WO2011013646A1 - Produit pharmaceutique pour la prévention et le traitement de la sclérose latérale amyotrophique - Google Patents
Produit pharmaceutique pour la prévention et le traitement de la sclérose latérale amyotrophique Download PDFInfo
- Publication number
- WO2011013646A1 WO2011013646A1 PCT/JP2010/062580 JP2010062580W WO2011013646A1 WO 2011013646 A1 WO2011013646 A1 WO 2011013646A1 JP 2010062580 W JP2010062580 W JP 2010062580W WO 2011013646 A1 WO2011013646 A1 WO 2011013646A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- adar2
- interferon
- site
- glur2
- gene
- Prior art date
Links
- 230000002265 prevention Effects 0.000 title claims abstract description 18
- 239000000825 pharmaceutical preparation Substances 0.000 title abstract 2
- 229940127557 pharmaceutical product Drugs 0.000 title abstract 2
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 title description 42
- 210000002161 motor neuron Anatomy 0.000 claims abstract description 45
- 235000019367 oleandomycin Nutrition 0.000 claims abstract description 19
- 239000004480 active ingredient Substances 0.000 claims abstract description 18
- 239000000126 substance Substances 0.000 claims abstract description 18
- 102000014150 Interferons Human genes 0.000 claims abstract description 13
- 108010050904 Interferons Proteins 0.000 claims abstract description 13
- 201000008257 amyotrophic lateral sclerosis type 1 Diseases 0.000 claims abstract description 8
- 208000019929 sporadic amyotrophic lateral sclerosis Diseases 0.000 claims abstract description 7
- 101000742223 Homo sapiens Double-stranded RNA-specific editase 1 Proteins 0.000 claims abstract 5
- 102100025372 Nuclear pore complex protein Nup98-Nup96 Human genes 0.000 claims abstract 4
- 238000010357 RNA editing Methods 0.000 claims description 42
- 230000026279 RNA modification Effects 0.000 claims description 42
- 239000003814 drug Substances 0.000 claims description 41
- 241001465754 Metazoa Species 0.000 claims description 28
- 108090000623 proteins and genes Proteins 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 22
- XJSFLOJWULLJQS-NGVXBBESSA-N josamycin Chemical compound CO[C@H]1[C@H](OC(C)=O)CC(=O)O[C@H](C)C\C=C\C=C\[C@H](O)[C@H](C)C[C@H](CC=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](N(C)C)[C@H](O[C@@H]2O[C@@H](C)[C@H](OC(=O)CC(C)C)[C@](C)(O)C2)[C@@H](C)O1 XJSFLOJWULLJQS-NGVXBBESSA-N 0.000 claims description 18
- 108010047761 Interferon-alpha Proteins 0.000 claims description 16
- 102000006992 Interferon-alpha Human genes 0.000 claims description 16
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 claims description 14
- 229960004397 cyclophosphamide Drugs 0.000 claims description 14
- 150000001875 compounds Chemical class 0.000 claims description 12
- 229940079322 interferon Drugs 0.000 claims description 12
- 229960004144 josamycin Drugs 0.000 claims description 12
- 150000003839 salts Chemical class 0.000 claims description 12
- 102100038418 Cytoplasmic FMR1-interacting protein 2 Human genes 0.000 claims description 11
- 101000956870 Homo sapiens Cytoplasmic FMR1-interacting protein 2 Proteins 0.000 claims description 11
- 150000002148 esters Chemical class 0.000 claims description 10
- 108010051219 Cre recombinase Proteins 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 6
- RXZBMPWDPOLZGW-XMRMVWPWSA-N (E)-roxithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=N/OCOCCOC)/[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 RXZBMPWDPOLZGW-XMRMVWPWSA-N 0.000 claims description 5
- -1 polyoxyethylene Polymers 0.000 claims description 5
- 229960005224 roxithromycin Drugs 0.000 claims description 5
- RANONBLIHMVXAJ-UHFFFAOYSA-N 4-hydroxycyclophosphamide Chemical compound OC1CCOP(=O)(N(CCCl)CCCl)N1 RANONBLIHMVXAJ-UHFFFAOYSA-N 0.000 claims description 4
- 102100040018 Interferon alpha-2 Human genes 0.000 claims description 3
- 108010079944 Interferon-alpha2b Proteins 0.000 claims description 3
- IEMDOFXTVAPVLX-YWQHLDGFSA-N Leucomycin A1 Chemical compound CO[C@H]1[C@H](O)CC(=O)O[C@H](C)C\C=C\C=C\[C@H](O)[C@H](C)C[C@H](CC=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](N(C)C)[C@H](O[C@@H]2O[C@@H](C)[C@H](OC(=O)CC(C)C)[C@](C)(O)C2)[C@@H](C)O1 IEMDOFXTVAPVLX-YWQHLDGFSA-N 0.000 claims description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 2
- 150000002334 glycols Chemical class 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 33
- 230000003213 activating effect Effects 0.000 abstract description 6
- 229940047124 interferons Drugs 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 26
- 241000699666 Mus <mouse, genus> Species 0.000 description 23
- 210000002569 neuron Anatomy 0.000 description 18
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 11
- 230000016273 neuron death Effects 0.000 description 11
- 230000009471 action Effects 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 10
- 239000003120 macrolide antibiotic agent Substances 0.000 description 10
- 229940079593 drug Drugs 0.000 description 9
- 210000000278 spinal cord Anatomy 0.000 description 8
- 238000011813 knockout mouse model Methods 0.000 description 7
- 238000007911 parenteral administration Methods 0.000 description 7
- 239000008194 pharmaceutical composition Substances 0.000 description 7
- 230000005856 abnormality Effects 0.000 description 6
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 6
- 230000003247 decreasing effect Effects 0.000 description 6
- 229940041033 macrolides Drugs 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 5
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Natural products O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 4
- 108010021188 Superoxide Dismutase-1 Proteins 0.000 description 4
- 102100038836 Superoxide dismutase [Cu-Zn] Human genes 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 4
