次抗体とのインキュベーションの後、細胞をPBSで3回洗浄し、そして適切な二次抗体と1時間室温でインキュベートした。BIOREVO BZ−9000(Keyence,Osaka,Japan)、Opera phenix(PerkinElmer,Waltham,MA,USA)又はIN Cell Analyzer 6000(GE Healthcare,Chicago,Illinois,USA)を用いて細胞イメージを獲得した。抗体を表3に列挙する。 Immunostained cells were fixed in PBS in 4% paraformaldehyde for 30 minutes at room temperature, washed with PBS, and in PBS containing 0.1% Triton X-100 and 20% Block Ace (Yukijirushi, Tokyo, Japan). Permeation treatment / blocking at room temperature for 60 minutes. After incubation with the primary antibody in PBS containing 0.1% Triton X100 overnight at 4 ° C., cells were washed 3 times with PBS and incubated with the appropriate secondary antibody for 1 hour at room temperature. BIOREVO BZ-9000 (Keyence, Osaka, Japan), Opera phenix (PerkinElmer, Waltham, MA, USA) or IN Cell Analyzer 6000 (GE Healthcare, Chicago, Illinois) acquired cells, Illinois. The antibodies are listed in Table 3.
神経突起の定量
上記のようにiPSCを皮質神経細胞に分化させた。25日目に、細胞をAccumaxを使用して単一細胞に解離し、次いでMatrigelコートプレート中に播種した。使用した培養培地は、1x B27 without Vitamin A、1x Glutamax、10ng/ml BDNF、10ng/ml GDNF及び10ng/ml NT−3を補充したNeurobasal Mediumであった。28日目に、細胞を4%PFAを用いて固定し、そして抗βIIIチューブリン及びDAPIで染色した。染色された細胞をOpera Phenix又はIN CELL6000を使用してイメージングし、そして神経突起長をHarmony High Content Imaging and Analysis Software(PerkinElmer)又はIN Cell Developer toolbox software 1.9(GE Healthcare)によって測定した。 Quantification of neurites iPSCs were differentiated into cortical neurons as described above. Onday 25, cells were dissociated into single cells using Accumax and then seeded in a Matrigel coated plate. The culture medium used was Neurobasal Medium supplemented with 1x B27 without Vitamin A, 1x Glutamax, 10 ng / ml BDNF, 10 ng / ml GDNF and 10 ng / ml NT-3. On day 28, cells were fixed with 4% PFA and stained with anti-βIII tubulin and DAPI. Stained cells were imaged using Opera Phenix or IN CELL 6000 and neurite length was measured by Harmony High Content Imaging and Analysis Software (PerkinElmer) or IN Cell Developer Tole.
上記のようにiPSCを皮質神経細胞に分化させた。25日目に、細胞をAccumaxを使用して単一細胞に解離し、次いでMatrigelコートプレート中に播種した。使用した培養培地は、1x B27 without Vitamin A、1x Glutamax、10ng/ml BDNF、10ng/ml GDNF及び10ng/ml NT−3を補充したNeurobasal Mediumであった。28日目に、細胞を4%PFAを用いて固定し、そして抗βIIIチューブリン及びDAPIで染色した。染色された細胞をOpera Phenix又はIN CELL6000を使用してイメージングし、そして神経突起長をHarmony High Content Imaging and Analysis Software(PerkinElmer)又はIN Cell Developer toolbox software 1.9(GE Healthcare)によって測定した。 Quantification of neurites iPSCs were differentiated into cortical neurons as described above. On
単一細胞RNA−seq
3つの健常対照iPSC及び3つのDup15q iPSCを使用した。皮質神経細胞をこれらのiPSCから上記の方法に基づいて分化させた。28日目に、細胞を収集し、そして単一細胞RNA−seqを行った。単一細胞トランスクリプトーム分析を10X genomics chromium platformを使用して行った。細胞懸濁液を10X Chromium System(10x Genomics,Pleasanton,CA,USA)上に単一細胞単離のために製造業者の説明書に従ってロードした。単一細胞トランスクリプトーム分析を10X genomics chromium platformを使用して行った。RNA配列決定ライブラリーを10X genomics Single Cell 3’ v2プロトコルに従って調製した。得られたcDNAの定量及び品質管理をBioanalyzer High Sensitivity DNA Assay(Aglient)を使用して行った。単一細胞cDNAライブラリーをIllumina HiSeq2500プラットフォーム上でGENEWIZ JAPAN(Saitama,Japan)を使用して配列決定した。分析はAmelieff Co.(Tokyo,Japan)によって行われた。リードを処理し、そしてヒトリファレンスGRCh38に対して10X Genomics Cell Ranger software(v 3.0.1)を使用してアラインし、そしてLoupe Cell browser(v3.0.1)によって可視化した。神経細胞クラスターの単一細胞データを遺伝子発現変動分析のためにscatter(v1.10.1)及びSeurat(v 2.3.0)を使用して分析した。健常神経細胞とDup15q神経細胞との間で差次的に発現される遺伝子をウィルコクソン検定(調整されたP値<0.05)によって同定した。Gene Ontology分析をDAVIDデータベース(http://david.abcc.ncifcrf.gov/)を使用して評価した。ASD関連遺伝子を自閉症関連遺伝子データベースSFARI gene(https://gene.sfari.org/autdb/Welcome.do)及びAutismKB(http://autismkb.cbi.pku.edu.cn/)によって定義した。 Single cell RNA-seq
Three healthy control iPSCs and three Dup15q iPSCs were used. Cortical neurons were differentiated from these iPSCs based on the method described above. On day 28, cells were harvested and single-cell RNA-seq was performed. Single cell transcriptome analysis was performed using the 10X genomics chromium plateform. Cell suspensions were loaded onto a 10X Chromium System (10x Genomics, Pleasanton, CA, USA) according to the manufacturer's instructions for single cell isolation. Single cell transcriptome analysis was performed using the 10X genomics chromium plateform. RNA sequencing libraries were prepared according to the 10X genomics Single Cell 3'v2 protocol. The quantification and quality control of the obtained cDNA were performed using the Bioanalyzer High Sensitivity DNA Assay (Aglient). A single-cell cDNA library was sequenced using GENEWIZ JAPAN (Saitama, Japan) on the Illumina HiSeq2500 platform. The analysis was performed by Amelieff Co., Inc. (Tokyo, Japan). Leads were processed and aligned to human reference GRCh38 using 10X Genomics Cell Ranger software (v 3.0.1) and visualized by Loope Cell browser (v 3.0.1). Single cell data of neuronal clusters were analyzed using scatter (v1.10.1) and Seurat (v 2.3.0) for gene expression variation analysis. Genes differentially expressed between healthy and Dup15q neurons were identified by the Wilcoxon assay (adjusted P-value <0.05). Gene Ontology analysis was evaluated using the DAVID database (http://david.abcc.ncifcrf.gov/). ASD-related genes were defined by the autism-related gene databases SFARI gene (https://gene.sfari.org/autdb/Welcome.do) and AutismKB (http://autismkb.cbi.pku.edu.cn/). ..
