US20200392489A1 - Uses for prevention or treatment of brain diseases using microrna - Google Patents
Uses for prevention or treatment of brain diseases using microrna Download PDFInfo
- Publication number
- US20200392489A1 US20200392489A1 US16/443,700 US201916443700A US2020392489A1 US 20200392489 A1 US20200392489 A1 US 20200392489A1 US 201916443700 A US201916443700 A US 201916443700A US 2020392489 A1 US2020392489 A1 US 2020392489A1
- Authority
- US
- United States
- Prior art keywords
- mir
- seq
- hsa
- inhibitor
- disease
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000014644 Brain disease Diseases 0.000 title abstract description 34
- 108091070501 miRNA Proteins 0.000 title description 41
- 230000002265 prevention Effects 0.000 title description 2
- 108091035982 miR-485 stem-loop Proteins 0.000 claims abstract description 106
- 230000014509 gene expression Effects 0.000 claims abstract description 71
- 238000000034 method Methods 0.000 claims abstract description 60
- 108010008795 ELAV-Like Protein 2 Proteins 0.000 claims abstract description 51
- 102000007303 ELAV-Like Protein 2 Human genes 0.000 claims abstract description 51
- 208000024827 Alzheimer disease Diseases 0.000 claims abstract description 49
- 239000003112 inhibitor Substances 0.000 claims abstract description 45
- 108010064539 amyloid beta-protein (1-42) Proteins 0.000 claims abstract description 27
- 239000000203 mixture Substances 0.000 claims abstract description 20
- 108091034117 Oligonucleotide Proteins 0.000 claims description 47
- 239000000074 antisense oligonucleotide Substances 0.000 claims description 29
- 238000012230 antisense oligonucleotides Methods 0.000 claims description 29
- 150000007523 nucleic acids Chemical group 0.000 claims description 18
- 102000039446 nucleic acids Human genes 0.000 claims description 17
- 108020004707 nucleic acids Proteins 0.000 claims description 17
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 claims description 16
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 claims description 16
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 claims description 16
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 claims description 16
- 230000000295 complement effect Effects 0.000 claims description 16
- 239000002773 nucleotide Substances 0.000 claims description 15
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- 239000004055 small Interfering RNA Substances 0.000 claims description 13
- 102000013498 tau Proteins Human genes 0.000 claims description 11
- 108010026424 tau Proteins Proteins 0.000 claims description 11
- 108091093037 Peptide nucleic acid Proteins 0.000 claims description 10
- 108020004414 DNA Proteins 0.000 claims description 8
- 108020004459 Small interfering RNA Proteins 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 6
- 230000026731 phosphorylation Effects 0.000 claims description 6
- 238000006366 phosphorylation reaction Methods 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 238000007913 intrathecal administration Methods 0.000 claims description 4
- 238000001990 intravenous administration Methods 0.000 claims description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 238000010254 subcutaneous injection Methods 0.000 claims description 3
- 239000007929 subcutaneous injection Substances 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 2
- 238000006467 substitution reaction Methods 0.000 claims description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 26
- 239000003814 drug Substances 0.000 abstract description 26
- 239000008194 pharmaceutical composition Substances 0.000 abstract description 24
- 201000010099 disease Diseases 0.000 abstract description 22
- 239000003795 chemical substances by application Substances 0.000 abstract description 21
- 230000003247 decreasing effect Effects 0.000 abstract description 18
- 208000024891 symptom Diseases 0.000 abstract description 14
- 238000012216 screening Methods 0.000 abstract description 12
- 229940124597 therapeutic agent Drugs 0.000 abstract description 7
- 208000036626 Mental retardation Diseases 0.000 abstract description 6
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 abstract description 6
- 208000029560 autism spectrum disease Diseases 0.000 abstract description 6
- 230000001939 inductive effect Effects 0.000 abstract description 3
- 239000002679 microRNA Substances 0.000 description 46
- 210000004027 cell Anatomy 0.000 description 37
- 230000000694 effects Effects 0.000 description 25
- 239000000126 substance Substances 0.000 description 25
- 210000001320 hippocampus Anatomy 0.000 description 23
- 230000007423 decrease Effects 0.000 description 22
- 241000699666 Mus <mouse, genus> Species 0.000 description 21
- 230000005764 inhibitory process Effects 0.000 description 19
- 210000003710 cerebral cortex Anatomy 0.000 description 18
- 229940079593 drug Drugs 0.000 description 18
- 210000004556 brain Anatomy 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 238000011160 research Methods 0.000 description 16
- 229920002477 rna polymer Polymers 0.000 description 15
- 230000001965 increasing effect Effects 0.000 description 14
- 150000001875 compounds Chemical class 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 108091053855 Homo sapiens miR-485 stem-loop Proteins 0.000 description 12
- 230000002401 inhibitory effect Effects 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 11
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 10
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 10
- 238000009825 accumulation Methods 0.000 description 9
- 108020004999 messenger RNA Proteins 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 230000003920 cognitive function Effects 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- 102000053602 DNA Human genes 0.000 description 7
- 108091036066 Three prime untranslated region Proteins 0.000 description 7
- 238000010171 animal model Methods 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 206010012289 Dementia Diseases 0.000 description 6
- 108091063991 Mus musculus miR-485 stem-loop Proteins 0.000 description 6
- 239000004480 active ingredient Substances 0.000 description 6
- 230000003542 behavioural effect Effects 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 210000002569 neuron Anatomy 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 108700011259 MicroRNAs Proteins 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 238000004445 quantitative analysis Methods 0.000 description 5
- -1 quisulate Chemical compound 0.000 description 5
- 150000003384 small molecules Chemical class 0.000 description 5
- 230000002269 spontaneous effect Effects 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 206010010904 Convulsion Diseases 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 208000018737 Parkinson disease Diseases 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 208000006011 Stroke Diseases 0.000 description 4
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 4
- 229960004373 acetylcholine Drugs 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000012790 confirmation Methods 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 230000000971 hippocampal effect Effects 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000011302 passive avoidance test Methods 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 208000020431 spinal cord injury Diseases 0.000 description 4
- CCEKAJIANROZEO-UHFFFAOYSA-N sulfluramid Chemical group CCNS(=O)(=O)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F CCEKAJIANROZEO-UHFFFAOYSA-N 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 108020005345 3' Untranslated Regions Proteins 0.000 description 3
- 102100034234 ELAV-like protein 2 Human genes 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- 101000925866 Homo sapiens ELAV-like protein 2 Proteins 0.000 description 3
- 108091053832 Homo sapiens miR-487a stem-loop Proteins 0.000 description 3
- 108091092227 Homo sapiens miR-489 stem-loop Proteins 0.000 description 3
- 108091064506 Homo sapiens miR-499a stem-loop Proteins 0.000 description 3
- 108091064367 Homo sapiens miR-509-1 stem-loop Proteins 0.000 description 3
- 108091086508 Homo sapiens miR-509-2 stem-loop Proteins 0.000 description 3
- 108091087072 Homo sapiens miR-509-3 stem-loop Proteins 0.000 description 3
- 108091092230 Homo sapiens miR-511 stem-loop Proteins 0.000 description 3
- 108091092274 Homo sapiens miR-512-1 stem-loop Proteins 0.000 description 3
- 108091092275 Homo sapiens miR-512-2 stem-loop Proteins 0.000 description 3
- 108091064470 Homo sapiens miR-518b stem-loop Proteins 0.000 description 3
- 108091063810 Homo sapiens miR-539 stem-loop Proteins 0.000 description 3
- 108091061616 Homo sapiens miR-652 stem-loop Proteins 0.000 description 3
- 108091067625 Homo sapiens miR-7-1 stem-loop Proteins 0.000 description 3
- 108091067630 Homo sapiens miR-7-2 stem-loop Proteins 0.000 description 3
- 108091067633 Homo sapiens miR-7-3 stem-loop Proteins 0.000 description 3
- 108091070380 Homo sapiens miR-92a-1 stem-loop Proteins 0.000 description 3
- 108091070381 Homo sapiens miR-92a-2 stem-loop Proteins 0.000 description 3
- 108091070377 Homo sapiens miR-93 stem-loop Proteins 0.000 description 3
- 108091070375 Homo sapiens miR-95 stem-loop Proteins 0.000 description 3
- 108091068856 Homo sapiens miR-98 stem-loop Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000004075 alteration Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 210000005013 brain tissue Anatomy 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000006735 deficit Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000002493 microarray Methods 0.000 description 3
- 210000003928 nasal cavity Anatomy 0.000 description 3
- 230000001537 neural effect Effects 0.000 description 3
- 230000007372 neural signaling Effects 0.000 description 3
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 206010000117 Abnormal behaviour Diseases 0.000 description 2
- 101710159080 Aconitate hydratase A Proteins 0.000 description 2
