WO2022065518A1 - Promoteur de croissance des neurites - Google Patents

Promoteur de croissance des neurites Download PDF

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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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dup15q
syndrome
salt
derivative
cells
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PCT/JP2021/036361
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English (en)
Japanese (ja)
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治久 井上
恵子 今村
剛史 仁木
剛司 日置
イニゴ ナルバイザ
哲 林
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国立大学法人京都大学
武田薬品工業株式会社
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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
    • 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

La présente invention concerne un promoteur de croissance des neurites contenant au moins un composé choisi dans le groupe constitué par : le rébamipide ou un dérivé ou un sel de ce dernier ; le sotalol ou un dérivé ou un sel de ce dernier ; et la rifabutine ou un dérivé ou un sel de cette dernière.
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