3つの健常対照iPSC及び3つのDup15q iPSCを使用した。皮質神経細胞をこれらのiPSCから上記の方法に基づいて分化させた。28日目に、細胞を収集し、そして単一細胞RNA−seqを行った。単一細胞トランスクリプトーム分析を10X genomics chromium platformを使用して行った。細胞懸濁液を10X Chromium System(10x Genomics,Pleasanton,CA,USA)上に単一細胞単離のために製造業者の説明書に従ってロードした。単一細胞トランスクリプトーム分析を10X genomics chromium platformを使用して行った。RNA配列決定ライブラリーを10X genomics Single Cell 3’ v2プロトコルに従って調製した。得られたcDNAの定量及び品質管理をBioanalyzer High Sensitivity DNA Assay(Aglient)を使用して行った。単一細胞cDNAライブラリーをIllumina HiSeq2500プラットフォーム上でGENEWIZ JAPAN(Saitama,Japan)を使用して配列決定した。分析はAmelieff Co.(Tokyo,Japan)によって行われた。リードを処理し、そしてヒトリファレンスGRCh38に対して10X Genomics Cell Ranger software(v 3.0.1)を使用してアラインし、そしてLoupe Cell browser(v3.0.1)によって可視化した。神経細胞クラスターの単一細胞データを遺伝子発現変動分析のためにscatter(v1.10.1)及びSeurat(v 2.3.0)を使用して分析した。健常神経細胞とDup15q神経細胞との間で差次的に発現される遺伝子をウィルコクソン検定(調整されたP値<0.05)によって同定した。Gene Ontology分析をDAVIDデータベース(http://david.abcc.ncifcrf.gov/)を使用して評価した。ASD関連遺伝子を自閉症関連遺伝子データベースSFARI gene(https://gene.sfari.org/autdb/Welcome.do)及びAutismKB(http://autismkb.cbi.pku.edu.cn/)によって定義した。 Single cell RNA-seq
Three healthy control iPSCs and three Dup15q iPSCs were used. Cortical neurons were differentiated from these iPSCs based on the method described above. On day 28, cells were harvested and single-cell RNA-seq was performed. Single cell transcriptome analysis was performed using the 10X genomics chromium plateform. Cell suspensions were loaded onto a 10X Chromium System (10x Genomics, Pleasanton, CA, USA) according to the manufacturer's instructions for single cell isolation. Single cell transcriptome analysis was performed using the 10X genomics chromium plateform. RNA sequencing libraries were prepared according to the 10X genomics Single Cell 3'v2 protocol. The quantification and quality control of the obtained cDNA were performed using the Bioanalyzer High Sensitivity DNA Assay (Aglient). A single-cell cDNA library was sequenced using GENEWIZ JAPAN (Saitama, Japan) on the Illumina HiSeq2500 platform. The analysis was performed by Amelieff Co., Inc. (Tokyo, Japan). Leads were processed and aligned to human reference GRCh38 using 10X Genomics Cell Ranger software (v 3.0.1) and visualized by Loope Cell browser (v 3.0.1). Single cell data of neuronal clusters were analyzed using scatter (v1.10.1) and Seurat (v 2.3.0) for gene expression variation analysis. Genes differentially expressed between healthy and Dup15q neurons were identified by the Wilcoxon assay (adjusted P-value <0.05). Gene Ontology analysis was evaluated using the DAVID database (http://david.abcc.ncifcrf.gov/). ASD-related genes were defined by the autism-related gene databases SFARI gene (https://gene.sfari.org/autdb/Welcome.do) and AutismKB (http://autismkb.cbi.pku.edu.cn/). ..
Dup15q神経細胞を使用した神経突起長を回復させる化合物のスクリーニング
Dup15q−1 iPSCから作製された皮質神経細胞を化合物スクリーニングのために使用した。アメリカ食品医薬局(FDA)承認化合物の1269個の化合物からなるPrestwick Chemical Library(Prestwick Chemical,Washington,DC,USA)を使用した。化合物をジメチルスルホキシド(DMSO)で希釈し、そしてDMSOの最終濃度を0.1%に維持した。細胞を化合物(最終濃度、3μM及び10μM)で26日目に処理し、そして28日目に固定及び染色した。DMSOを陰性対照に使用した。βIIIチューブリンで染色された細胞をOpera Phenix(PerkinElmer)を使用してイメージングし、そして化合物の効果をHarmony Analysis Softwareによって算出された神経突起長測定値によって評価した。二次スクリーニングのための化合物を表4に列挙する。 Screening of compounds that restore neurite length using Dup15q neurons Cortical neurons prepared from Dup15q-1 iPSC were used for compound screening. A Presswick Chemical Library (Prestwick Chemical, Washington, DC, USA) consisting of 1269 compounds approved by the US Food and Drug Administration (FDA) was used. The compound was diluted with dimethyl sulfoxide (DMSO) and the final concentration of DMSO was maintained at 0.1%. Cells were treated with compound (final concentrations 3 μM and 10 μM) on day 26 and fixed and stained on day 28. DMSO was used as a negative control. Cells stained with βIII tubulin were imaged using Opera Phenix (PerkinElmer) and the effect of the compound was assessed by neurite length measurements calculated by Harmony Analysis Software. The compounds for secondary screening are listed in Table 4.