- 101710159078 Aconitate hydratase B Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 208000037259 Amyloid Plaque Diseases 0.000 description 2
- 108091023037 Aptamer Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 229920001661 Chitosan Polymers 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101000617536 Homo sapiens Presenilin-1 Proteins 0.000 description 2
- 108091070514 Homo sapiens let-7b stem-loop Proteins 0.000 description 2
- 108091070508 Homo sapiens let-7e stem-loop Proteins 0.000 description 2
- 108091068840 Homo sapiens miR-101-1 stem-loop Proteins 0.000 description 2
- 108091065458 Homo sapiens miR-101-2 stem-loop Proteins 0.000 description 2
- 108091068943 Homo sapiens miR-105-1 stem-loop Proteins 0.000 description 2
- 108091068938 Homo sapiens miR-105-2 stem-loop Proteins 0.000 description 2
- 108091065165 Homo sapiens miR-106b stem-loop Proteins 0.000 description 2
- 108091069085 Homo sapiens miR-126 stem-loop Proteins 0.000 description 2
- 108091069005 Homo sapiens miR-128-1 stem-loop Proteins 0.000 description 2
- 108091065160 Homo sapiens miR-128-2 stem-loop Proteins 0.000 description 2
- 108091069022 Homo sapiens miR-130a stem-loop Proteins 0.000 description 2
- 108091069024 Homo sapiens miR-132 stem-loop Proteins 0.000 description 2
- 108091066990 Homo sapiens miR-133b stem-loop Proteins 0.000 description 2
- 108091069094 Homo sapiens miR-134 stem-loop Proteins 0.000 description 2
- 108091066895 Homo sapiens miR-135b stem-loop Proteins 0.000 description 2
- 108091069092 Homo sapiens miR-138-1 stem-loop Proteins 0.000 description 2
- 108091069015 Homo sapiens miR-138-2 stem-loop Proteins 0.000 description 2
- 108091067617 Homo sapiens miR-139 stem-loop Proteins 0.000 description 2
- 108091092238 Homo sapiens miR-146b stem-loop Proteins 0.000 description 2
- 108091067009 Homo sapiens miR-148b stem-loop Proteins 0.000 description 2
- 108091067014 Homo sapiens miR-151a stem-loop Proteins 0.000 description 2
- 108091070507 Homo sapiens miR-15a stem-loop Proteins 0.000 description 2
- 108091069045 Homo sapiens miR-15b stem-loop Proteins 0.000 description 2
- 108091067469 Homo sapiens miR-181a-1 stem-loop Proteins 0.000 description 2
- 108091067618 Homo sapiens miR-181a-2 stem-loop Proteins 0.000 description 2
- 108091092213 Homo sapiens miR-181d stem-loop Proteins 0.000 description 2
- 108091068998 Homo sapiens miR-191 stem-loop Proteins 0.000 description 2
- 108091068960 Homo sapiens miR-195 stem-loop Proteins 0.000 description 2
- 108091070519 Homo sapiens miR-19b-1 stem-loop Proteins 0.000 description 2
- 108091070495 Homo sapiens miR-19b-2 stem-loop Proteins 0.000 description 2
- 108091067483 Homo sapiens miR-203a stem-loop Proteins 0.000 description 2
- 108091067466 Homo sapiens miR-212 stem-loop Proteins 0.000 description 2
- 108091070494 Homo sapiens miR-22 stem-loop Proteins 0.000 description 2
- 108091070397 Homo sapiens miR-28 stem-loop Proteins 0.000 description 2
- 108091070398 Homo sapiens miR-29a stem-loop Proteins 0.000 description 2
- 108091065168 Homo sapiens miR-29c stem-loop Proteins 0.000 description 2
- 108091065459 Homo sapiens miR-302a stem-loop Proteins 0.000 description 2
- 108091067650 Homo sapiens miR-30d stem-loop Proteins 0.000 description 2
- 108091067005 Homo sapiens miR-328 stem-loop Proteins 0.000 description 2
- 108091067008 Homo sapiens miR-342 stem-loop Proteins 0.000 description 2
- 108091066970 Homo sapiens miR-346 stem-loop Proteins 0.000 description 2
- 108091067619 Homo sapiens miR-34a stem-loop Proteins 0.000 description 2
- 108091053844 Homo sapiens miR-376b stem-loop Proteins 0.000 description 2
- 108091067554 Homo sapiens miR-381 stem-loop Proteins 0.000 description 2
- 108091032537 Homo sapiens miR-409 stem-loop Proteins 0.000 description 2
- 108091032638 Homo sapiens miR-431 stem-loop Proteins 0.000 description 2
- 108091092306 Homo sapiens miR-432 stem-loop Proteins 0.000 description 2
- 108091032636 Homo sapiens miR-433 stem-loop Proteins 0.000 description 2
- 108091053854 Homo sapiens miR-484 stem-loop Proteins 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 108091028141 MiR-203 Proteins 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 238000009004 PCR Kit Methods 0.000 description 2
- 102100022033 Presenilin-1 Human genes 0.000 description 2
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 2
- 101710105008 RNA-binding protein Proteins 0.000 description 2
- 101710133283 RNA-binding protein 2 Proteins 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 101150084233 ago2 gene Proteins 0.000 description 2
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000001713 cholinergic effect Effects 0.000 description 2
- 230000001149 cognitive effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000003413 degradative effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- ADEBPBSSDYVVLD-UHFFFAOYSA-N donepezil Chemical compound O=C1C=2C=C(OC)C(OC)=CC=2CC1CC(CC1)CCN1CC1=CC=CC=C1 ADEBPBSSDYVVLD-UHFFFAOYSA-N 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 206010015037 epilepsy Diseases 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- ASUTZQLVASHGKV-JDFRZJQESA-N galanthamine Chemical compound O1C(=C23)C(OC)=CC=C2CN(C)CC[C@]23[C@@H]1C[C@@H](O)C=C2 ASUTZQLVASHGKV-JDFRZJQESA-N 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000008897 memory decline Effects 0.000 description 2
- 230000009149 molecular binding Effects 0.000 description 2
- 210000002850 nasal mucosa Anatomy 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 108091027963 non-coding RNA Proteins 0.000 description 2
- 102000042567 non-coding RNA Human genes 0.000 description 2
- 239000003961 penetration enhancing agent Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000011200 topical administration Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- 125000006273 (C1-C3) alkyl group Chemical group 0.000 description 1
- MGRVRXRGTBOSHW-UHFFFAOYSA-N (aminomethyl)phosphonic acid Chemical compound NCP(O)(O)=O MGRVRXRGTBOSHW-UHFFFAOYSA-N 0.000 description 1
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- OGYGFUAIIOPWQD-UHFFFAOYSA-N 1,3-thiazolidine Chemical compound C1CSCN1 OGYGFUAIIOPWQD-UHFFFAOYSA-N 0.000 description 1
- WJFKNYWRSNBZNX-UHFFFAOYSA-N 10H-phenothiazine Chemical compound C1=CC=C2NC3=CC=CC=C3SC2=C1 WJFKNYWRSNBZNX-UHFFFAOYSA-N 0.000 description 1
- WDJHALXBUFZDSR-UHFFFAOYSA-N Acetoacetic acid Natural products CC(=O)CC(O)=O WDJHALXBUFZDSR-UHFFFAOYSA-N 0.000 description 1
- 102000012440 Acetylcholinesterase Human genes 0.000 description 1
- 108010022752 Acetylcholinesterase Proteins 0.000 description 1
- 208000000044 Amnesia Diseases 0.000 description 1
- 102000002659 Amyloid Precursor Protein Secretases Human genes 0.000 description 1
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 1
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 229940123239 Cholesterol synthesis inhibitor Drugs 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- MQJKPEGWNLWLTK-UHFFFAOYSA-N Dapsone Chemical compound C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=C1 MQJKPEGWNLWLTK-UHFFFAOYSA-N 0.000 description 1
- 108091027757 Deoxyribozyme Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- WOISDAHQBUYEAF-UHFFFAOYSA-N Ebelactone A Natural products CCC(C)C(O)C(C)C(=O)C(C)C=C(C)CC(C)C1OC(=O)C1C WOISDAHQBUYEAF-UHFFFAOYSA-N 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101000780643 Homo sapiens Protein argonaute-2 Proteins 0.000 description 1
- 108091070511 Homo sapiens let-7c stem-loop Proteins 0.000 description 1
- 108091070512 Homo sapiens let-7d stem-loop Proteins 0.000 description 1
- 108091069047 Homo sapiens let-7i stem-loop Proteins 0.000 description 1
- 108091068928 Homo sapiens miR-107 stem-loop Proteins 0.000 description 1
- 108091069019 Homo sapiens miR-124-1 stem-loop Proteins 0.000 description 1
- 108091069008 Homo sapiens miR-124-2 stem-loop Proteins 0.000 description 1
- 108091069007 Homo sapiens miR-124-3 stem-loop Proteins 0.000 description 1
- 108091069006 Homo sapiens miR-125b-1 stem-loop Proteins 0.000 description 1
- 108091069087 Homo sapiens miR-125b-2 stem-loop Proteins 0.000 description 1
- 108091069017 Homo sapiens miR-140 stem-loop Proteins 0.000 description 1
- 108091069089 Homo sapiens miR-146a stem-loop Proteins 0.000 description 1
- 108091068997 Homo sapiens miR-152 stem-loop Proteins 0.000 description 1
- 108091092301 Homo sapiens miR-193b stem-loop Proteins 0.000 description 1
- 108091070496 Homo sapiens miR-20a stem-loop Proteins 0.000 description 1
- 108091070373 Homo sapiens miR-24-1 stem-loop Proteins 0.000 description 1
- 108091070374 Homo sapiens miR-24-2 stem-loop Proteins 0.000 description 1
- 108091070399 Homo sapiens miR-26b stem-loop Proteins 0.000 description 1
- 108091070400 Homo sapiens miR-27a stem-loop Proteins 0.000 description 1
- 108091086636 Homo sapiens miR-298 stem-loop Proteins 0.000 description 1
- 108091068837 Homo sapiens miR-29b-1 stem-loop Proteins 0.000 description 1
- 108091068845 Homo sapiens miR-29b-2 stem-loop Proteins 0.000 description 1
- 108091067250 Homo sapiens miR-302b stem-loop Proteins 0.000 description 1
- 108091067013 Homo sapiens miR-337 stem-loop Proteins 0.000 description 1
- 108091067010 Homo sapiens miR-338 stem-loop Proteins 0.000 description 1
- 108091066993 Homo sapiens miR-339 stem-loop Proteins 0.000 description 1
- 108091063813 Homo sapiens miR-455 stem-loop Proteins 0.000 description 1
- 108091092234 Homo sapiens miR-488 stem-loop Proteins 0.000 description 1
- 108091069003 Homo sapiens miR-9-1 stem-loop Proteins 0.000 description 1
- 108091068996 Homo sapiens miR-9-2 stem-loop Proteins 0.000 description 1
- 108091069001 Homo sapiens miR-9-3 stem-loop Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 108091008051 MIR27A Proteins 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 238000000585 Mann–Whitney U test Methods 0.000 description 1
- 108091093073 MiR-134 Proteins 0.000 description 1
- 108091030146 MiRBase Proteins 0.000 description 1