- 238000001990 intravenous administration Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 230000000750 progressive effect Effects 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- ACTOXUHEUCPTEW-CEUOBAOPSA-N 2-[(4r,5s,6s,7r,9r,10r,11e,13e,16r)-6-[(2s,3r,4r,5s,6r)-5-[(2s,4r,5s,6s)-4,5-dihydroxy-4,6-dimethyloxan-2-yl]oxy-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-10-[(2r,5s,6r)-5-(dimethylamino)-6-methyloxan-2-yl]oxy-4-hydroxy-5-methoxy-9,16-dimethyl-2-o Chemical compound O([C@H]1/C=C/C=C/C[C@@H](C)OC(=O)C[C@@H](O)[C@@H]([C@H]([C@@H](CC=O)C[C@H]1C)O[C@H]1[C@@H]([C@H]([C@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(O)C2)[C@@H](C)O1)N(C)C)O)OC)[C@H]1CC[C@H](N(C)C)[C@@H](C)O1 ACTOXUHEUCPTEW-CEUOBAOPSA-N 0.000 description 3
- 102100030651 Glutamate receptor 2 Human genes 0.000 description 3
- 108091006774 SLC18A3 Proteins 0.000 description 3
- 102000045965 Vesicular Acetylcholine Transport Proteins Human genes 0.000 description 3
- 210000002226 anterior horn cell Anatomy 0.000 description 3
- 210000001130 astrocyte Anatomy 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 108010089305 glutamate receptor type B Proteins 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 239000012453 solvate Substances 0.000 description 3
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 description 2
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 2
- DMUAPQTXSSNEDD-QALJCMCCSA-N Midecamycin Chemical compound C1[C@](O)(C)[C@@H](OC(=O)CC)[C@H](C)O[C@H]1O[C@H]1[C@H](N(C)C)[C@@H](O)[C@H](O[C@@H]2[C@H]([C@H](OC(=O)CC)CC(=O)O[C@H](C)C/C=C/C=C/[C@H](O)[C@H](C)C[C@@H]2CC=O)OC)O[C@@H]1C DMUAPQTXSSNEDD-QALJCMCCSA-N 0.000 description 2
- 101100468236 Mus musculus Adarb1 gene Proteins 0.000 description 2
- 208000010428 Muscle Weakness Diseases 0.000 description 2
- 206010028372 Muscular weakness Diseases 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000004187 Spiramycin Substances 0.000 description 2
- 239000000935 antidepressant agent Substances 0.000 description 2
- 229940005513 antidepressants Drugs 0.000 description 2
- 210000003050 axon Anatomy 0.000 description 2
- 229930192649 bafilomycin Natural products 0.000 description 2
- XDHNQDDQEHDUTM-UHFFFAOYSA-N bafliomycin A1 Natural products COC1C=CC=C(C)CC(C)C(O)C(C)C=C(C)C=C(OC)C(=O)OC1C(C)C(O)C(C)C1(O)OC(C(C)C)C(C)C(O)C1 XDHNQDDQEHDUTM-UHFFFAOYSA-N 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 229960003276 erythromycin Drugs 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 125000005842 heteroatom Chemical class 0.000 description 2
- 150000004677 hydrates Chemical class 0.000 description 2
- 238000007901 in situ hybridization Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 229960005292 josamycin propionate Drugs 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 210000000274 microglia Anatomy 0.000 description 2
- 229960002757 midecamycin Drugs 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 239000008177 pharmaceutical agent Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229960001294 spiramycin Drugs 0.000 description 2
- 235000019372 spiramycin Nutrition 0.000 description 2
- 229930191512 spiramycin Natural products 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 229960005041 troleandomycin Drugs 0.000 description 2
- LQCLVBQBTUVCEQ-QTFUVMRISA-N troleandomycin Chemical compound O1[C@@H](C)[C@H](OC(C)=O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](C)C(=O)O[C@H](C)[C@H](C)[C@H](OC(C)=O)[C@@H](C)C(=O)[C@@]2(OC2)C[C@H](C)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)OC(C)=O)[C@H]1C LQCLVBQBTUVCEQ-QTFUVMRISA-N 0.000 description 2
- ZZVDXRCAGGQFAK-UHFFFAOYSA-N 2h-oxazaphosphinine Chemical class N1OC=CC=P1 ZZVDXRCAGGQFAK-UHFFFAOYSA-N 0.000 description 1
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 1
- 108090000078 AMPA Receptors Proteins 0.000 description 1
- 102000003678 AMPA Receptors Human genes 0.000 description 1
- 102100036664 Adenosine deaminase Human genes 0.000 description 1
- 101100490566 Arabidopsis thaliana ADR2 gene Proteins 0.000 description 1
- 101100244969 Arabidopsis thaliana PRL1 gene Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 101150050114 CTL1 gene Proteins 0.000 description 1
- 101150099461 CTL2 gene Proteins 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229930006677 Erythromycin A Natural products 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108010032606 Fragile X Mental Retardation Protein Proteins 0.000 description 1
- 102000007338 Fragile X Mental Retardation Protein Human genes 0.000 description 1
- 102100039558 Galectin-3 Human genes 0.000 description 1
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 1
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 1
- 101100454448 Homo sapiens LGALS3 gene Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 101150051246 MAC2 gene Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- GQNZGCARKRHPOH-GSSUJARLSA-N Midecamycin acetate Chemical compound CCC(=O)O[C@H]1[C@H](C)O[C@H](C[C@@]1(C)OC(C)=O)O[C@@H]1[C@@H](C)O[C@@H](OC2[C@@H](CC=O)C[C@@H](C)[C@@H](OC(C)=O)\C=C\C=C\C[C@@H](C)OC(=O)C[C@@H](OC(=O)CC)[C@@H]2OC)[C@H](O)[C@H]1N(C)C GQNZGCARKRHPOH-GSSUJARLSA-N 0.000 description 1
- 206010028289 Muscle atrophy Diseases 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 239000004104 Oleandomycin Substances 0.000 description 1
- RZPAKFUAFGMUPI-UHFFFAOYSA-N Oleandomycin Natural products O1C(C)C(O)C(OC)CC1OC1C(C)C(=O)OC(C)C(C)C(O)C(C)C(=O)C2(OC2)CC(C)C(OC2C(C(CC(C)O2)N(C)C)O)C1C RZPAKFUAFGMUPI-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 208000035977 Rare disease Diseases 0.000 description 1
- 101100269260 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ADH2 gene Proteins 0.000 description 1
- 108050007496 Shikimate kinase 2 Proteins 0.000 description 1
- 101150116431 Slc44a2 gene Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000001430 anti-depressive effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 230000003376 axonal effect Effects 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 229960002626 clarithromycin Drugs 0.000 description 1