Dup15q−1 iPSCから作製された皮質神経細胞を化合物スクリーニングのために使用した。アメリカ食品医薬局(FDA)承認化合物の1269個の化合物からなるPrestwick Chemical Library(Prestwick Chemical,Washington,DC,USA)を使用した。化合物をジメチルスルホキシド(DMSO)で希釈し、そしてDMSOの最終濃度を0.1%に維持した。細胞を化合物(最終濃度、3μM及び10μM)で26日目に処理し、そして28日目に固定及び染色した。DMSOを陰性対照に使用した。βIIIチューブリンで染色された細胞をOpera Phenix(PerkinElmer)を使用してイメージングし、そして化合物の効果をHarmony Analysis Softwareによって算出された神経突起長測定値によって評価した。二次スクリーニングのための化合物を表4に列挙する。 Screening of compounds that restore neurite length using Dup15q neurons Cortical neurons prepared from Dup15q-1 iPSC were used for compound screening. A Presswick Chemical Library (Prestwick Chemical, Washington, DC, USA) consisting of 1269 compounds approved by the US Food and Drug Administration (FDA) was used. The compound was diluted with dimethyl sulfoxide (DMSO) and the final concentration of DMSO was maintained at 0.1%. Cells were treated with compound (
細胞毒性試験
皮質神経細胞を化合物(濃度、3μM又は10μM)で26日目に処理し、そして28日目に4%PFAを用いて固定した。βIIIチューブリン抗体及びDAPIで染色された細胞をCell Analyzer 6000を使用してイメージングし、そして神経細胞の数をIN Cell Developer toolbox software 1.9(GE Healthcare)によって定量して、細胞毒性を評価した。 Cytotoxicity test Cortical neurons were treated with compound (concentration, 3 μM or 10 μM) on day 26 and fixed with 4% PFA on day 28. Cells stained with βIII tubulin antibody and DAPI were imaged using Cell Analyzer 6000, and the number of neurons was quantified by IN Cell Developer toolbox software 1.9 (GE Healthcare) to assess cytotoxicity. ..
皮質神経細胞を化合物(濃度、3μM又は10μM)で26日目に処理し、そして28日目に4%PFAを用いて固定した。βIIIチューブリン抗体及びDAPIで染色された細胞をCell Analyzer 6000を使用してイメージングし、そして神経細胞の数をIN Cell Developer toolbox software 1.9(GE Healthcare)によって定量して、細胞毒性を評価した。 Cytotoxicity test Cortical neurons were treated with compound (concentration, 3 μM or 10 μM) on day 26 and fixed with 4% PFA on day 28. Cells stained with βIII tubulin antibody and DAPI were imaged using Cell Analyzer 6000, and the number of neurons was quantified by IN Cell Developer toolbox software 1.9 (GE Healthcare) to assess cytotoxicity. ..
定量的RT−PCR
全RNAをRNeasy kit(QIAGEN)を使用して抽出した。1μgのRNAをReverTra Ace(TOYOBO,Osaka,Japan)を使用して逆転写した。定量的PCR分析を、Step One Plus(Applied Biosystems,Waltham,MA,USA)を使用するSYBR Premix Ex TaqII(TAKARA)又はTaqmanプローブを用いる逆転写反応によって行った。プライマーセットを表2に列挙する。 Quantitative RT-PCR
Total RNA was extracted using RNeasy kit (QIAGEN). 1 μg of RNA was reverse transcribed using RiverTra Ace (TOYOBO, Osaka, Japan). Quantitative PCR analysis was performed by reverse transcriptase using a SYBR Premix Ex TaqII (TAKARA) or Taqman probe using Step One Plus (Applied Biosystems, Waltham, MA, USA). The primer sets are listed in Table 2.
全RNAをRNeasy kit(QIAGEN)を使用して抽出した。1μgのRNAをReverTra Ace(TOYOBO,Osaka,Japan)を使用して逆転写した。定量的PCR分析を、Step One Plus(Applied Biosystems,Waltham,MA,USA)を使用するSYBR Premix Ex TaqII(TAKARA)又はTaqmanプローブを用いる逆転写反応によって行った。プライマーセットを表2に列挙する。 Quantitative RT-PCR
Total RNA was extracted using RNeasy kit (QIAGEN). 1 μg of RNA was reverse transcribed using RiverTra Ace (TOYOBO, Osaka, Japan). Quantitative PCR analysis was performed by reverse transcriptase using a SYBR Premix Ex TaqII (TAKARA) or Taqman probe using Step One Plus (Applied Biosystems, Waltham, MA, USA). The primer sets are listed in Table 2.
統計分析
結果をスチューデントt検定、ウィルコクソン順位和検定及び一元配置分散分析(ANOVA)及び続くダネット検定によって分析して、統計的有意性を決定した。p<0.05で差を有意であるとみなした。R software(version 3.5.1.,R Foundation for stastical computing,Vienna,Austria)又はSPSS(IBM,Chicago,IL,USA)を使用して分析を行った。全ての棒グラフは平均±SEMを表す。 Statistical analysis results were analyzed by Student's t-test, Wilcoxon rank sum test and one-way analysis of variance (ANOVA) followed by Danette test to determine statistical significance. The difference was considered significant at p <0.05. Analysis was performed using R software (version 3.5.1., R Foundation for static computing, Vienna, USA) or SPSS (IBM, Chicago, IL, USA). All bar graphs represent mean ± SEM.
結果をスチューデントt検定、ウィルコクソン順位和検定及び一元配置分散分析(ANOVA)及び続くダネット検定によって分析して、統計的有意性を決定した。p<0.05で差を有意であるとみなした。R software(version 3.5.1.,R Foundation for stastical computing,Vienna,Austria)又はSPSS(IBM,Chicago,IL,USA)を使用して分析を行った。全ての棒グラフは平均±SEMを表す。 Statistical analysis results were analyzed by Student's t-test, Wilcoxon rank sum test and one-way analysis of variance (ANOVA) followed by Danette test to determine statistical significance. The difference was considered significant at p <0.05. Analysis was performed using R software (version 3.5.1., R Foundation for static computing, Vienna, USA) or SPSS (IBM, Chicago, IL, USA). All bar graphs represent mean ± SEM.