- 229940122938 MicroRNA inhibitor Drugs 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- HOKKHZGPKSLGJE-GSVOUGTGSA-N N-Methyl-D-aspartic acid Chemical compound CN[C@@H](C(O)=O)CC(O)=O HOKKHZGPKSLGJE-GSVOUGTGSA-N 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010043958 Peptoids Proteins 0.000 description 1
- 101710124239 Poly(A) polymerase Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 102100034207 Protein argonaute-2 Human genes 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 102000004389 Ribonucleoproteins Human genes 0.000 description 1
- 108010081734 Ribonucleoproteins Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- XSVMFMHYUFZWBK-NSHDSACASA-N Rivastigmine Chemical compound CCN(C)C(=O)OC1=CC=CC([C@H](C)N(C)C)=C1 XSVMFMHYUFZWBK-NSHDSACASA-N 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical group OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 229940022698 acetylcholinesterase Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- VLSMHEGGTFMBBZ-UHFFFAOYSA-N alpha-Kainic acid Natural products CC(=C)C1CNC(C(O)=O)C1CC(O)=O VLSMHEGGTFMBBZ-UHFFFAOYSA-N 0.000 description 1
- 102000038380 alpha-secretases Human genes 0.000 description 1
- 108091007736 alpha-secretases Proteins 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229940049706 benzodiazepine Drugs 0.000 description 1
- 150000001557 benzodiazepines Chemical class 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-QZABAPFNSA-N beta-D-glucosamine Chemical compound N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-QZABAPFNSA-N 0.000 description 1
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical class OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 1
- 108091007737 beta-secretases Proteins 0.000 description 1
- 150000005347 biaryls Chemical class 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229910052797 bismuth Inorganic materials 0.000 description 1
- JCXGWMGPZLAOME-UHFFFAOYSA-N bismuth atom Chemical compound [Bi] JCXGWMGPZLAOME-UHFFFAOYSA-N 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000002867 ciliostatic effect Effects 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- WOWBFOBYOAGEEA-UHFFFAOYSA-N diafenthiuron Chemical compound CC(C)C1=C(NC(=S)NC(C)(C)C)C(C(C)C)=CC(OC=2C=CC=CC=2)=C1 WOWBFOBYOAGEEA-UHFFFAOYSA-N 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 125000004925 dihydropyridyl group Chemical group N1(CC=CC=C1)* 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 229960003530 donepezil Drugs 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 150000002081 enamines Chemical class 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 230000003492 excitotoxic effect Effects 0.000 description 1
- 231100000063 excitotoxicity Toxicity 0.000 description 1
- 210000000887 face Anatomy 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000003188 fatty acid synthesis inhibitor Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 229960003980 galantamine Drugs 0.000 description 1
- ASUTZQLVASHGKV-UHFFFAOYSA-N galanthamine hydrochloride Natural products O1C(=C23)C(OC)=CC=C2CN(C)CCC23C1CC(O)C=C2 ASUTZQLVASHGKV-UHFFFAOYSA-N 0.000 description 1
- 108091007739 gamma-secretases Proteins 0.000 description 1
- 102000038383 gamma-secretases Human genes 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 210000004295 hippocampal neuron Anatomy 0.000 description 1
- 150000001469 hydantoins Chemical class 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 230000002584 immunomodulator Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- VLSMHEGGTFMBBZ-OOZYFLPDSA-N kainic acid Chemical compound CC(=C)[C@H]1CN[C@H](C(O)=O)[C@H]1CC(O)=O VLSMHEGGTFMBBZ-OOZYFLPDSA-N 0.000 description 1
- 229950006874 kainic acid Drugs 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 231100000863 loss of memory Toxicity 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 150000004667 medium chain fatty acids Chemical class 0.000 description 1
- 230000015654 memory Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 108091063796 miR-206 stem-loop Proteins 0.000 description 1
- 108091059501 miR-320a stem-loop Proteins 0.000 description 1
- 108091088570 miR-320a-1 stem-loop Proteins 0.000 description 1
- 108091070041 miR-320a-2 stem-loop Proteins 0.000 description 1
- 108091065447 miR-320a-3 stem-loop Proteins 0.000 description 1
- 108091054114 miR-320a-4 stem-loop Proteins 0.000 description 1
- 108091089992 miR-9-1 stem-loop Proteins 0.000 description 1
- 108091071572 miR-9-2 stem-loop Proteins 0.000 description 1
- 108091076838 miR-9-3 stem-loop Proteins 0.000 description 1
- 108091060187 miR-9-5 stem-loop Proteins 0.000 description 1
- 108091058972 miR-9-6 stem-loop Proteins 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 230000000510 mucolytic effect Effects 0.000 description 1
- ACTNHJDHMQSOGL-UHFFFAOYSA-N n',n'-dibenzylethane-1,2-diamine Chemical compound C=1C=CC=CC=1CN(CCN)CC1=CC=CC=C1 ACTNHJDHMQSOGL-UHFFFAOYSA-N 0.000 description 1
- 239000007923 nasal drop Substances 0.000 description 1
- 229940100652 nasal gel Drugs 0.000 description 1
- 229940100657 nasal ointment Drugs 0.000 description 1
- 229940052404 nasal powder Drugs 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 210000000492 nasalseptum Anatomy 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 230000010807 negative regulation of binding Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000007472 neurodevelopment Effects 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- UMRZSTCPUPJPOJ-KNVOCYPGSA-N norbornane Chemical compound C1C[C@H]2CC[C@@H]1C2 UMRZSTCPUPJPOJ-KNVOCYPGSA-N 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 210000002475 olfactory pathway Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000011368 organic material Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 210000000578 peripheral nerve Anatomy 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229950000688 phenothiazine Drugs 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 125000005642 phosphothioate group Chemical group 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 230000007505 plaque formation Effects 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 125000003367 polycyclic group Chemical group 0.000 description 1
- 238000011886 postmortem examination Methods 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 230000007859 posttranscriptional regulation of gene expression Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000009822 protein phosphorylation Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 229960004136 rivastigmine Drugs 0.000 description 1
- 150000003872 salicylic acid derivatives Chemical class 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000006104 solid solution Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 230000005062 synaptic transmission Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 229960001685 tacrine Drugs 0.000 description 1
- YLJREFDVOIBQDA-UHFFFAOYSA-N tacrine Chemical compound C1=CC=C2C(N)=C(CCCC3)C3=NC2=C1 YLJREFDVOIBQDA-UHFFFAOYSA-N 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- CBXCPBUEXACCNR-UHFFFAOYSA-N tetraethylammonium Chemical compound CC[N+](CC)(CC)CC CBXCPBUEXACCNR-UHFFFAOYSA-N 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000759 toxicological effect Toxicity 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000001521 two-tailed test Methods 0.000 description 1
- 229940124549 vasodilator Drugs 0.000 description 1
- 239000003071 vasodilator agent Substances 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 230000003936 working memory Effects 0.000 description 1
- 238000001086 yeast two-hybrid system Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
- C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
Definitions
- the present disclosure relates to a use of miR-485-3p for preventing or treating a brain disease, more particularly to a pharmaceutical composition for preventing or treating a brain disease, which contains a miR-485-3p inhibitor, and a method for screening an agent for preventing or treating a brain disease, which includes a step of measuring the expression level of miR-485-3p.
- Alzheimer's disease is the most common form of dementia. 75% of patients with dementia have Alzheimer's disease. In most cases, Alzheimer's disease begins in people over 65 years of age, although it can occur earlier in rare cases. In the United States, about 3% of the population aged 65-74 years, about 19% of the population aged 75-84 years, and 50% of the population aged over 85 years suffer from this disease. In Korea, according to a recently reported study on a rural region, about 21% of the population aged over 60 years in the rural region showed dementia, and 63% of them had Alzheimer's dementia. In 2006, 266,000 people around the world had the disease. It is expected that the disease will occur in one out of every 85 people in 2050.
- Alzheimer's disease has recently focused on the fact that Alzheimer's disease may be caused by impaired cholinergic signaling and transmission in the cerebral cortex and hippocampus (Bartus et al., Science. 217(4558): 408-14(1982) and Coyle et al., Science. 219(4589): 1184-90(1983)).
- NFT neurofibrillary tangles
- Alzheimer's disease is a notable feature of this disease, and Alzheimer's disease can be confirmed by post-mortem examination (Khachaturian, Arch. Neurol. 42(11): 1097-105(1985)).
- Alzheimer's disease As a way of treating Alzheimer's disease, a method of increasing the amount of acetylcholine to inhibit the impairment of cholinergic signaling or causing acetylcholine to act more effectively on transmission of neuronal cells has been proposed.
- patients with Alzheimer's disease use a variety of compounds for increasing the activity of acetylcholine.
- Some compounds are aimed at improving the general state of neurons and maintaining aged cells in good function.
- some drugs such as NGF or estrogen act as neuroprotecting agents to delay neurodegeneration, and other drugs such as antioxidants decrease cell damage caused by oxidation of cells resulting from normal aging.