- AGOYDEPGAOXOCK-KCBOHYOISA-N clarithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@](C)([C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)OC)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 AGOYDEPGAOXOCK-KCBOHYOISA-N 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- TYQXKHPOXXXCTP-CSLYCKPJSA-N erythromycin A 2'-propanoate Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)OC(=O)CC)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 TYQXKHPOXXXCTP-CSLYCKPJSA-N 0.000 description 1
- 229950001028 erythromycin propionate Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 229960001101 ifosfamide Drugs 0.000 description 1
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 1
- 229960004801 imipramine Drugs 0.000 description 1
- BCGWQEUPMDMJNV-UHFFFAOYSA-N imipramine Chemical compound C1CC2=CC=CC=C2N(CCCN(C)C)C2=CC=CC=C21 BCGWQEUPMDMJNV-UHFFFAOYSA-N 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000012750 in vivo screening Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229960001388 interferon-beta Drugs 0.000 description 1
- 229950007634 kitasamycin Drugs 0.000 description 1
- XYJOGTQLTFNMQG-KJHBSLKPSA-N leucomycin V Chemical compound CO[C@H]1[C@H](O)CC(=O)O[C@H](C)C\C=C\C=C\[C@H](O)[C@H](C)C[C@H](CC=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](N(C)C)[C@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(O)C2)[C@@H](C)O1 XYJOGTQLTFNMQG-KJHBSLKPSA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 229940049920 malate Drugs 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 229960000931 miocamycin Drugs 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000000472 morula Anatomy 0.000 description 1
- 108700007949 mouse ADAR2 Proteins 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000020763 muscle atrophy Effects 0.000 description 1
- 201000000585 muscular atrophy Diseases 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 229960002351 oleandomycin Drugs 0.000 description 1
- RZPAKFUAFGMUPI-KGIGTXTPSA-N oleandomycin Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](C)C(=O)O[C@H](C)[C@H](C)[C@H](O)[C@@H](C)C(=O)[C@]2(OC2)C[C@H](C)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C RZPAKFUAFGMUPI-KGIGTXTPSA-N 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000001050 pharmacotherapy Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 210000003019 respiratory muscle Anatomy 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 101150052401 slc44a1 gene Proteins 0.000 description 1
- 229950006796 spiramycin ii Drugs 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 210000001364 upper extremity Anatomy 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
- A61K38/212—IFN-alpha
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- 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
- A61K31/66—Phosphorus compounds
- A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/20—Animal model comprising regulated expression system
- A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
Definitions
- the present invention relates to a medicament for the prevention and / or treatment of amyotrophic lateral sclerosis.
- Amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, which may be abbreviated as ⁇ ALS '' in this specification) is a neurodegenerative disease whose cause of motor neuron selective degeneration has not been clarified. It causes progressive and severe muscle atrophy and weakness that begins without trigger. The disease is extremely fast, with more than half of patients dying from respiratory muscle paralysis within 3 years of onset. About 2 people per 100,000 people a year (especially 20-30 people over 60 years old) develop and it is not a rare disease, but effective treatment has been established at present. Absent.
- causative ALS which accounts for about 10% of ALS
- the causative gene has been identified as part of it.
- autosomal dominant ALS1 has a mutation in SOD1 (superoxide dismutase 1 gene) on chromosome 21, and this causative gene is said to occupy about 20% of hereditary ALS.
- SOD1 superoxide dismutase 1 gene
- the development of drugs for ALS has been carried out mainly using mutant SOD1 transgenic animals, which are animal models of familial ALS.
- about 90% of ALS is sporadic ALS that is not heritable, and this sporadic ALS may develop by a mechanism that is essentially different from familial ALS. Research is unlikely to produce effective medicines for sporadic ALS, which accounts for the majority of ALS.
- the Q / of GluR2 one of the subunits that form the AMPA receptor glutamate receptor subtype by reducing the activity of the RNA editing enzyme ADAR2 (adenosine deaminase acting on RNA type 2) Since RNA editing that should not occur naturally at the R site does not occur, this sporadic ALS disease-specific molecular change is not found in familial ALS due to mutation SOD1, but is found disease-specific in sporadic ALS It has been suggested that this decrease in ADAR2 activity is a direct cause of motor neuron death. In motor neurons, it was also revealed that when the RNA editing rate of the GluR2 Q / R region is less than 100%, the neuron falls slowly. Therefore, it is considered that sporadic ALS can be treated by activating ADAR2 activity in motor neurons and restoring the RNA editing rate of the GluR2 Q / R site to 100%.
- ADAR2 adenosine deaminase acting on RNA type 2
- the present inventors have succeeded in establishing a cultured cell TetHeLaG2m cell capable of efficiently screening a substance having an ADAR2 activity stimulating action, and using this cell, several antidepressants such as imipramine have been used for ADAR2 activity in motor neurons. (Neurosci. Res., 64, pp.251-258, 2009). However, there is no suggestion or teaching in the above publications about the possibility that other drugs have the activity of activating ADAR2. In addition, the present inventors have reported that K / E sites of cytoplasmic fragile X mental retardation protein interacting protein 2 (CYFIP2) RNA undergo ADAR2-specific RNA editing using cultured cells (Neurosci.