結果:
Dup15q患者由来iPSCの特徴
本発明者らは、母性染色体15q11−13領域のint dup(15)を有する2人のDup15q患者及び母性idic(15)を有する1人の患者由来のヒトiPSCを分析した(表1)。多能性マーカーNANOG及びSSEA−4によりiPSCの免疫染色を行った(図1A)。Dup15q iPSCは、Gバンド分染法及びそれぞれユビキチンタンパク質リガーゼE3A(UBE3A)遺伝子用のプローブ及び15番染色体における対照プローブを用いるDNA FISH分析によって、Dup15q−1では47,XX,+idic(15)ish 15q11.2(D15S10 x2)idic(15;15)(D15Z1++,D15S10++);Dup15q−2では46,XY,dup(15)ish dup(15)(q11.2q13.1)(D15S10++);及びDup15q−3では46,XX,dup(15)ish dup(15)(q11.2q13.1)(D15S10++)の核型及び重複を有することが示された(図6A及び図1B)。iPSCゲノムDNAを使用してアレイ比較ゲノムハイブリダイゼーション(aCGH)を行ったところ、Dup15q−1では15q11.2q13.2(2010254−30652489)の四重複及び15q11.2q13.2(23208842−28535051)の三重複、Dup15q−2では15q11.2q13.2(23208842−28535051)の三重複、Dup15q−3では15q11.2q13.2(22765628−28535051)の三重複が確認された(図1C及び図6B)。本発明者らはまた、メチル化特異的PCR及びゲノムqPCRを使用し、母性15番染色体のコピー数又は15番染色体の全コピー数を調べるために、プラダー・ウィリー症候群インプリンティング対照領域(PWS−ICR)のメチル化状態を分析した。Dup15q iPSCは約2又は3コピーの母性15q11.2−13、及び、3又は4コピーの15q11.2−13を有していた(図1D)。図1E及び表5は、それぞれ15番染色体構造及びDup15q iPSCの遺伝情報を示す。以上の結果から、15番染色体上で増加したコピー数及びPWS−ICRにおけるDNAメチル化が、Dup15q患者由来iPSCで予測されるCNVと一致することが示された。 result:
Characteristics of iPSC derived from Dup15q patients We analyzed human iPSCs derived from two Dup15q patients with int dup (15) in the maternal chromosome 15q11-13 region and one patient with maternal idic (15). (Table 1). Immunostaining of iPSCs was performed with pluripotent markers NANOG and SSEA-4 (Fig. 1A). Dup15q iPSC was found to be 47, XX, + idic (15) ish 15q11 on Dup15q-1 by G-banding and DNA FISH analysis using a probe for the ubiquitin protein ligase E3A (UBE3A) gene and a control probe onchromosome 15, respectively. .2 (D15S10 x2) idic (15; 15) (D15Z1 ++, D15S10 ++); 46, XY, dup (15) issh dup (15) (q11.2q13.1) (D15S10 ++); and Dup15q-3 for Dup15q-2. 46, XX, dup (15) is dup (15) (q11.2q13.1) (D15S10 ++) was shown to have karyotypes and overlaps (FIGS. 6A and 1B). Array comparative genomic hybridization (aCGH) was performed using iPSC genomic DNA, and Dup15q-1 had four duplications of 15q11.2q13.2 (2010254-30562489) and three of 15q11.2q13.2. Overlapping, three duplications of 15q11.2q13.2 (23208842-28535051) for Dup15q-2 and three duplications of 15q11.2q13.2. (22765628-28535051) for Dup15q-3 were confirmed (FIGS. 1C and 6B). We also use methylation-specific PCR and genomic qPCR to determine the number of copies of maternal chromosome 15 or the total number of copies of chromosome 15 in the Prader-Willi syndrome imprinting control region (PWS-). The methylation status of ICR) was analyzed. The Dup15q iPSC had about 2 or 3 copies of maternal 15q 11.2-13 and 3 or 4 copies of 15q 11.2-13 (FIG. 1D). 1E and Table 5 show the genetic information of chromosome 15 structure and Dup15q iPSC, respectively. These results indicate that the increased copy number on chromosome 15 and DNA methylation in PWS-ICR are consistent with the CNV predicted for iPSC from Dup15q patients.
Dup15q患者由来iPSCの特徴
本発明者らは、母性染色体15q11−13領域のint dup(15)を有する2人のDup15q患者及び母性idic(15)を有する1人の患者由来のヒトiPSCを分析した(表1)。多能性マーカーNANOG及びSSEA−4によりiPSCの免疫染色を行った(図1A)。Dup15q iPSCは、Gバンド分染法及びそれぞれユビキチンタンパク質リガーゼE3A(UBE3A)遺伝子用のプローブ及び15番染色体における対照プローブを用いるDNA FISH分析によって、Dup15q−1では47,XX,+idic(15)ish 15q11.2(D15S10 x2)idic(15;15)(D15Z1++,D15S10++);Dup15q−2では46,XY,dup(15)ish dup(15)(q11.2q13.1)(D15S10++);及びDup15q−3では46,XX,dup(15)ish dup(15)(q11.2q13.1)(D15S10++)の核型及び重複を有することが示された(図6A及び図1B)。iPSCゲノムDNAを使用してアレイ比較ゲノムハイブリダイゼーション(aCGH)を行ったところ、Dup15q−1では15q11.2q13.2(2010254−30652489)の四重複及び15q11.2q13.2(23208842−28535051)の三重複、Dup15q−2では15q11.2q13.2(23208842−28535051)の三重複、Dup15q−3では15q11.2q13.2(22765628−28535051)の三重複が確認された(図1C及び図6B)。本発明者らはまた、メチル化特異的PCR及びゲノムqPCRを使用し、母性15番染色体のコピー数又は15番染色体の全コピー数を調べるために、プラダー・ウィリー症候群インプリンティング対照領域(PWS−ICR)のメチル化状態を分析した。Dup15q iPSCは約2又は3コピーの母性15q11.2−13、及び、3又は4コピーの15q11.2−13を有していた(図1D)。図1E及び表5は、それぞれ15番染色体構造及びDup15q iPSCの遺伝情報を示す。以上の結果から、15番染色体上で増加したコピー数及びPWS−ICRにおけるDNAメチル化が、Dup15q患者由来iPSCで予測されるCNVと一致することが示された。 result:
Characteristics of iPSC derived from Dup15q patients We analyzed human iPSCs derived from two Dup15q patients with int dup (15) in the maternal chromosome 15q11-13 region and one patient with maternal idic (15). (Table 1). Immunostaining of iPSCs was performed with pluripotent markers NANOG and SSEA-4 (Fig. 1A). Dup15q iPSC was found to be 47, XX, + idic (15) ish 15q11 on Dup15q-1 by G-banding and DNA FISH analysis using a probe for the ubiquitin protein ligase E3A (UBE3A) gene and a control probe on
Dup15q神経細胞は神経突起長の有意な減少を示した
皮質機能不全は、ASDにおける中心的な症状を引き起こすと考えられている。また、自閉症患者神経細胞が神経分化能力及び神経細胞形態(例えば神経突起長)の変化を呈することが報告されている。本発明者らは、改変SFEBq(迅速な凝集をともなう胚様体様凝集体の無血清培養)分化法を使用してiPSC株から皮質神経細胞を作製し(図2A)、βIIIチューブリン免疫染色のイメージを使用してDup15q神経細胞の神経細胞分化能及び形態を分析した(図2B)。健常対照とDup15q神経細胞との分化効率を比較したところ、有意な差は認められなかった(図2C)。Dup15q神経細胞は、健常対照神経細胞と比較して神経突起長が有意に短縮していることが確認された(図2D及び図7)。 Dup15q neurons showed a significant reduction in neurite length Cortical dysfunction is thought to cause central symptoms in ASD. It has also been reported that nerve cells of autistic patients exhibit changes in nerve differentiation ability and nerve cell morphology (for example, neurite length). We generated cortical neurons from iPSC strains using a modified SFEBq (serum-free culture of embryoid body-like aggregates with rapid aggregation) differentiation method (FIG. 2A) and βIII tubulin immunostaining. The neuronal differentiation potential and morphology of Dup15q neurons were analyzed using the image of (Fig. 2B). When the differentiation efficiency of the healthy control and the Dup15q neuron was compared, no significant difference was observed (Fig. 2C). It was confirmed that the Dup15q neurons had a significantly shorter neurite length as compared with the healthy control neurons (FIGS. 2D and 7).