- Alzheimer's disease becomes serious as the amyloid beta peptide is accumulated in the neuritic space. It is thought that the progress of Alzheimer's disease can be delayed by reducing the accumulation of amyloid beta.
- amyloid precursor protein APP
- APP amyloid precursor protein
- amyloid beta acts on neuronal signaling.
- Abnormally cleaved APP induces amyloid beta generation, and plaque formation is induced by the accumulation of amyloid beta in the neuritic space.
- various factors involved in this cleavage reaction e.g., inflammation reaction, etc.
- PHF paired helical filaments
- ELAVL2 or ELAVL-like neuron-specific RNA binding protein 2
- nELAVL2 is an RNA-binding protein expressed specifically in the brain and is known to be associated with neurodegenerative diseases.
- RNA sequencing using brain tissue after post-mortem of patients with Alzheimer's disease, it was found out that ELAVL2 was expressed with low levels.
- U.S. Pat. No. 5,532,219 discloses a composition for treating Alzheimer's disease containing 4,4′-diaminodiphenylsulfone, etc.
- U.S. Pat. No. 5,506,097 discloses a composition for treating Alzheimer's disease containing para-amidinophenylmethanesulfonyl fluoride or ebelactone A
- U.S. Pat. No. 6,136,861 discloses a composition for treating Alzheimer's disease containing bicyclo[2.2.1]heptane.
- WO 2013/045652 discloses a treatment of epilepsy using a miR-134 inhibitor
- WO 2015/025995 discloses treatment of epilepsy using a miR-203 inhibitor
- European Patent Registration No. 2436784 discloses diagnosis and treatment of colon cancer using miR-203.
- Alzheimer's disease Although the development of therapeutic methods to reduce the effect of Alzheimer's disease is carried out actively, temporary improvement of symptoms is the current strategy. In conclusion, the current treatment of Alzheimer's disease is just focused on improvement of symptoms instead of slowing or reversing the progress of the disease. Despite the biological knowledge about the disease, clinical application is still not successful.
- the inventors of the present disclosure have made efforts to develop an agent for preventing or treating brain diseases including Alzheimer's disease.
- the inhibition of miR-485-3p expression or the inhibition of interaction between miR-485-3p and ELAVL2 leads to inhibition of A ⁇ 42 production, inhibition of APP expression or inhibition of tau protein phosphorylation, thereby being useful in treating brain diseases, and have completed the present disclosure.
- the present disclosure is directed to providing a pharmaceutical composition for preventing or treating a brain disease using a microRNA.
- the present disclosure is also directed to providing a method for screening an agent for preventing or treating a brain disease by measuring the expression level of a microRNA.
- the present disclosure provides a pharmaceutical composition for preventing or treating a brain disease, which contains a miR-485-3p inhibitor.
- the present disclosure also provides a method for preventing or treating a brain disease, which includes a step of administering a pharmaceutically effective amount of a miR-485-3p inhibitor.
- the present disclosure also provides a use of a miR-485-3p inhibitor for preventing or treating a brain disease.
- the present disclosure also provides a use of a miR-485-3p inhibitor for preparing a medication for preventing or treating a brain disease.
- the present disclosure also provides a method for screening an agent for preventing or treating a brain disease, which includes: (A) a step of treating a cell expressing miR-485-3p with a candidate substance and measuring the expression level of miR-485-3p; and (B) a step of screening the candidate substance as an agent for preventing or treating a brain disease if the expression level of miR-485-3p measured in the step (A) is decreased as compared to a control group not treated with the candidate substance.
- FIG. 1 summarizes a procedure of cDNA synthesis and detection.
- FIGS. 2A-2B show a miRNA expression pattern analysis result (volcano plot) for a patient group as compared to a normal group ( FIG. 2A ), and a miRNA expression pattern analysis result (scatter plot) for a patient group as compared to a normal group ( FIG. 2B ).
- FIG. 3 compares the expression of miR-485-3p in the hippocampus and the cortex.
- FIG. 4 shows a list of the 3′-untranslated region (UTR) mRNAs of ELAVL2.
- FIGS. 5A-5B shows a comparative quantitative analysis result of A ⁇ 42 in the cerebral cortex of 5 ⁇ FAD ( FIG. 5A ), and a comparative quantitative analysis result of A ⁇ 42 in the hippocampus ( FIG. 5B ).
- FIG. 6 shows a result of comparing the expression of ELAVL2 in the cerebral cortex and the hippocampus of 5 ⁇ FAD.
- FIGS. 7A and 7B show results of comparing the expression of ELAVL2 ( FIG. 7A ) and A ⁇ ( FIG. 7B ) in hippocampal primary cells depending on transfection with antagomir (AM)-485-3p.
- FIG. 8A-8B show an photograph of a mouse brain ( FIG. 8A ) and an imaging analysis of drug delivery after intranasal administration of Cy3-AM-485-3p ( FIG. 8B ).
- FIGS. 9A-9B show comparative quantitative analysis results of ELAVL2 ( FIG. 9A ) and A ⁇ ( FIG. 9B ) for 5 ⁇ FAD intranasally treated with AM-485-3p.
- FIGS. 10A-10B show a results of comparing the expression of APP ( FIGS. 10A-10B ), tau ( FIG. 10B ), and p-tau ( FIG. 10B ) in HeLa cells depending on AM-485-3p transfection.
- FIGS. 11A-11B show results of comparing the cognitive function of 5 ⁇ FAD intranasally treated with AM-485-3p.
- the present disclosure relates to a pharmaceutical composition for preventing or treating a brain disease, which contains a miR-485-3p inhibitor.
- the ‘miR’ or ‘microRNA (miRNA)’ refers to a non-coding RNA consisting of 21-23 nucleotides, which is known to be involved in post-transcriptional regulation of gene expression by suppressing the translation of target RNA or promoting degradation thereof.
- the mature sequence of the miRNA can be obtained from the miRNA database (http://www.mirbase.org). As of Aug. 13, 2012, 25,141 mature miRNAs derived from 193 species are listed in the miRNA database (19th edition, miRBase).
- a pre-miRNA which has a hairpin structure and is about 70-80 nt (nucleotides) in length
- a mature form of miRNA is produced as the pre-miRNA is cleaved by the RNAse III enzyme Dicer.
- the miRNA forms a ribonucleoprotein complex called a miRNP and cleaves a target gene or inhibits its translation through complementary binding to the target site. 30% or more of human miRNAs exist in the form of a cluster.
- the miR-485-3p may be expressed in the brain, particularly in the hippocampus and the cortex, although not being limited thereto.
- ELAVL2 mRNA which encodes ELAVL2 (ELAV-like RNA binding protein 2), it inhibits its expression, thereby lowering the concentration of the ELAVL2 protein in the brain.
- the sequence of miR-485-3p may be derived from a mammal, for example, a human, mouse or rat.
- the sequence of miR-485-3p is derived from a human, and includes not only a mature sequence [5′-GUCAUACACGGCUCUCCUCUCU-3′ (SEQ ID NO 1)] but also a precursor sequence [ 5 ′-ACUUGGAGAGAGGCUGGCCGUGAUGAAUUCGAUUCAUCAAAGCGAGUCAUAC ACGGCUCUCCUCUCUUUUAGU-3′ (SEQ ID NO 2)].
- the miR-485-3p inhibitor may inhibit the expression of miR-485-3p. Alternatively, it may inhibit the interaction between miR-485-3p and the 3′-UTR of ELAVL2 (ELAV-like neuron-specific RNA binding protein 2).
- the miR-485-3p inhibitor may inhibit or interfere with the action or function of miR-485-3p in cells.
- the inhibition of miR-485-3p includes direct inhibition of binding of miR-485-3p to its target, e.g., an mRNA molecule encoding the ELAVL2 protein.
- direct inhibition of the function of miR-485-3p using a small molecule inhibitor, an antibody or an antibody fragment, or indirect regulation using an inhibitor or a small interfering RNA molecule is included.
- the miR-485-3p inhibitor may be a nucleic acid molecule binding to all or a part of the base sequence of SEQ ID NO 1 or SEQ ID NO 2.
- the nucleic acid molecule binding to a part of the base sequence of SEQ ID NO 1 or SEQ ID NO 2 may be 7-50 nt (nucleotides), specifically 10-40 nt, more specifically 15-30 nt, further more specifically 15-25 nt, particularly 16-19 nt, in length, although not being limited thereto.
- the nucleic acid molecule may bind to the 1st or 2nd through the 7th or 8th base sequence of SEQ ID NO 1.
- the nucleic acid molecule may be selected from a group consisting of DNA, RNA, an antagomir (antisense oligonucleotide of miRNA), siRNA, shRNA and an oligonucleotide.
- the activity of the precursor sequence (SEQ ID NO 2) and the mature sequence (SEQ ID NO 1) is inhibited directly or indirectly for the interference with or inhibition of the activity of miR-485-3p.
- the inhibition of the activity of miR-485-3p includes lowering its cellular level by inhibiting the transcription of miR-485-3p and/or the binding of miR-485-3p to its target mRNA.
- the miR-485-3p inhibitor includes any substance capable of inhibiting the expression and/or activity of miR-485-3p.
- the substance includes a low-molecular-weight compound, an antagomir, an antisense molecule, a small hairpin RNA (shRNA) molecule, a small interfering RNA (siRNA) molecule, a seed target LNA (locked nucleic acid) oligonucleotide, a decoy oligonucleotide, an aptamer, a ribozyme, or an antibody that recognizes a DNA:RNA hybrid, although not being limited thereto.