- CYFIP2 cytoplasmic fragile X mental retardation protein interacting protein 2
- ADAR2 knockout mouse brain homogenate has also been reported to specifically reduce editing at this site, and it has been confirmed that in vivo has similar activity. (RNA, 14, pp.1110-1118, 2008).
- An object of the present invention is to provide a medicament having ADAR2 activity activation action in motor neurons and useful for prevention and / or treatment of sporadic ALS.
- the present inventors conducted intensive research to solve the above problems, screened substances having an action to activate ADAR2 activity in motor neurons using TetHeLaG2m cells, and obtained several new candidate substances.
- the present inventors have shown that ADAR2 activity in motor neurons increases when an antidepressant having ADAR2 activity stimulating action is systemically administered to wild-type mice, using ADAR2 -specific substrate CYFIP2 to RNA at K / E site It was clarified from the increase in the editing rate, and by applying this method to the above-mentioned candidate compounds, it was confirmed that these candidate compounds have an ADR2 activity activating action even in an in vivo test.
- the present inventors have found that a 50% decrease in ADAR2 activity causes motoneuron death via RNA editing abnormalities in the GluR2 Q / R region based on analysis of mice that are conditionally knocked out so that the ADAR2 gene becomes heterozygous. Based on this finding, it was confirmed that the above candidate compounds obtained by screening increase the RNA editing rate of GluR2 Q / R site in ADAR2 conditional mouse heterozygotes. Is useful as an active ingredient of a medicine for prevention and / or treatment of sporadic ALS. The present invention has been completed based on the above findings.
- sporadic muscle comprising as an active ingredient a substance selected from the group consisting of (a) leucomycins and oleandomycins, (b) cyclophosphamides, and (c) interferon ⁇ s.
- a medicament for the treatment and / or prevention of amyotrophic lateral sclerosis is provided.
- the aforementioned medicament wherein the oleandomycins are roxithromycin, a salt thereof, or an ester thereof; the leucomycins are leucomycin A 3 (josamycin), a salt thereof, or an ester thereof.
- the above drugs the above drug in which the cyclophosphamide is cyclophosphamide or 4-hydroxycyclophosphamide; and the interferon ⁇ is interferon ⁇ , interferon ⁇ -2a, interferon ⁇ -2b, or
- the above-mentioned medicament which is a polyoxyethylene glycol derivative is provided.
- the present invention provides a method for screening a substance useful as an active ingredient of a medicament for the treatment and / or prevention of sporadic amyotrophic lateral sclerosis, comprising heterozygous ADAR2 conditional
- a method comprising the step of selecting, as a candidate compound, a substance that increases the RNA editing rate of a GluR2 Q / R site in a knockout animal, preferably a heterozygous ADAR2 conditional knockout mouse.
- a substance that increases the RNA editing rate at the K / E site of ADAR2 -specific substrate CYFIP2 in a wild-type animal, preferably a wild-type mouse is selected as a candidate compound.
- a method comprising the steps of:
- the above heterozygous ADAR2 conditional knockout animal is also provided by the present invention. According to a preferred embodiment of this animal, it has a heterozygous gene having a LoxP site inserted so as to sandwich exons 7, 8, and 9 encoding the active group of mouse ADAR2 gene adarb1, There is provided the above conditional knockout animal expressing Cre recombinase; the above conditional knockout animal expressing Cre recombinase in a timed manner specifically in motor neurons.
- the animal is preferably a mouse, and more preferably a mouse that expresses Cre recombinase depending on the promoter of the vesicular acetylcholine transporter.
- the present invention also provides heterozygous ADAR2 conditional knockout animals for use in the above screening.
- the medicament provided by the present invention has ADAR2 activity activation action in motor neurons and is useful for prevention and / or treatment of sporadic amyotrophic lateral sclerosis.
- ADAR2 activity activation action in motor neurons and is useful for prevention and / or treatment of sporadic amyotrophic lateral sclerosis.
- This figure shows the results of measuring the RNA editing rate of RK / E site of ADAR2-specific substrate CYFIP2 in spinal single motor neurons after whole-body administration of josamycin, cyclophosphamide, and interferon ⁇ for 7 days to wild-type mice. is there.
- 1) is the average value of the editing rate of all samples
- 2) is the percentage of samples whose editing rate was 100%
- 3) is the neuron whose editing rate is 100% calculated from 2) above.
- the predicted value of the percentage of It is the conceptual diagram which showed the production method of a heterojunction ADAR2 conditional knockout mouse.
- the upper figure of 1) shows the gene structure in which the LoxP site is inserted into intron 6 and intron 9 with the active group (deaminase) part of the adarb1 gene in between, and the result of confirming the genotype of the resulting genetically modified mouse by PCR Is shown in the figure below.
- Fig. 2 (1) Bottom 2) Mutant mouse VAChT-Cre., Which expresses Cre recombinase depending on the promoter of vesicular acetylcholine transporter. Fast and VAChT-Cre. It is the figure which showed the expression of the motor neuron specific Cre recombinase in Slow.
- FIG. 6 shows progressive motor neuron death in Fast mice. It is the figure which showed that a motor neuron death was blocked
- FIG. 4 shows that GluR2 RNA editing is reduced and motor neuron death is caused by halving ADAR2 activity.
- the medicament of the present invention is a medicament for the treatment and / or prevention of ALS, and comprises (a) leucomycins and oleandomycins, (b) cyclophosphamides, and (c) interferon ⁇ s. It contains a substance selected from the group as an active ingredient.
- the medicament of the present invention can be applied to sporadic ALS, but may be effective when administered to familial ALS.
- Natural or semi-synthetic leucomycins can be used as the leucomycins, but typically, for example, leucomycin (LM: sometimes referred to as kitasamycin) A 1 , A 3 (josamycin), A 4 , A 5 , A 6 , A 7 , A 8 , and A 9 , and any mixtures thereof.