皮質機能不全は、ASDにおける中心的な症状を引き起こすと考えられている。また、自閉症患者神経細胞が神経分化能力及び神経細胞形態(例えば神経突起長)の変化を呈することが報告されている。本発明者らは、改変SFEBq(迅速な凝集をともなう胚様体様凝集体の無血清培養)分化法を使用してiPSC株から皮質神経細胞を作製し(図2A)、βIIIチューブリン免疫染色のイメージを使用してDup15q神経細胞の神経細胞分化能及び形態を分析した(図2B)。健常対照とDup15q神経細胞との分化効率を比較したところ、有意な差は認められなかった(図2C)。Dup15q神経細胞は、健常対照神経細胞と比較して神経突起長が有意に短縮していることが確認された(図2D及び図7)。 Dup15q neurons showed a significant reduction in neurite length Cortical dysfunction is thought to cause central symptoms in ASD. It has also been reported that nerve cells of autistic patients exhibit changes in nerve differentiation ability and nerve cell morphology (for example, neurite length). We generated cortical neurons from iPSC strains using a modified SFEBq (serum-free culture of embryoid body-like aggregates with rapid aggregation) differentiation method (FIG. 2A) and βIII tubulin immunostaining. The neuronal differentiation potential and morphology of Dup15q neurons were analyzed using the image of (Fig. 2B). When the differentiation efficiency of the healthy control and the Dup15q neuron was compared, no significant difference was observed (Fig. 2C). It was confirmed that the Dup15q neurons had a significantly shorter neurite length as compared with the healthy control neurons (FIGS. 2D and 7).
Dup15q神経細胞における神経突起関連遺伝子の発現解析
Dup15q神経細胞の分子表現型を調べるために、健常対照及びDup15q患者iPSC由来神経細胞を使用して単一細胞RNAシークエンシング(scRNA−seq)を行った。細胞型/状態を同定するためにデータをK平均法クラスタリングに供し、これを10x Genomics softwareを使用してt分布型確立的近傍埋め込み(t−SNE)プロットに投影した。scRNA−seqデータのK平均クラスタリングにより、9つの主要なクラスターを同定した(図3A、B)。9つのクラスターのうちクラスター4は神経細胞特異的遺伝子の発現を有する神経細胞クラスターとして、クラスター8はグリア細胞特異的遺伝子の発現を含むグリア細胞クラスターとして、その他のクラスターは神経幹細胞特異的遺伝子の発現を有する神経幹細胞クラスターであることが見出された(図3C~E)。また、クラスター4は、TUBB3、ELAVL4、MAPT及びTBR1を、クラスター8はS100B及びGFRA2を、その他のクラスターはPAX6及びSOX2をそれぞれ含んでいた。
神経細胞クラスターのデータを使用して、本発明者らは、Dup15q神経細胞の網羅的遺伝子発現を、健常対照神経細胞のものと比較した。結果は、健常対照神経細胞と比較して、Dup15q神経細胞において、染色体15q11−13内に位置するUBE3A、HERC2(HECT及びRLDドメイン含有E3ユビキチンタンパク質リガーゼ)及びNIPA2(NIPAマグネシウムトランスポーター2)を含む114個の発現が増加した遺伝子と、13個の発現が低下した遺伝子が見出された(図4A、B及び図8)。さらに、Gene Ontology(GO)分析からは、発現が増加した遺伝子が、Neuron projection developmentのGOターム及び他の発生関連タームにおいて富化されることが示された(図4C、図9A、及び表6)。Neuron projection developmentのGOに含まれる遺伝子の発現レベルを図4Dに示す。興味深いことに、RTN4(Reticulon 4)、SEMA6A(Semaphorin 6A)及びDCC(Deleted in colorectal cancer)が神経突起発生を負に調節することがこれまでに報告されている。これらの遺伝子の誘導は、神経突起長の減少を生じ得、そしてDup15q神経細胞において検出される形態異常を支持し得る。 Expression analysis of neurite-related genes in Dup15q neurons Single-cell RNA sequencing (scRNA-seq) was performed using healthy controls and Dup15q patient iPSC-derived neurons to investigate the molecular phenotype of Dup15q neurons. .. Data were subjected to K-means clustering to identify cell types / states and projected onto a t-distributed established neighborhood embedding (t-SNE) plot using a 10x Genomics software. Nine major clusters were identified by K-means clustering of scRNA-seq data (FIGS. 3A, B). Of the nine clusters,cluster 4 is a neuron cluster having neural cell-specific gene expression, cluster 8 is a glial cell cluster containing glial cell-specific gene expression, and the other clusters are neural stem cell-specific gene expression. It was found to be a neural stem cell cluster with (FIGS. 3C to E). In addition, cluster 4 contained TUBB3, ELAVL4, MAPT and TBR1, cluster 8 contained S100B and GFRA2, and other clusters contained PAX6 and SOX2, respectively.