- a low-molecular-weight compound an antagomir, an antisense molecule, a small hairpin RNA (shRNA) molecule, a small interfering RNA (siRNA) molecule, a seed target LNA (locked nucleic acid) oligonucleotide, a decoy oligonucleotide, an apt
- the miR-485-3p inhibitor may be an antisense oligonucleotide which can inhibit the activity of miR-485-3p by complementarily binding to all or a part of the precursor and/or mature sequence, particularly the seed sequence.
- the ‘seed sequence’ is a sequence which is very important in recognition of the target molecule of miRNA and is conserved in a variety of species (Krenz, M. et al., J. Am. Coll. Cardiol. 44: 2390-2397 (2004); H. Kiriazis, et al., Annu. Rev. Physiol. 62: 321 (2000)). Because miRNA binds to its target via the sequence seed, the translation, etc. of the target mRNA may be inhibited effectively by inhibiting the interaction between the seed sequence and the target.
- the nucleic acid molecule may be an antisense oligonucleotide containing a sequence all or a part of which is complementary to the base sequence of SEQ ID NO 1.
- the antisense oligonucleotide may be represented by a base sequence selected from a group consisting of SEQ ID NO 3 to SEQ ID NO 7.
- the antisense oligonucleotide may include a sequence all or a part of which is complementary to the 1st or 2nd through the 7th or 8th base sequence of the base sequence of SEQ ID NO 1, although not being limited thereto.
- the antisense oligonucleotide may be represented by a base sequence selected from a group consisting of 5′-GUGUAUGAC-3′ (SEQ ID NO 3), 5′-UGUAUGAC-3′ (SEQ ID NO 4), 5′-GUGUAUGA-3′ (SEQ ID NO 5), 5′-UGUAUGA-3′ (SEQ ID NO 6) or 5′-AGAGAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO 7).
- the antisense oligonucleotide includes a nucleic acid-based molecule having a sequence complementary to all or a part of a target miRNA, particularly the seed sequence of the miRNA, and thus capable of forming a duplex with the miRNA.
- the antisense oligonucleotide may be referred to as a complementary nucleic acid-based inhibitor.
- the antisense oligonucleotide includes a variety of molecules, for example, a ribonucleic acid (RNA), a deoxyribonucleic acid (DNA), an antagomir, a 2′-O-modified oligonucleotide, a phosphorothioate-backbone deoxyribonucleotide, a phosphorothioate-backbone ribonucleotide, a PNA (peptide nucleic acid) oligonucleotide or an LNA (locked nucleic acid) oligonucleotide.
- RNA ribonucleic acid
- DNA deoxyribonucleic acid
- an antagomir an antagomir
- a 2′-O-modified oligonucleotide a phosphorothioate-backbone deoxyribonucleotide
- a phosphorothioate-backbone ribonucleotide a PNA
- the ribonucleic acid includes a double-stranded small hairpin RNA (shRNA) molecule, a small interfering RNA (siRNA) molecule and a ribozyme.
- shRNA small hairpin RNA
- siRNA small interfering RNA
- the LNA has a locked conformation due to further modification between the 2 ‘ and 4’ carbon of the ribose moiety of the oligonucleotide and, thus, ensures thermal stability.
- the PNA peptide nucleic acid
- the 2′-O-modified oligonucleotide is specifically a 2′-O-alkyl oligonucleotide, more specifically a 2′-O—C 1-3 alkyl oligonucleotide, and most specifically a 2′-O-methyl oligonucleotide.
- the antisense oligonucleotide includes an antisense oligonucleotide in a narrow sense, an antagomir and an inhibitory RNA molecule.
- the antagomir is a chemically modified single-stranded oligonucleotide and is used to silence an endogenous microRNA.
- the antagomir contains a sequence that is not complementary at the Argonaute 2 (Ago2) cleavage site, or inhibits cleavage of Ago2 such that the base is modified with, for example, a 2-′methoxy group, a 3′-cholesterol group or a phosphorothioate. There is a complementary sequence to the target sequence.
- the antagomir has a sequence which is at least partially or completely complementary to miR-485-3p.
- the antagomir may include one or more modification (e.g., 2′-O-methyl-sugar modification or 3′-cholesterol modification).
- the antagomir may contain one or more phosphorothioate linkage and have a phosphorothioate backbone at least in part.
- the appropriate length of the antagomir for inhibiting the expression of miR-485-3p is 7-50 nt (nucleotides), specifically 10-40 nt, more specifically 15-30 nt, more specifically 15-25 nt, more specifically 16-19 nt, although not being limited thereto.
- the term ‘complementary’ as used the present disclosure means that the antisense oligonucleotide is sufficiently complementary to the miR-485-3p target under predetermined hybridization conditions or annealing conditions, specifically under physiological conditions, such that it can selectively hybridize to the target, and encompasses both partially or substantially complementary and completely (perfectly) complementary. Specifically, it means being completely complementary.
- substantially complementary means that, although not completely complementary, it has complementarity sufficient to bind to the target sequence and exert an effect according to the present disclosure, i.e., interference with the activity of miR-485-3p.
- the ‘nucleic acid’ includes an oligonucleotide, a DNA, an RNA, a polynucleotide, and analogs and derivatives thereof.
- a PNA or a mixture thereof is included.
- the nucleic acid may be single- or double-stranded and can encode molecules including an mRNA, a microRNA, a siRNA, a polypeptides, etc.
- the antisense oligonucleotide may include one or more modification selected from: 1) modification to a LNA (locked nucleic acid) or PNA (peptide nucleic acid) form; 2) substitution of the —OH group at the 2′ carbon of a nucleotide with —CH 3 (methyl); and 3) modification of a nucleotide bond to phosphorothioate.
- One or more nucleotide constituting the antisense oligonucleotide may be a LNA or a PNA.
- the sugar of at least one nucleotide constituting the same may be 2′-O-methylated or methoxylated, or one or more phosphothioate may be contained in the backbone, although not being limited thereto.
- the miR-485-3p inhibitor may have one or more of the following features: 1) recovery of the expression level of ELAVL2; 2) inhibition of the production of amyloid beta 42 (A ⁇ 42); 3) inhibition of the expression of amyloid precursor protein (APP); and 4) inhibition of the phosphorylation of tau protein.
- the miR-485-3p inhibitor has the features of recovery of the expression level of ELAVL2, inhibition of the production of A ⁇ 42, inhibition of the expression of APP and inhibition of the phosphorylation of tau protein using 5 ⁇ FAD mouse, which is an animal model of Alzheimer disease which exhibits severe accumulation of intraneuronal A ⁇ 42 from about 6 weeks due to overexpression of mutant forms of APP and PSEN1.
- the decreased expression level of ELAVL2 is associated with the onset of Alzheimer's disease, autism spectrum disorder, mental retardation and amyotrophic lateral sclerosis.
- the level of the ELAVL2 protein is decreased by substances inducing excitotoxicity such as kainic acid, NMDA, quisulate, AMPA, glutamate, etc., resulting in neuronal cell death and disturbance of brain function, causing a number of brain diseases such as seizure, stroke, Parkinson's disease, spinal cord injury, etc. (Kaminska, B. et al., Acta Biochim Pol. 44: 781-789).
- the recovery of the ELAVL2 protein through the inhibition of the activity of miR-485-3p can be used in the treatment of various brain diseases such as Alzheimer's disease, autism spectrum disorder, mental retardation, amyotrophic lateral sclerosis, seizure, stroke, Parkinson's disease, spinal cord injury, etc.
- the brain disease may be selected from a group consisting of Alzheimer's disease, autism spectrum disorder, mental retardation, amyotrophic lateral sclerosis, seizure, stroke, Parkinson's disease and spinal cord injury, although not being limited thereto.
- the pharmaceutical composition may further contain, in addition to the miR-485-3p inhibitor, one or more active ingredient exhibiting the same, similar or synergistic function for the treatment of related diseases or a compound which maintains/increases the solubility and/or absorbency of the miR-485-3p inhibitor or the active ingredient. And, optionally, it may further contain an immunomodulator and/or a chemotherapeutic agent.
- the pharmaceutical composition may further contain one or more pharmaceutically acceptable diluent, carrier and/or adjuvant in addition to the above-mentioned active ingredient.
- a pharmaceutically acceptable carrier saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and a mixture of one or more of these components may be used. If necessary, other common additives such as an antioxidant, a buffer, a bacteriostatic agent, etc. may be added.
- an injectable formulation such as an aqueous solution, a suspension, an emulsion, etc., a pill, a capsule, a granule or a tablet by additionally adding a diluent, a dispersant, a surfactant, a binder and a lubricant, and it can be used by binding a target organ-specific antibody or other ligand with the carrier.
- it can be suitably formulated depending on the particular disease or ingredient by using appropriate methods in the art or using the methods disclosed in the Remington's literature (Remington's Pharmaceutical Science (newest edition), Mack Publishing Company, Easton Pa.).
- it can be formulated into one of a suspension, a liposomal formulation, an emulsion, a tablet, a capsule, a gel, a syrup or a suppository.
- the pharmaceutical composition may be prepared into a suspension using an aqueous, nonaqueous or mixed medium.
- An aqueous suspension may further contain a material increasing the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol and/or dextran.
- the pharmaceutical composition may be formulated into a formulation for intranasal administration, intravenous administration, subcutaneous injection, intrathecal injection, inhalation administration or oral administration.
- the administration method of the pharmaceutical composition according to the present disclosure is not particularly limited and any known administration method of inhibitors may be applied.
- parenteral administration e.g., intranasal, intravenous, subcutaneous, intraperitoneal or topical administration
- oral administration may be employed.
- administration by intranasal injection may be selected to achieve a quick therapeutic effect.
- the pharmaceutical composition may be delivered via various routes, e.g., via infusion, bolus injection, transdermal or transmucosal administration (via buccal, anal or intestinal mucosa), or systemic or topical administration.