- leucomycin sometimes referred to as kitasamycin
- SPM spiramycin
- MDM midecamycin
- physiologically acceptable salts of these leucomycins or their esters can be used.
- ester of leucomycin examples include josamycin propionate, 3 "-O-propionylleucomomycin (rokitamicin: RKM), acetylspiramycin I, II, or III, and midecamycin acetate.
- Physiologically acceptable salts include, for example, mineral salts such as hydrochloride, sulfate and phosphate, organic acid salts such as tartrate, maleate and malate.
- the leucomomycins may be hydrates or solvates of a free form substance or a salt thereof or an ester thereof.
- leucomycin A 3 josamycin
- josamycin propionate particularly preferably leucomycin A 3 It is.
- leucomycins that can be used as the active ingredient of the medicament of the present invention is shown below, but the active ingredient of the medicament of the present invention is not limited to these leucomycins (Source: “Today's New Drug— “Transition of modern pharmaceuticals", by Saburo Fukai, published by Yakuho Hokpo, Inc., March 15, 1995, p. 903).
- Leucomycins are classified as 16-membered ring macrolides, but macrolides (but excluding bafilomycin) included in 16-membered ring macrolides may be used as the active ingredient of the medicament of the present invention. .
- oleandomycins natural or semi-synthetic oleandomycins can be used. Typical examples include, but are not limited to, erythromycins (such as erythromycin A), roxithromycin, clarithromycin, and oleandomycin. Furthermore, physiologically acceptable salts of these oleandomycins, or esters thereof can be used.
- esters of oleandomycins include, but are not limited to, erythromycin propionate, erythromycin estrate, triacetyloleandomycin (troleandomycin), and the like.
- physiologically acceptable salts include, but are not limited to, the salts mentioned for the above leucomycins.
- oleandomycins a hydrate or solvate of a free form substance or a salt thereof or an ester thereof may be used.
- roxithromycin is preferable.
- oleandomycins that can be used as the active ingredient of the pharmaceutical agent of the present invention is shown below, but the active ingredient of the pharmaceutical agent of the present invention is not limited to these oleandomycins (Source: “Today's New drug-transition of modern pharmaceuticals ", Saburo Fukai, published by Yakuho Hokpo, Inc., March 15, 1995, page 899). Although oleandomycins are classified as 14-membered ring macrolides, the macrolides included in the 14-membered ring macrolides may be used as the active ingredient of the medicament of the present invention.
- cyclophosphamides include oxaazaphosphorine compounds having a bis (2-chloroethyl) amino group, such as cyclophosphamide or its active metabolite 4-hydroxycyclophosphamide, ifosfamide. Of these, cyclophosphamide is preferred. Hydrates or solvates may be used as the cyclophosphamides.
- interferon ⁇ s isoforms IFN- ⁇ 1, ⁇ 2, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 10, ⁇ 13, ⁇ 14, ⁇ 16, ⁇ 17, and ⁇ 21 and their are known, for example, interferon ⁇ , interferon ⁇ 2a, interferon ⁇ 2b and the like are preferably used.
- interferon ⁇ s in addition to interferon ⁇ derived from cultured lymphocytes, interferon ⁇ s produced by gene recombination may be used.
- polyethylene glycol (PEG) derivatives of interferon ⁇ are also provided for the purpose of extending the action time in the body, but such derivatives may be used as the active ingredient of the medicament of the present invention.
- PEG polyethylene glycol
- the medicament when a medicament containing the above active ingredient is already marketed, the medicament can be used as it is as a medicament of the present invention.
- the active ingredient can be combined with an appropriate formulation additive, and can be prepared and administered as a pharmaceutical composition for oral administration or parenteral administration depending on the type of the active ingredient.
- leucomycins are preferably provided as a pharmaceutical composition for oral administration or as a pharmaceutical composition for parenteral administration (for example, intravenous administration), and for cyclophosphamides, a pharmaceutical composition for oral administration.
- the interferon ⁇ is preferably provided as a pharmaceutical composition for parenteral administration (preferably for intramuscular administration).
- a pharmaceutical composition for parenteral administration for example, intravenous administration
- the interferon ⁇ is preferably provided as a pharmaceutical composition for parenteral administration (preferably for intramuscular administration).
- the kind of pharmaceutical composition for oral administration is not specifically limited, For example, a tablet, a capsule, etc. can be mentioned.
- the medicament of the present invention has ADAR2 activity activation action in motor neurons, and is particularly useful for the prevention and / or treatment of sporadic amyotrophic lateral sclerosis among amyotrophic lateral sclerosis.
- the medicament of the present invention suppresses the reduction in RNA editing rate of GluR2 Q / R site due to decreased ADAR2 activity and / or increases the RNA editing rate of GluR2 Q / R site Can be made.
- prevention mainly means prevention of the onset of ALS
- treatment mainly refers to prevention of progression of various symptoms in ALS, or relief or remission of various symptoms in ALS. Although meant, these terms should not be construed as limiting in any way, but in the broadest sense.
- the medicament of the present invention can be applied to, for example, ALS with dementia.
- the action of the medicament of the present invention can be confirmed, for example, by the method specifically shown in the following examples.
- the method for confirming in vitro the effect of the pharmaceutical of the present invention on the RNA editing rate of the GluR2 Q / R site was specifically shown in Neurosci.NRes., 64, pp.251-258, 2009, for example.
- a method using cultured cells TetHeLaG2m cells can be employed.
- ADAR2 activity activation action in vivo is, for example, (a) that the ADAR2 activity in motor neurons is increased when the pharmaceutical of the present invention is systemically administered using a wild-type mouse, the ADAR2 specific substrate CYFIP2 A method of confirming from an increase in the RNA editing rate at the K / E site; and (b) the ADAR2 activity of spinal motor neurons is increased by the medicament of the present invention using an animal that is conditionally knocked out to be heterozygous for ADAR2. And a method for confirming that motor neuron death is suppressed can be employed.