Using data from neural clusters, we compared the exhaustive gene expression of Dup15q neurons with that of healthy control neurons. Results include UBE3A, HERC2 (HECT and RLD domain-containing E3 ubiquitin protein ligase) and NIPA2 (NIPA magnesium transporter 2) located within chromosome 15q11-13 in Dup15q neurons compared to healthy control neurons. 114 genes with increased expression and 13 genes with decreased expression were found (FIGS. 4A, B and 8). In addition, Gene Ontology (GO) analysis showed that genes with increased expression were enriched in the GO term and other developmental related terms of the Neuron projection development (FIGS. 4C, 9A, and 6). ). The expression level of the gene contained in the GO of Neuron projection development is shown in FIG. 4D. Interestingly, it has been previously reported that RTN4 (Reticulon 4), SEMA6A (Semaphorin 6A) and DCC (Deleted in colorectal canceler) negatively regulate neurite development. Induction of these genes can result in a decrease in neurite length and can support morphological abnormalities detected in Dup15q neurons.
Dup15q神経細胞の分子表現型を調べるために、健常対照及びDup15q患者iPSC由来神経細胞を使用して単一細胞RNAシークエンシング(scRNA−seq)を行った。細胞型/状態を同定するためにデータをK平均法クラスタリングに供し、これを10x Genomics softwareを使用してt分布型確立的近傍埋め込み(t−SNE)プロットに投影した。scRNA−seqデータのK平均クラスタリングにより、9つの主要なクラスターを同定した(図3A、B)。9つのクラスターのうちクラスター4は神経細胞特異的遺伝子の発現を有する神経細胞クラスターとして、クラスター8はグリア細胞特異的遺伝子の発現を含むグリア細胞クラスターとして、その他のクラスターは神経幹細胞特異的遺伝子の発現を有する神経幹細胞クラスターであることが見出された(図3C~E)。また、クラスター4は、TUBB3、ELAVL4、MAPT及びTBR1を、クラスター8はS100B及びGFRA2を、その他のクラスターはPAX6及びSOX2をそれぞれ含んでいた。
神経細胞クラスターのデータを使用して、本発明者らは、Dup15q神経細胞の網羅的遺伝子発現を、健常対照神経細胞のものと比較した。結果は、健常対照神経細胞と比較して、Dup15q神経細胞において、染色体15q11−13内に位置するUBE3A、HERC2(HECT及びRLDドメイン含有E3ユビキチンタンパク質リガーゼ)及びNIPA2(NIPAマグネシウムトランスポーター2)を含む114個の発現が増加した遺伝子と、13個の発現が低下した遺伝子が見出された(図4A、B及び図8)。さらに、Gene Ontology(GO)分析からは、発現が増加した遺伝子が、Neuron projection developmentのGOターム及び他の発生関連タームにおいて富化されることが示された(図4C、図9A、及び表6)。Neuron projection developmentのGOに含まれる遺伝子の発現レベルを図4Dに示す。興味深いことに、RTN4(Reticulon 4)、SEMA6A(Semaphorin 6A)及びDCC(Deleted in colorectal cancer)が神経突起発生を負に調節することがこれまでに報告されている。これらの遺伝子の誘導は、神経突起長の減少を生じ得、そしてDup15q神経細胞において検出される形態異常を支持し得る。 Expression analysis of neurite-related genes in Dup15q neurons Single-cell RNA sequencing (scRNA-seq) was performed using healthy controls and Dup15q patient iPSC-derived neurons to investigate the molecular phenotype of Dup15q neurons. .. Data were subjected to K-means clustering to identify cell types / states and projected onto a t-distributed established neighborhood embedding (t-SNE) plot using a 10x Genomics software. Nine major clusters were identified by K-means clustering of scRNA-seq data (FIGS. 3A, B). Of the nine clusters,
Using data from neural clusters, we compared the exhaustive gene expression of Dup15q neurons with that of healthy control neurons. Results include UBE3A, HERC2 (HECT and RLD domain-containing E3 ubiquitin protein ligase) and NIPA2 (NIPA magnesium transporter 2) located within chromosome 15q11-13 in Dup15q neurons compared to healthy control neurons. 114 genes with increased expression and 13 genes with decreased expression were found (FIGS. 4A, B and 8). In addition, Gene Ontology (GO) analysis showed that genes with increased expression were enriched in the GO term and other developmental related terms of the Neuron projection development (FIGS. 4C, 9A, and 6). ). The expression level of the gene contained in the GO of Neuron projection development is shown in FIG. 4D. Interestingly, it has been previously reported that RTN4 (Reticulon 4), SEMA6A (Semaphorin 6A) and DCC (Deleted in colorectal canceler) negatively regulate neurite development. Induction of these genes can result in a decrease in neurite length and can support morphological abnormalities detected in Dup15q neurons.
本発明者らは、Dup15q神経細胞において差次的に発現される遺伝子と公的なASD関連遺伝子データベースであるSFARI及びAutismKBにおいて列挙される遺伝子との共通性を分析した。差次的に発現される127個の遺伝子の中で、33個の遺伝子が公的ASD関連遺伝子データベース中に存在していた(図9B)。さらに、Dup15q神経細胞のRNA−seqにおいて、Neuron projection developmentのGOタームが付与された、富化された遺伝子の約53%(17遺伝子中9個)はASD関連遺伝子であった(図9C)。一方、Nervous system developmentの42%(33遺伝子中14遺伝子)、Adherens junctionの50%(14遺伝子中7遺伝子)、及びDup15q神経細胞において発現が低下した遺伝子の38%(13遺伝子中5遺伝子)がASD関連遺伝子と重複していた(図9B及びC)。
分子表現型アッセイの結果から、Dup15q神経細胞の細胞表現型であるより短い神経突起を再現できること、そして神経突起成長阻害分子の発現誘導もまたDup15q神経細胞において神経突起長の減少の原因となり得ることが示唆された(図4E)。 We analyzed the commonality between genes that are differentially expressed in Dup15q neurons and the genes listed in the public ASD-related gene databases SFARI and AutismKB. Of the 127 genes that are differentially expressed, 33 genes were present in the public ASD-related gene database (Fig. 9B). Furthermore, in RNA-seq of Dup15q neurons, about 53% (9 out of 17 genes) of enriched genes endowed with the GO term of Neuron projection development were ASD-related genes (FIG. 9C). On the other hand, 42% of Nervous system development (14 genes out of 33 genes), 50% of Adherens junction (7 genes out of 14 genes), and 38% of genes whose expression was reduced in Dup15q nerve cells (5 genes out of 13 genes). It overlapped with the ASD-related gene (FIGS. 9B and C).