- routes e.g., via infusion, bolus injection, transdermal or transmucosal administration (via buccal, anal or intestinal mucosa), or systemic or topical administration.
- the pharmaceutical composition may be delivered to the brain.
- the pharmaceutical composition may be introduced to the central or peripheral nerves via an appropriate route.
- the appropriate route includes intraventricular or intrathecal administration.
- the administration may be achieved using a catheter connected to a reservoir.
- the pharmaceutical composition may be formulated as an aerosol and may be administered to the lungs using an inhaler or a nebulizer.
- the appropriate route is not limited as long as the effect of the present disclosure is achieved, and includes intravenous administration, subcutaneous injection, intrathecal injection, inhalation administration or oral administration.
- the pharmaceutical composition can be prepared into a variety of unit dosage forms.
- Such forms include a nasal drop, a nasal spray, a nasal gel, a nasal ointment and a nasal powder, although not being limited thereto.
- the composition could be administered intranasally.
- the effect of the pharmaceutical composition can be enhanced when it is administered intranasally because it is delivered to the brain through the olfactory pathway.
- the nasal cavity refers to a space in the nose, which is divided into left and right fossae by the nasal septum, and the intranasal administration refers to delivery of the composition of the present disclosure to any tissue of the nasal epithelium.
- an intranasally acceptable carrier may be contained.
- the carrier refers to one or more solid or liquid filler, diluent or encapsulating material which is suitable for administration to any portion of the nasal epithelium of a mammal, specifically human.
- the carrier may be a liquid, a solution, a suspension, a gel, an ointment, a lotion, or a combination thereof.
- the carrier may be a pharmaceutically acceptable aqueous carrier.
- an intranasal delivery-enhancing agent may include an aggregation-inhibiting agent, a dosage-changing agent, a pH control agent, a degradative enzyme-inhibiting agent, a mucolytic or mucus-clearing agent, a ciliostatic agent, a membrane penetration-enhancing agent, a surfactant, a bile salt, a phospholipid or fatty acid additive, a mixed micelle, a liposome or carrier, an alcohol, an enamine, a nitric oxide-donating compound, a long-chain amphiphilic molecule, a small hydrophobic penetration enhancer, a sodium or salicylic acid derivative, a glycerol ester of acetoacetic acid, a cyclodextrin or beta-cyclodextrin derivative, a medium-chain fatty acid, a chelating agent, an amino acid or a salt thereof, a N-acetylamino acid
- the pharmaceutical composition may be administered in a pharmaceutically or therapeutically effective amount.
- the pharmaceutically or therapeutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and an effective dose level will depend on factors including the type and severity of the disease, the activity of a drug, sensitivity to the drug, the time of administration, the route of administration, the rate of excretion, the duration of the treatment, and drugs used together, and other factors well known in the medical field.
- the pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or multiply. It is important that the pharmaceutical composition is administered in such an amount that the maximum effect can be obtained with a minimum amount without side effects considering all of the above-mentioned factors, which can be easily determined by those skilled in the art.
- the dosage may vary depending on the patient's body weight, age, sex, health condition and diet, administration time, administration method, excretion rate, the severity of the disease, etc., and a proper dosage may also vary depending on the amount of the drug accumulated in the patient's body and/or the specific efficacy of the polynucleotide used. In general, it can be calculated on the basis of EC 50 measured as effective from an in-vivo animal model and in vitro. For example, it may be from 0.01 ⁇ g to 1 g per 1 kg of body weight, and may be administered once to several times per unit period in a daily, weekly, monthly, or annual unit period. Also, it can be administered continuously for a long period of time using an infusion pump. The number of repeated administrations is determined in consideration of the time during which the drug remains in the body, the drug concentration in the body, and the like. Even after treatment according to the course of disease treatment, the pharmaceutical composition can be continuously administered to prevent the recurrence of the disease.
- the active ingredient of the pharmaceutical composition e.g., the antisense oligonucleotide
- the pharmaceutically acceptable salt refers to a salt that retains the desired biological activity of the oligonucleotide according to the present disclosure and exhibits minimal undesired toxicological effect.
- the salt includes, for example, a base addition salt formed with a metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, etc., or a salt formed with a cation derived from ammonia, N,N-dibenzylethylenediamine, D-glucosamine, tetraethylammonium or ethylenediamine, although not being limited thereto.
- a base addition salt formed with a metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, etc.
- a salt formed with a cation derived from ammonia, N,N-dibenzylethylenediamine, D-glucosamine, tetraethylammonium or ethylenediamine although not being limited thereto.
- the antisense oligonucleotide which is the active ingredient of the pharmaceutical composition, may be negatively charged due to the characteristic of the nucleotide.
- the cellular uptake of the antisense oligonucleotide may be reduced due to the lipophilic nature of cell membranes.
- the hindered uptake due to polarity can be avoided by using the prodrug approach described in Crooke, R. M. (1998) in Crooke, S. T. Antisense research and Application. Springer-Verlag, Berlin, Germany, vol. 131, pp. 103-140.
- improvement means any action to change favorably or improve the symptoms of related diseases by administering the composition.
- treatment means any action to change favorably or improve the symptoms of related diseases by administering the composition.
- Those of ordinary skill in the art to which the present disclosure belongs will know the exact criteria of diseases by referring to the data presented, for example, by the Korean Academy of Medical Sciences and will be able to judge the degree of improvement, progress and treatment.
- prevention used in the present disclosure means any action to inhibit or delay the onset of related diseases. It will be apparent to those skilled in the art that the related diseases can be prevented if the pharmaceutical composition according to the present disclosure is administered when or before early symptoms appear.
- miR-485-3p is increased in Alzheimer's patients and that behavioral disorder and decline in cognitive function, which are the main symptoms of Alzheimer's disease, can be improved by an oligonucleotide which inhibits the expression or activity of miR-485-3p.
- the present disclosure relates to a method for preventing or treating a brain disease, which includes a step of administering a pharmaceutically effective amount of a miR-485-3p inhibitor.
- the method for preventing or treating a brain disease inhibits the activity of miR-485-3p in the cells or tissues, particularly in the brain cells or brain tissues, of a subject.
- the present disclosure relates to a use of a miR-485-3p inhibitor for preventing or treating a brain disease.
- the present disclosure relates to a use of a miR-485-3p inhibitor for preparing a medication for preventing or treating a brain disease.
- the present disclosure relates to a method for screening an agent for preventing or treating a brain disease, which includes: (A) a step of treating a cell expressing miR-485-3p with a candidate substance and measuring the expression level of miR-485-3p; and (B) a step of screening the candidate substance as an agent for preventing or treating a brain disease if the expression level of miR-485-3p measured in the step (A) is decreased as compared to a control group not treated with the candidate substance.
- the activity of miR-485-3p may be determined by analyzing the interaction between miR-485-3p and the 3′-UTR of ELAVL2 (ELAV-like neuron-specific RNA binding protein 2).
- the brain disease may be selected from a group consisting of Alzheimer's disease, autism spectrum disorder, mental retardation, amyotrophic lateral sclerosis, seizure, stroke, Parkinson's disease and spinal cord injury.
- the change in the expression level of miR-485-3p may be compared with that before the contacting or with a control group cell not in contact with the test substances and the substance which shows change, particularly decrease, in the expression level may be selected as an agent for preventing or treating a brain disease.
- the expression level of miR-485-3p may be measured by performing a known method such as northern blot, RT-PCR, a hybridization method using a microarray, etc.
- the miR-485-3p is provided in the form of a cell expressing the same, and the activity is determined by analyzing the interaction between miR-485-3p and the 3′-UTR of its target ELAVL2 protein. For example, after contacting a cell expressing the miR-485-3p according to the present disclosure with candidate substances, the change in the expression level of miR-485-3p may be compared with that before the contacting or with a control group cell not in contact with the test substances and the substance which shows change, particularly decrease, in the expression level may be selected as an agent for preventing or treating a brain disease.
- the type of the cell and the amount and kind of the candidate substance used in the screening method will vary depending on the particular test method and candidate substance used, and those skilled in the art will be able to select the suitable type, amount and/or condition of the cell. Based on the test result, the substance which leads to decreased activity of miR-485-3p in the presence of the test substance as compared to the control group not in contact with the test substance is selected as a therapeutic agent.
- the decrease means decrease by about 99% or less, decrease by about 95% or less, decrease by about 90% or less, decrease by about 85% or less, decrease by about 80% or less, decrease by about 75% or less, decrease by about 70% or less, decrease by about 65% or less, decrease by about 60% or less, decrease by about 55% or less, decrease by about 50% or less, decrease by about 45% or less, decrease by about 40% or less, decrease by about 30% or less, or decrease by about 20% or less, as compared to the control group, although not being limited thereto.
- RNA-RNA interaction used in the screening method according to the present disclosure may be detected by a method known in the art, for example, RNA walk (Lusting et al., Nucleic Acids Res. 2010; 38 (1): e5) or yeast two-hybrid system (Piganeau et al, RNA 2006; 12: 177-184, and RNA: A Laboratory Manual (Cold Spring Harbor Laboratory Press 2011)).
- the candidate substance means a substance which is expected to inhibit the activity of miR-485-3p, and includes a low-molecular-weight compound, a high-molecular-weight compound, a mixture of compounds (e.g., a natural extract or a cell or tissue culture), a biomedicine (e.g., a protein, an antibody, a peptide, DNA, RNA, an antisense oligonucleotide, RNAi, an aptamer, RNAzyme and DNAzyme), a sugar and a lipid, although not being limited thereto.
- the candidate substance can be a polypeptide having two or more amino acid residues, for example, 6, 10, 12, 20 or fewer, or more than 20, e.g., 50, amino acid residues.