- the type of model animal is not particularly limited, but preferably a heterozygous ADAR2 conditional knockout mouse or the like can be mentioned, and the specific production method is specifically shown in the Examples.
- the dose of the medicament of the present invention is not particularly limited, but for leucomycins and oleandomycins, about 100 to 2,000 mg for oral administration and about 50 to 1,000 mg for parenteral administration such as intravenous administration.
- cyclophosphamide is about 10 to 500 mg for oral administration, about 1 to 200 mg for parenteral administration such as intravenous administration, and 100 mg for parenteral administration such as intramuscular administration for interferon ⁇ . About 10 to 20 million international units.
- the dose is not limited to the above range, and can be appropriately selected according to the age and weight of the patient, the purpose of prevention or treatment, the degree of symptoms, and the like.
- the administration route is not limited to the routes exemplified above, and it is needless to say that an appropriate administration route can be selected according to the type of active ingredient.
- in vivo screening for a test compound using an animal that has been conditionally knocked out to be heterozygous for ADAR2, preferably a heterozygous ADAR2 conditional knockout mouse Can be used to select candidate compounds that increase ADAR2 activity in neurons, preferably brain, spinal cord, and motor neurons, by increasing the RNA editing rate of the GluR2 Q / R site. Therefore, it becomes possible to obtain a new active pharmaceutical ingredient having a preventive and / or therapeutic effect.
- this heterozygous knockout animal in addition to the above-mentioned measurement of the editing rate, it is possible to measure the inhibitory effect of the test compound on neuronal cell death. It is also possible to confirm the morphological measurement of the number of axons, the reduced expression of specific cell death markers by Western blotting, and the like.
- a gene in which a LoxP site is inserted so as to sandwich an exon of the ADAR2 gene is introduced into an animal embryo, and a heterozygous gene-modified animal is prepared through a chimeric animal, for example, exercise.
- a conditional knockout animal can be created by crossing a mutant animal that expresses Crerecombinase (Cre) in neurons in a timely manner with the above gene mutant animal. This technique is well known to those skilled in the art, and can be performed with appropriate alterations or modifications.
- the test compound was administered to wild-type mice, and RNA at the K / E site of CYFIP2 which is a specific substrate for ADAR2
- RNA at the K / E site of CYFIP2 which is a specific substrate for ADAR2
- RNA editing rate of the GluR2 Q / R site is specifically catalyzed by ADAR2.
- TetHeLaG2m cells (Neurosci. Res., 64, pp.251-258, 2009) were used as a culture system for measuring the RNA editing activity of the GluR2 Q / R site of ADAR2.
- This cell is obtained by introducing a mini GluR2 gene consisting of exon 11 containing the Q / R site of the GluR2 gene, a part of intron 11 containing the sequence ECS complementary to the Q / R site, and exon 12 into TetHeLa cells.
- This is a cell line obtained by cloning a cell line that stably expresses the mini-GluR2 gene and has an editing rate of about 50% in its Q / R site.
- Example 2 Wild-type mice (C57Bl / 6J strain, male, 16 weeks old, average body weight 20 g) were administered systemically for 7 days with josamycin, cyclophosphamide, and interferon ⁇ , and then the ADAR2-specific substrate CYFIP2 The K / E site RNA editing rate was measured. Each test drug is administered subcutaneously to mice (100 mg, 20 mg, and 1,000,000 units per kg body weight as daily dose), and the brain and spinal cord of the mouse is removed after decapitation, and total RNA is extracted by a standard method did. After RT-PCR, the RNA editing rate of the K / E site was calculated by quantifying the digested fragment obtained by treating the product with the restriction enzyme MseI.
- Table 2 shows the results of measuring the RNA K / E site RNA editing rate of the ADAR2-specific substrate CYFIP2 for the mouse brain administered with each drug.
- a single motor neuron was excised from the spinal cord of the same individual using a laser microdissector, and the RNA obtained from three neurons was taken as one sample.
- the result of editing rate in 20 samples) is shown in Fig. 1 as a scatter diagram.
- 1) is the average editing rate for all samples
- 2) is the percentage of samples where the editing rate was 100%
- 3) is the neuron whose editing rate was calculated from 2) above.
- the predicted value of the percentage of The arrow indicates the sample whose edit rate was 100%.
- Example 3 (a) Preparation of a conditional ADAR2 knockout mouse A LoxP site ( ⁇ ) was inserted into intron 6 and intron 9 so as to sandwich the active group (deaminase) part of the adarb1 gene, which is a 129 / SvEv mouse homologous gene of ADAR2 gene (Fig. 2 (1) top) and a vector in which a neomycin resistance gene (Neo) sandwiched between FRT sites as a selection cassette was inserted into intron 9 was constructed using pBluescript SKII (-). The nucleic acid sequence of the mouse adarb1 gene into which LoxP and Neo-PLUS selection cassete are inserted is shown in FIG.
- the obtained gene was introduced into morulas derived from C57BL / 6 mice by a conventional method, and knockout mice were prepared via chimeric mice. The genotype was confirmed by PCR (FIG. 2 (1) lower diagram).
- VAChT-Cre that expresses Cre recombinase (Cre) in a promoter-dependent manner in the vesicular acetylcholine transporter. Fast and VAChT-Cre.
- the ADAR2 flox / flox / VAChT-Cre.Fast mouse and the ADAR2 flox / flox / VAChT-Cre.Slow mouse were obtained by crossing Slow with the ADAR2 flox / flox mouse obtained above.
- VAChT-Cre mice express Cre recombinase selectively in motor neurons, but in the Fast system, it is expressed in 40-70% of motor neurons by 5 weeks of age, and in the Slow system, about 50% of motor neurons at 8 months of age. It is expressed (Fig. 2 (2)).