From the results of the molecular phenotype assay, it is possible to reproduce shorter neurites that are the cell phenotype of Dup15q neurons, and induction of expression of neurite growth inhibitory molecules can also cause a decrease in neurite length in Dup15q neurons. Was suggested (Fig. 4E).
分子表現型アッセイの結果から、Dup15q神経細胞の細胞表現型であるより短い神経突起を再現できること、そして神経突起成長阻害分子の発現誘導もまたDup15q神経細胞において神経突起長の減少の原因となり得ることが示唆された(図4E)。 We analyzed the commonality between genes that are differentially expressed in Dup15q neurons and the genes listed in the public ASD-related gene databases SFARI and AutismKB. Of the 127 genes that are differentially expressed, 33 genes were present in the public ASD-related gene database (Fig. 9B). Furthermore, in RNA-seq of Dup15q neurons, about 53% (9 out of 17 genes) of enriched genes endowed with the GO term of Neuron projection development were ASD-related genes (FIG. 9C). On the other hand, 42% of Nervous system development (14 genes out of 33 genes), 50% of Adherens junction (7 genes out of 14 genes), and 38% of genes whose expression was reduced in Dup15q nerve cells (5 genes out of 13 genes). It overlapped with the ASD-related gene (FIGS. 9B and C).
From the results of the molecular phenotype assay, it is possible to reproduce shorter neurites that are the cell phenotype of Dup15q neurons, and induction of expression of neurite growth inhibitory molecules can also cause a decrease in neurite length in Dup15q neurons. Was suggested (Fig. 4E).
短い神経突起長を回復させる化合物のスクリーニング
Dup15−q−1 iPSC由来神経細胞、及び1269個の化合物からなるFDA承認薬物ライブラリーを使用して、本発明者らは、神経突起長をリードアウトとして神経突起伸長誘導物質のスクリーニングを行った。Dup15−q−1 iPSCを28日間神経細胞に分化させた後、1269個のFDA承認化合物で2日間処理し、次いで神経突起長をβIIIチューブリンの免疫染色を使用するハイコンテント分析によって評価した(図5A)。スクリーニングの模式的ストラテジーを図5Bに示す。化合物一次スクリーニングの結果を図5Cに示す。これら18個の化合物の再現性をチェックした後にヒット基準を3標準偏差以上と定義した(表4)。本発明者らは、レバミピド、ソタロール及びリファブチンが、Dup15q神経細胞において神経突起長を有意に増加させることを確認した(図5D及びE)。他方、レバミピド、ソタロール及びリファブチンは神経細胞生存性に対する効果を有しなかった(図10B)。
次に、本発明者らは、神経突起成長阻害分子の遺伝子発現に対するこれらの化合物の効果を調べた。RTN4及びSEMA6A遺伝子の発現は、ソタロール又はレバミピドによって減少させられたが、リファブチンによっては減少させられなかった。DCC mRNAの発現は、レバミピドによってのみ抑制された(図5F)。本発明者らはまた、UBE3A遺伝子(15q11−13に位置する)の発現に対するこれらの化合物の効果を調べた。結果は、UBE3A mRNAの発現がリファブチン処理によって減少したが、ソタロール及びレバミピド処理によっては減少しないことが確認された(図10C)。 Screening for Compounds to Restore Short Neurite Length Using an FDA-approved drug library consisting of Dup15-q-1 iPSC-derived neurons and 1269 compounds, we used neurite length as a lead-out. We screened for neurite outgrowth inducers. Dup15-q-1 iPSC was differentiated into neurons for 28 days, then treated with 1269 FDA approved compounds for 2 days, then neurite length was assessed by high content analysis using βIII tubulin immunostaining ( FIG. 5A). A schematic screening strategy is shown in FIG. 5B. The results of the primary compound screening are shown in FIG. 5C. After checking the reproducibility of these 18 compounds, the hit criterion was defined as 3 standard deviations or greater (Table 4). We confirmed that rebamipide, sotalol and rifabutin significantly increased neurite length in Dup15q neurons (FIGS. 5D and E). On the other hand, rebamipide, sotalol and rifabutin had no effect on neuronal viability (Fig. 10B).
Next, we investigated the effect of these compounds on the gene expression of neurite growth inhibitory molecules. Expression of the RTN4 and SEMA6A genes was reduced by sotalol or rebamipide, but not by rifabutin. Expression of DCC mRNA was suppressed only by rebamipide (Fig. 5F). We also investigated the effect of these compounds on the expression of the UBE3A gene (located at 15q11-13). The results confirmed that UBE3A mRNA expression was reduced by rifabutin treatment but not by sotalol and rebamipide treatment (FIG. 10C).
Dup15−q−1 iPSC由来神経細胞、及び1269個の化合物からなるFDA承認薬物ライブラリーを使用して、本発明者らは、神経突起長をリードアウトとして神経突起伸長誘導物質のスクリーニングを行った。Dup15−q−1 iPSCを28日間神経細胞に分化させた後、1269個のFDA承認化合物で2日間処理し、次いで神経突起長をβIIIチューブリンの免疫染色を使用するハイコンテント分析によって評価した(図5A)。スクリーニングの模式的ストラテジーを図5Bに示す。化合物一次スクリーニングの結果を図5Cに示す。これら18個の化合物の再現性をチェックした後にヒット基準を3標準偏差以上と定義した(表4)。本発明者らは、レバミピド、ソタロール及びリファブチンが、Dup15q神経細胞において神経突起長を有意に増加させることを確認した(図5D及びE)。他方、レバミピド、ソタロール及びリファブチンは神経細胞生存性に対する効果を有しなかった(図10B)。
次に、本発明者らは、神経突起成長阻害分子の遺伝子発現に対するこれらの化合物の効果を調べた。RTN4及びSEMA6A遺伝子の発現は、ソタロール又はレバミピドによって減少させられたが、リファブチンによっては減少させられなかった。DCC mRNAの発現は、レバミピドによってのみ抑制された(図5F)。本発明者らはまた、UBE3A遺伝子(15q11−13に位置する)の発現に対するこれらの化合物の効果を調べた。結果は、UBE3A mRNAの発現がリファブチン処理によって減少したが、ソタロール及びレバミピド処理によっては減少しないことが確認された(図10C)。 Screening for Compounds to Restore Short Neurite Length Using an FDA-approved drug library consisting of Dup15-q-1 iPSC-derived neurons and 1269 compounds, we used neurite length as a lead-out. We screened for neurite outgrowth inducers. Dup15-q-1 iPSC was differentiated into neurons for 28 days, then treated with 1269 FDA approved compounds for 2 days, then neurite length was assessed by high content analysis using βIII tubulin immunostaining ( FIG. 5A). A schematic screening strategy is shown in FIG. 5B. The results of the primary compound screening are shown in FIG. 5C. After checking the reproducibility of these 18 compounds, the hit criterion was defined as 3 standard deviations or greater (Table 4). We confirmed that rebamipide, sotalol and rifabutin significantly increased neurite length in Dup15q neurons (FIGS. 5D and E). On the other hand, rebamipide, sotalol and rifabutin had no effect on neuronal viability (Fig. 10B).