- the candidate substance may be obtained from a library of synthetic or natural compounds, and a method for obtaining a library of such compounds is known in the art.
- the libraries of synthetic compounds are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and the libraries of natural compounds are commercially available from Pan Laboratories (USA) and MycoSearch (USA).
- the test substance may be obtained by a variety of combinatorial library methods known in the art, for example, a biological library, a spatially addressable parallel solid-phase or solution-phase library, a synthetic library requiring deconvolution, a “one-bead/one-compound” library, and a synthetic library using affinity chromatography selection.
- a low-molecular-weight compound exhibiting a therapeutic effect may be used for the screening purpose of a drug which treats a brain.
- a compound with a molecular weight of about 1000 Da e.g., 400 Da, 600 Da or 800 Da
- these compounds can form a part of a compound library, and the number of compounds that make up the library can also vary from dozens to millions.
- the compound library may contain peptides, peptoids, other cyclic or linear oligomeric compounds, template-based low-molecular-weight compounds, e.g., benzodiazepines, hydantoins, biaryls, carbocycles and polycyclic compounds (e.g., naphthalene, phenothiazine, acridine, steroid, etc.), carbohydrates, amino acid derivatives, dihydropyridines, benzhydryls and heterocycles (e.g. triazine, indole, thiazolidine, etc.), although not being limited thereto.
- template-based low-molecular-weight compounds e.g., benzodiazepines, hydantoins, biaryls, carbocycles and polycyclic compounds (e.g., naphthalene, phenothiazine, acridine, steroid, etc.), carbohydrates, amino acid derivatives, dihydr
- biologics may be used for the screening.
- the biologics refers to use of a cell or a biomolecule
- the biomolecule refers to a protein, a nucleic acid, a carbohydrate, a lipid or a material produced in vivo or in vitro using a cellular system.
- the biomolecule may be provided either alone or in combination with other biomolecules or cells.
- the biomolecule includes polynucleotides, peptides, antibodies or other proteins or biological organic materials found in the plasma.
- Example 1 Analysis of miRNA Expression Pattern in Alzheimer's Patients Using Microarray
- Table 1 shows the characteristics of the patients used in the study. About 3 mL of blood was collected in blood tubes (Becton Dickinson, Germany) containing sodium citrate (3.2% w/v) from 4 patients diagnosed with Alzheimer's dementia by physicians. Four healthy adults of corresponding ages ( ⁇ 4 years) were included as a control group.
- RNA extraction was extracted using the miRNAeasy Serum/Plasma kit (Qiagen, USA) according to the manufacturer's recommendations. The concentration and purity of the extracted RNA were analyzed using Bioanalyzer 2100 (Agilent, USA). Eight groups including a normal group satisfied the quality criteria and were used in the study.
- Table 2 shows a list of genes used in microarray assay.
- the mature sequence of each miRNA is available from the miRNA database (http://www.mirbase.org).
- the extracted RNA was screened using a miRNA array containing 84 different miRNAs known to be associated with human neurological development and the progress of neurological disease.
- FIG. 1 summarizes the procedure of cDNA synthesis and detection.
- the quantitative PCR assay method can be summarized as follows.
- a mature miRNA is generally a 22-nt, non-coding RNA and is responsible for post-transcriptional regulation. Polyadenylation of mature miRNA was induced by poly(A) polymerase, and cDNA was synthesized using oligo-dT primers. The oligo-dT primer enables the amplification of the mature miRNA during the real-time PCR process because it has a 3′ degenerate anchor and a universal tag sequence at the 5′ end. The mature miRNA was quantified during the real-time PCR process using the miScript SYBR Green PCR kit (Qiagen).
- FIG. 2A shows a miRNA expression pattern analysis result (volcano plot) for the patient group as compared to the normal group
- FIG. 2B shows a miRNA expression pattern analysis result (scatter plot) for the patient group as compared to the normal group.
- the expression pattern of 84 miRNAs was analyzed as compared to the normal group.
- the x axis represents fold-change and the y axis represents ⁇ log 10 of the p value.
- the horizontal black line shows where the p value is 0.05 or smaller.
- hsa-miR-485-3p the regulation of miRNA was not statistically significant except for hsa-miR-485-3p.
- the expression of hsa-485-3p was significantly increased as compared to the normal group, with a p value of 0.00439. Therefore, hsa-miR-485-3p can be used as a marker for treatment of Alzheimer's disease.
- Table 3 shows the base sequence of has-miR-485-3p. Based on the above result, a functional study was conducted to elucidate the physiological functions of has-miR-485-3p on cells by synthesizing the sequence.
- the 5 ⁇ FAD transgenic mouse is an animal model of Alzheimer disease obtained by overexpressing mutant forms of APP and PSEN1, which exhibits severe accumulation of intraneuronal A ⁇ 42 from about 6 weeks.
- RT-qPCR was performed to confirm the expression of miR-485-3p in the dementia animal model.
- 5 ⁇ FAD transgenic mice and wild-type (WT) mice were deeply anesthetized and sacrificed by decapitation. After excising the brain immediately, the hippocampus and cerebral cortex were dissected from the remaining brain structure.
- Total miRNA was isolated from the hippocampus using the PAXgene Tissue miRNA kit (Qiagen, USA) according to the manufacturer's instructions.
- cDNA was synthesized using the miScript II RT kit (Qiagen, USA), and qPCR was performed using the mmu_miR-485-3p miScript Primer Assay kit and the miScript SYBR Green PCR kit. The miRNA level was quantified by normalizing to snoRNA202 (control mouse).
- FIG. 3 compares the expression of miR-485-3p in the hippocampus and the cortex. RT-PCR was conducted to investigate the expression pattern of miR-485-3p in the hippocampus and the cerebral cortex of 5 ⁇ FAD. The result showed that the expression of miR-485-3p was increased in the hippocampus of 5 ⁇ FAD as compared to WT. This, together with the results of Example 1, shows that the expression of miR-485-3p is increased in Alzheimer's dementia. Therefore, the neuronal target mRNA or protein that may be affected by miR-485-3p was investigated.
- hsa-miR-485-3p In order to analyze the base sequence and target location of hsa-miR-485-3p, it was confirmed using a target prediction software (miRDB) that the 3′-untranslated region (UTR) of human-derived ELAVL2 is the target of hsa-miR-485-3p. It was confirmed that the identified seed sequence was conserved also in mmu-miR-485-3p and the 3′-untranslated region of mouse-derived ELAVL2.
- miRDB target prediction software
- FIG. 4 shows a list of the 3′-untranslated region (UTR) mRNAs of ELAVL2, and shows the target 3′-untranslated region (UTR) mRNAs of miR485-3p.
- the 5′ seed sequence of miR-485-3p (ELAVL2) is shown in blue color.
- Table 4 shows the base sequence and target location of mmu-miR485-3p. It was confirmed using a target prediction software (miRDB) that the 3′-untranslated region (UTR) of human-derived ELAVL2 is the target of mmu-miR-485-3p.
- miRDB target prediction software
- Example 3 Given the results of Example 3, the expression of A ⁇ 42 and ELAVL2 in the hippocampus and the cerebral cortex of 5 ⁇ FAD was investigated. After sacrificing an anesthetized mouse by decapitation, the brain was extracted immediately. After preparing a homogenate of the brain (hippocampus and cerebral cortex), western blot was conducted using anti-ELAVL2 antibody (Abcam, USA). The immunoreactive protein was visualized with a chemiluminescence reagent (GE Healthcare, UK) and was measured and quantified using a chemiluminescence analyzer (Fusion SL). A ⁇ 42 in the hippocampus and the cerebral cortex was quantified by using the mouse/rat amyloid beta (1-42) ELISA kit (IBL) according to the manufacturer's instructions.
- IBL mouse/rat amyloid beta
- FIGS. 5A-5B show results of quantitatively comparing the expression of A ⁇ 42 in the cerebral cortex ( FIG. 5A ) and the hippocampus ( FIG. 5B ) of 5 ⁇ FAD. It was confirmed that A ⁇ 42 was significantly increased as compared towild-typ (WT) both in the cerebral cortex and in the hippocampus.
- FIG. 6 shows the expression of ELAVL2 in the cerebral cortex and hippocampus of 5 ⁇ FAD.
- ELAVL2 an ELAV-like RNA-binding protein
- a protein that regulates neural functions such as neuronal excitation or synaptic transmission, which are directly associated with cognitive and behavioral functions.
- FIG. 6 it was confirmed that the expression of ELAVL2 in the hippocampus and cerebral cortex of 5 ⁇ FAD was decreased as compared to WT. This suggests that the dementia induced in 5 ⁇ FAD is associated with the decline in cognitive and behavioral functions caused by decreased ELAVL2.
- a cell homogenate was obtained 48 hours after the transfection, which was subjected to western blot using ELAVL2 antibody (Abcam, UK).
- the immunoreactive protein was visualized with a chemiluminescence reagent (GE Healthcare, UK) and was measured and quantified using a chemiluminescence analyzer (Fusion SL).
- the amyloid beta 42 protein was measured by using the mouse/rat amyloid beta (1-42) ELISA kit (IBL) according to the manufacturer's instructions.
- FIGS. 7A-7B show the results of comparing the expression of ELAVL2 and AP depending on transfection of hippocampal primary cells with AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)).
- ELAVL2 was expressed in the hippocampal primary cells of 5 ⁇ FAD, and the expression of ELAVL2 was increased in the cells transfected with antagomir (AM)-485-3p as compared to the control ( FIG. 7 A). This means that miR-485-3p inhibits the expression of ElAVL2 in the cells treated with the antagomir. Because ELAVL2 is an important factor affecting cognitive function by being involved in excitation of neurons, the development of a drug or a composition that increases ELAVL2, such as a miR-485-3p inhibitor, can be a key strategy in preventing or treating Alzheimer's disease.