- ADAR2 flox / flox / VAChT-Cre mice When the anterior horn of the spinal cord of 6-month-old ADAR2 flox / flox / VAChT-Cre mice was examined by in situ hybridization, in the wild type (Ctl), the ADAR2 gene was expressed in all motor neurons. ADAR2 flox / flox / VAChT-Cre mice were deficient in ADAR2 gene expression in some motor neurons (upper left in FIG. 4). Furthermore, when the motor neuron was cut out and the RNA editing rate of the Q / R region of GluR2 mRNA was measured, the editing rate was 0% for the motor neuron lacking ADAR2, and the editing rate was 100% for the motor neuron expressing ADAR2. It was confirmed that ADAR2 activity was inactivated. We also confirmed that this gene knockout is Cre-dependent.
- Figure 5 shows the RNA editing rate of the GluR2 Q / R region measured by RT-PCR and restriction enzyme treatment using three 2-month-old ADAR2 flox / flox /VAChT-Cre.Fast mouse motoneurons as one sample. Shown in The horizontal axis represents the RNA editing rate of the GluR2 Q / R site, and the vertical axis represents the number of samples. In addition to samples with 0% editing rate and 100% samples, the presence of around 30% and around 70% samples was also observed. Since the edit rate of neurons lacking ADAR2 is 0%, the 0% group means that ADAR2 is missing in all three neurons in one sample (0: 3), and 100% The group means that ADAR2 is expressed in all neurons (3: 0).
- the middle sample means that ADAR2 is expressed only in 2 out of 3 neurons (1: 2) or 1 neuron (2: 1).
- ADAR2 was knocked out Cre-dependently in the group with an editing rate of 0%.
- Ctl1 shows the results of ADAR2 flox / flox mice
- Ctl2 shows the results of VAChT-Cre.Fast mice.
- FIG. 6 shows ADAR2 flox / flox / VAChT-Cre. It showed progressive motor neuron death in Fast mice.
- spinal cord morphology ADAR2 expression was deficient in some large anterior horn cells, and motor neurons with air bubbles in the cytoplasm were observed.
- the anterior spinal cord atrophy, axonal degeneration and a decrease in the number of axons were observed, the number of large anterior horn cells (AHC) decreased progressively after 2 months of age, especially only those cells that did not express ADAR2.
- Figure 6, lower left The immune activity of GFAP (astrocyte marker) increased with age, indicating that astrocytes are reactively proliferating with the loss of neurons.
- the microglia marker MAC2 is most prominent at 6 months, indicating that the response of microglia accompanying cell death is strongest at this time.
- the Q / R site is glutamine (CAG) in the GluR2 gene that expresses edited GluR2 without ADAR2 activity.
- CAG glutamine
- GluR-B mice whose genes were modified to encode arginine (CGG)
- ADAR2 flox / flox / VAChT-Cre.Fast mice were crossed.
- ADAR2 flox / flox / VAChTCre.Fast / GluR-B (R / R) mice were examined.At 6 months of age, ADAR2 flox / flox / VAChT-Cre.Fast mice (Fig. In A) of 7), more than 40% of motor neurons dropped out, and Rotarod score and arm grip strength decreased significantly, whereas ADAR2 flox / flox x / VAChT-Cre.Fast / GluR-B (R / R) Mice (A / G) showed no changes in the number of motor neurons and behavior compared to control mice. Therefore, it became clear that the GluR2 Q / R site was the only RNA editing abnormality caused by ADAR2 deficiency that involved motor neuron death.
- RNA editing rate of GluR2 Q / R site in motor neurons of heterozygous ADAR2 flox / + /VAChT-Cre.Fast mice knocked out only one of ADAR2 gene was less than 100% in about 20% motor neurons Met.
- the control group shows 100% editing rate in all motor neurons, so about half of the motor neurons expressing only one ADAR2 gene in heterozygous mice have decreased ADAR2 activity, and complete RNA editing at this site. It became clear that there was no.
- this hetero mouse is a model reflecting the molecular pathology of sporadic ALS, and was shown to be useful as a drug development tool for the prevention and / or treatment of ALS aimed at activating ADAR2 activity.
- Example 4 The ADAR2 heterozygote ADAR2 flox / + /VAChT-Cre.Fast mouse obtained in Example 3 was administered systemically with josamycin, cyclophosphamide, and interferon ⁇ in the same manner as in Example 2 for 7 days.
- the RNA editing rate at the Q / R site was measured.