Next, we investigated the effect of these compounds on the gene expression of neurite growth inhibitory molecules. Expression of the RTN4 and SEMA6A genes was reduced by sotalol or rebamipide, but not by rifabutin. Expression of DCC mRNA was suppressed only by rebamipide (Fig. 5F). We also investigated the effect of these compounds on the expression of the UBE3A gene (located at 15q11-13). The results confirmed that UBE3A mRNA expression was reduced by rifabutin treatment but not by sotalol and rebamipide treatment (FIG. 10C).
レバミピド類、ソタロール類及びリファブチン類は、神経細胞、特にASD患者の神経細胞において、神経突起の伸長を促進するので、ASDの治療、特にDup15q症候群の治療に有用である。特に、他の疾患に対する医薬品として既に上市されているレバミピド、ソタロール及びリファブチンは、安全性等の臨床及び非臨床データが蓄積されており、低コストにかつ迅速に、ASDを治療可能な医薬品を開発できる可能性がある。
Revamipids, sotalols and rifabutins promote neurite outgrowth in nerve cells, especially those in ASD patients, and are therefore useful in the treatment of ASD, especially in the treatment of Dup15q syndrome. In particular, rebamipide, sotalol, and rifabutin, which have already been marketed as drugs for other diseases, have accumulated clinical and non-clinical data such as safety, and develop drugs that can treat ASD at low cost and quickly. There is a possibility that it can be done.
本出願は、2020年9月23日に米国に出願された仮特許出願第63/082,189号を基礎としており、ここで参照することによりその内容のすべてが本明細書に組み込まれる。
This application is based on provisional patent application No. 63 / 082,189 filed in the United States on September 23, 2020, the entire contents of which are incorporated herein by reference.
This application is based on provisional patent application No. 63 / 082,189 filed in the United States on September 23, 2020, the entire contents of which are incorporated herein by reference.
Claims (9)
- レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物を含有してなる、神経突起伸長促進剤。 A neurite outgrowth promoter comprising one or more compounds selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof.
- レバミピドもしくはその塩、及び/又はソタロールもしくはその塩を含有してなる、請求項1に記載の剤。 The agent according to claim 1, which comprises rebamipide or a salt thereof and / or sotalol or a salt thereof.
- リファブチン又はその塩を含有してなる、請求項1に記載の剤。 The agent according to claim 1, which contains rifabutin or a salt thereof.
- 自閉症スペクトラム障害の治療のための、請求項1に記載の剤。 The agent according to claim 1, for the treatment of autism spectrum disorders.
- 自閉症スペクトラム障害が、Dup15q症候群、脆弱X症候群、レット症候群、結節性硬化症、コステロ症候群及びクレーフストラ症候群からなる群より選択される疾患を伴うか、あるいは非症候性の自閉症スペクトラム障害である、請求項4に記載の剤。 Autism spectrum disorders are associated with or with non-symptomatic autism spectrum disorders selected from the group consisting of Dup15q syndrome, Fragile X syndrome, Rett syndrome, nodular sclerosis, Costello syndrome and Clefstra syndrome. The agent according to claim 4.
- 自閉症スペクトラム障害がDup15q症候群を伴う、請求項4に記載の剤。 The agent according to claim 4, wherein the autism spectrum disorder is accompanied by Dup15q syndrome.
- 対象における神経突起伸長を促進する方法であって、レバミピドもしくはその誘導体又はその塩、ソタロールもしくはその誘導体又はその塩、及びリファブチンもしくはその誘導体又はその塩からなる群から選択される1以上の化合物の有効量を、該対象に投与することを含む、方法。 Effectiveness of one or more compounds selected from the group consisting of rebamipide or a derivative thereof or a salt thereof, sotalol or a derivative thereof or a salt thereof, and rifabutin or a derivative thereof or a salt thereof, which is a method for promoting nerve protrusion elongation in a subject. A method comprising administering an amount to the subject.
- 対象が自閉症スペクトラム障害を有する、請求項7に記載の方法。 The method according to claim 7, wherein the subject has an autism spectrum disorder.
- 自閉症スペクトラム障害の治療薬のスクリーニング方法であって、
(1)自閉症スペクトラム障害患者由来の人工多能性幹細胞から分化誘導した神経細胞に被検物質を接触させる工程、
(2)該神経細胞におけるRNT4、SEMA6A及びDCCからなる群より選択される1以上の遺伝子、並びに/或いはUBE3A遺伝子の発現レベルを測定する工程、及び
(3)工程(2)において1以上の遺伝子の発現レベルを低下させた被検物質を、自閉症スペクトラム障害の治療薬の候補として選択する工程
を含む方法。 It is a screening method for therapeutic agents for autism spectrum disorders.
(1) A step of contacting a test substance with a nerve cell that has been induced to differentiate from an induced pluripotent stem cell derived from a patient with autism spectrum disorder.
(2) One or more genes selected from the group consisting of RNT4, SEMA6A and DCC in the nerve cell, and / or a step of measuring the expression level of the UBE3A gene, and (3) one or more genes in step (2). A method comprising the step of selecting a test substance having a reduced expression level as a candidate for a therapeutic agent for autism spectrum disorder.
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