- the results are shown in Table 3 below as the average value ⁇ standard deviation of 60 individuals in each group of 3 individuals. From these results, it was confirmed that josamycin, cyclophosphamide, and interferon ⁇ all have an action of increasing the RNA editing rate of the GluR2 Q / R site in mouse neurons.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Environmental Sciences (AREA)
- Neurology (AREA)
- Cell Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Neurosurgery (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
- Orthopedic Medicine & Surgery (AREA)
Abstract
L'invention porte sur un produit pharmaceutique pour la prévention et le traitement de la sclérose latérale amyotrophique sporadique, lequel produit contient, comme principe actif, une substance qui est choisie dans le groupe constitué par (a) les leucomycines et les oléandomycines, (b) les cyclophosphamides et (c) les interférons a, ladite substance ayant un effet d'activation de l'activité de l'ADAR2 dans les motoneurones.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2009175128A JP2012197229A (ja) | 2009-07-28 | 2009-07-28 | 筋萎縮性側索硬化症の予防及び治療のための医薬 |
JP2009-175128 | 2009-07-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011013646A1 true WO2011013646A1 (fr) | 2011-02-03 |
Family
ID=43529301
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2010/062580 WO2011013646A1 (fr) | 2009-07-28 | 2010-07-27 | Produit pharmaceutique pour la prévention et le traitement de la sclérose latérale amyotrophique |
Country Status (2)
Country | Link |
---|---|
JP (1) | JP2012197229A (fr) |
WO (1) | WO2011013646A1 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019099368A1 (fr) * | 2017-11-16 | 2019-05-23 | Levetan Claresa | Compositions et méthodes pour traiter ou prévenir le diabète de type 1 en utilisant un modificateur de la réponse biologique en association avec une ou plusieurs thérapies de type régénération ou remplacement d'îlots de langerhans ou de cellules bêta |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002047206A (ja) * | 2000-07-28 | 2002-02-12 | Taisho Pharmaceut Co Ltd | Gdnf産生促進剤、パーキンソン病治療剤およびals治療剤 |
WO2004035053A1 (fr) * | 2002-10-15 | 2004-04-29 | Fujisawa Pharmaceutical Co., Ltd. | Promoteur de production du facteur neurotrophique |
-
2009
- 2009-07-28 JP JP2009175128A patent/JP2012197229A/ja active Pending
-
2010
- 2010-07-27 WO PCT/JP2010/062580 patent/WO2011013646A1/fr active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002047206A (ja) * | 2000-07-28 | 2002-02-12 | Taisho Pharmaceut Co Ltd | Gdnf産生促進剤、パーキンソン病治療剤およびals治療剤 |
WO2004035053A1 (fr) * | 2002-10-15 | 2004-04-29 | Fujisawa Pharmaceutical Co., Ltd. | Promoteur de production du facteur neurotrophique |
Non-Patent Citations (4)
Title |
---|
SHIN KAKU: "Kokoro no Kenko Kagaku Kenkyu Jigyo 'Kin Ishukusei Sokusaku Kokasho ni Taisuru Tokui Chiryoho no Kaihatsu'", KOSEI RODO KAGAKU KENKYU SEIKA DATABASE GAIYOBAN, 2008, Retrieved from the Internet <URL:http://mhlw-grants.niph.go.jp/niph/search/NIDDOO.do> [retrieved on 20100831] * |
YASUHIRO NAKAHARA: "Zaitaku Nanbyo Kanja san no Tameno Kusuri no Chishiki [Dai 2 Kai] Kin Ishukusei Sokusaku Kokasho Chiryozai Riluzole", NANBYO TO ZAITAKU CARE, 2001 NEN 2 GATSU, vol. 6, pages 40 - 41 * |
YASUO IWASAKI ET AL.: "Tokushu/Kin Ishukusei Sokusaku Kokasho no Saishin Chiryo Joho [Dai 4 Bu] Riluzole Ninka Go no Saishin'yaku Joho", NANBYO TO ZAITAKU CARE, 2000 NEN 10 GATSU, vol. 6, no. 7, pages 20 - 23 * |
YUYA YAMASHITA ET AL.: "'ALS Kikin' Kenkyu Shoreikin Kenkyu Seika Hokokusho ''Kohatsusei ALS no Hassho ni Kansuru RNA Henshu Koso ADAR 2 Kassei no Chosetsu Kiko no Kaiseki''", NIPPON ALS KYOKAI HEISEI 20 NENDO 'ALS KIKIN' KENKYU SHOREIKIN KOFU TAISHO KENKYU SEIKA HOKOKUSHO (1), 3 July 2009 (2009-07-03), pages 1 - 22, Retrieved from the Internet <URL:http://www.alsjapan.org/contents2/info0/downloads/20_hokoku02.pdf> [retrieved on 20100813] * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019099368A1 (fr) * | 2017-11-16 | 2019-05-23 | Levetan Claresa | Compositions et méthodes pour traiter ou prévenir le diabète de type 1 en utilisant un modificateur de la réponse biologique en association avec une ou plusieurs thérapies de type régénération ou remplacement d'îlots de langerhans ou de cellules bêta |
Also Published As
Publication number | Publication date |
---|---|
JP2012197229A (ja) | 2012-10-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7592314B2 (en) | Use of VEGF and homologues to treat neuron disorders | |
Kim et al. | Axon regeneration in young adult mice lacking Nogo-A/B | |
Von Koch et al. | Generation of APLP2 KO mice and early postnatal lethality in APLP2/APP double KO mice | |
Andressoo et al. | An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria | |
US10918697B2 (en) | Co-activation of mTOR and STAT3 pathways to promote neuronal survival and regeneration | |
JP2021169461A (ja) | 寿命に関する動物モデル並びに寿命を延ばす及び腫瘍化を阻害する関連方法 | |
AU2001268968A1 (en) | Use of VEGF and homologues to treat neuron disorders | |
US11351225B2 (en) | Methods for modulating development and function of photoreceptor cells | |
Dragatsis et al. | Huntingtin-associated protein 1 (Hap1) mutant mice bypassing the early postnatal lethality are neuroanatomically normal and fertile but display growth retardation | |
EP2471916A1 (fr) | Marqueur pour diagnostic, procédé de diagnostic et agent thérapeutique pour la sclérose latérale amyotrophique, et modèle animal et modèle cellulaire développant une sclérose latérale amyotrophique | |
WO2011013646A1 (fr) | Produit pharmaceutique pour la prévention et le traitement de la sclérose latérale amyotrophique | |
Ishiyama et al. | Genetic transfer of the wobbler gene to a C57BL/6J× NZB hybrid stock: natural history of the motor neuron disease and response to CNTF and BDNF cotreatment | |
WO1996035785A9 (fr) | ANIMAUX TRANSGENIQUES PRESENTANT UN GENE DU RECEPTEUR BETA DE L'HORMONE THYROïDIENNE DEFECTUEUX | |
WO1996035785A2 (fr) | ANIMAUX TRANSGENIQUES PRESENTANT UN GENE DU RECEPTEUR BETA DE L'HORMONE THYROïDIENNE DEFECTUEUX | |
Ostedgaard et al. | EXPRESSION AND LOCALIZATION OF PORCINE AIRWAY MUCINS | |
WO2002097079A2 (fr) | Gene codant une nouvelle proteine de type moteur moleculaire et procede diagnostique pour une maladie liee a ce gene |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10804392 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 10804392 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: JP |