- a ⁇ 42 was decreased in the cells transfected with AM-485-3p ((2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9); FIG. 7B ).
- the inhibition of miR-485-3p was induced by intranasally administering a sequence-specific antagomir.
- the intranasal administration of the antagomir was carried out according to a method targeting the brain without anesthetizing the mouse (Leah R. T., et al. (2013) Intranasal Administration of CNS Therapeutics to Awake Mice. J Vis Exp. 2013; (74): 4440).
- Example 7 Comparative Quantitative Analysis of ELAVL2 and AO in 5 ⁇ FAD Intranasally Treated with Antagomir (AM)-485-3p
- AM-485 (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9); 5 nmol in 24 ⁇ L of distilled water treated with 0.1% v/v diethylpyrocarbonate; Bioneer, Korea) was administered with a pipette while alternating nares every 2 minutes, with 4 ⁇ L per each administration (6 times in total).
- a vehicle of the same volume was administered to a control mouse.
- the anesthetized mouse was sacrificed by decapitation and the brain was excised immediately. After preparing a homogenate of the brain (hippocampus and cerebral cortex), western blot was conducted using ELAVL2 antibody (Abcam, USA).
- the immunoreactive protein was visualized with a chemiluminescence reagent (GE Healthcare, UK) and was measured and quantified using a chemiluminescence analyzer (Fusion SL).
- a ⁇ 42 was measured by using the mouse/rat amyloid beta (1-42) ELISA kit (IBL) according to the manufacturer's instructions.
- FIGS. 9A-9B show the comparative quantitative analysis of ELAVL2 and A ⁇ in 5 ⁇ FAD intranasally treated with the AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)). Because it was confirmed that the treatment of a mouse primary cell line with AM-485-3p induces change in ELAVL2 and A ⁇ (Example 5), the effect of AM-485-3p in vivo was investigated by intranasally treating 5 ⁇ FAD with AM-485-3p. The AM-485-3p group showed increased expression of ELAVL2 as compared to the control group ( FIG. 9 A). This suggests that the expression of ELAVL2 is decreased as the expression of miR-485-3p is increased, and that the decreased level of ELAVL2 can be increased by treating with a miR-485-3p inhibitor such as AM-485-3p.
- a miR-485-3p inhibitor such as AM-485-3p.
- Example 8 Expression Pattern of APP and Pattern of Tau and p-Tau in HeLa Cells Stably Transfected with Swedish Mutant Form of A ⁇ PP (A ⁇ PPsw) Depending on Treatment with AM-485-3p
- HeLa cells in which A ⁇ PPsw was expressed stably were transfected with 5-500 or 50 nM of miR-485-3p duplex (or scrambled miRNA duplex; Bioneer, Daej on, South Korea) and 50 nM of antagomir (AM)-485-3p in vitro using Lipofectamine 2000.
- a cell homogenate was obtained 48 hours after the transfection, which was subjected to western blot using APP antibody (Cell Signaling, USA), Tau (Thermofisher Scientific) and p-Tau (Thermofisher Scientific).
- the immunoreactive protein was visualized with a chemiluminescence reagent (GE Healthcare, UK) and was measured and quantified using a chemiluminescence analyzer (Fusion SL).
- FIGS. 10A-10B The expression of APP and pattern of Tau and p-Tau in the HeLa cells depending on the treatment with AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)) was compared ( FIGS. 10A-10B ). It was confirmed that the expression of APP is decreased in the cells transfected with AM-485-3p in a concentration-dependent manner as compared to the control group. In addition, it was confirmed that the HeLa cell treated with 50 nM of AM-485-3p showed decreased phosphorylation of tau protein, which is known as another cause of Alzheimer's disease.
- a drug such as a miR-485-3p inhibitor or a composition thereof can be a key strategy in preventing or treating Alzheimer's disease by inhibiting the precursor of amyloid beta and the phosphorylation of tau protein, which are known as main causes of Alzheimer's disease, at the same time.
- a Y-maze test apparatus is composed of Y-shaped maze prepared with black acrylic plates (10 cm wide, 41 cm long, 25 cm high). The maze is arranged with an angle of 120°. After dividing each maze into A, B and C zones, the experimental animals were placed carefully in each zone and allowed to move freely for 8 minutes. Spontaneous alternation (%) was evaluated by measuring the number and sequence of entries into each maze. The entrance into the three different zones in sequence was given one point (actual alternation, e.g., A-B—C, B—C-A, C-A-B, etc.). No point was given to discontinuous entrance. The spontaneous alternation (%) was calculated by the following formula.
- the passive avoidance test is a widely used method for measuring the working memory ability of rodents.
- a passive avoidance test apparatus is a shuttle box divided into two chambers, one equipped with a light bulb to create a bright environment that the test animals dislike, and the other with light blocked to create an environment which is comfort for the animals. After two hours of stress application, the passive avoidance response was tested (training test). Aluminum grids were placed on the floor of the dark chamber at regular intervals so as to apply electric shocks to the sole of the animals. The experimental animals tend to enter the dark chamber. After keeping the animal in the bright chamber and then allowing to enter the dark chamber, electric shock (5 V, 0.5 mA, 10 sec) was applied so that it could remember it. 24 hours later, the time (latency time) lapsed until the entry into the dark chamber was measured up to 90 seconds without applying electric shock (retention tests 1, 2 and 3).
- FIGS. 11A-11B show the results of comparing the cognitive function of the 5 ⁇ FAD intranasally treated with AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)).
- AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)
- both the spontaneous alteration and the latency time were decreased in the 5 ⁇ FAD and the control mouse as compared to WT.
- the typical symptoms of Alzheimer's dementia are behavior disorder and memory decline
- the behavior disorder of 5 ⁇ FAD seems to be due to the excessive accumulation and pathology of A ⁇ .
- the group intranasally treated with AM-485-3p showed significant increase in both the spontaneous alteration ( FIG. 11 A) and the latency time ( FIG. 11 B) as compared to 5 ⁇ FAD.
- the treatment with AM-485-3p can improve the main symptoms of Alzheimer's by relieving the pathological symptoms such as behavioral disorder and memory decline caused by the production of A ⁇ 42 facilitated by miR-485-3p. Therefore, the preparation of a drug that regulates miR-485-3p or a composition thereof can be a new strategy to improve the main symptoms of Alzheimer's dementia, i.e., behavioral disorder and cognitive function.
- a composition for treating a brain disease which contains a miR-485-3p inhibitor, can restore the ELAVL2 protein unlike the exiting therapeutic agents for Alzheimer's disease, which are limited only to alleviating symptoms by inducing decreased expression of amyloid beta 42. Therefore, it can fundamentally treat various diseases caused by decreased expression of ELAVL2, such as Alzheimer's disease, autism spectrum disorder, mental retardation, amyotrophic lateral sclerosis, etc. Accordingly, the present disclosure is useful for treating brain diseases including Alzheimer's disease fundamentally.
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Psychiatry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
- The content of the electronically submitted sequence listing in ASCII text file (Name: 4366_0120000_SeqListing_ST25.txt; Size: 1,985 bytes; and Date of Creation: Jun. 13, 2019) filed with the application is herein incorporated by reference in its entirety.
- The present disclosure relates to a use of miR-485-3p for preventing or treating a brain disease, more particularly to a pharmaceutical composition for preventing or treating a brain disease, which contains a miR-485-3p inhibitor, and a method for screening an agent for preventing or treating a brain disease, which includes a step of measuring the expression level of miR-485-3p.
- Alzheimer's disease is the most common form of dementia. 75% of patients with dementia have Alzheimer's disease. In most cases, Alzheimer's disease begins in people over 65 years of age, although it can occur earlier in rare cases. In the United States, about 3% of the population aged 65-74 years, about 19% of the population aged 75-84 years, and 50% of the population aged over 85 years suffer from this disease. In Korea, according to a recently reported study on a rural region, about 21% of the population aged over 60 years in the rural region showed dementia, and 63% of them had Alzheimer's dementia. In 2006, 266,000 people around the world had the disease. It is expected that the disease will occur in one out of every 85 people in 2050.
- The treatment of Alzheimer's disease has recently focused on the fact that Alzheimer's disease may be caused by impaired cholinergic signaling and transmission in the cerebral cortex and hippocampus (Bartus et al., Science. 217(4558): 408-14(1982) and Coyle et al., Science. 219(4589): 1184-90(1983)).
- Because these regions of the brain are associated with memory and intelligence, functional deficit in these regions may cause loss of memory and intelligence. Although the process of impairment in neuronal signaling is still controversial, senile plaques and neurofibrillary tangles (NFT) are considered as main pathological causes.
- In particular, development of senile plaques due to the accumulation of amyloid beta (Aβ) is a notable feature of this disease, and Alzheimer's disease can be confirmed by post-mortem examination (Khachaturian, Arch. Neurol. 42(11): 1097-105(1985)).
- As a way of treating Alzheimer's disease, a method of increasing the amount of acetylcholine to inhibit the impairment of cholinergic signaling or causing acetylcholine to act more effectively on transmission of neuronal cells has been proposed. Thus, patients with Alzheimer's disease use a variety of compounds for increasing the activity of acetylcholine.
- Currently, the most effective way is to rapidly decompose acetylcholine in synapses, thus inhibiting the activity of acetylcholinesterase that prevents neuronal signaling. These inhibitors (e.g., tacrine, donepezil, galantamine and rivastigmine) are approved by the United States Food and Drug Administration (FDA) and are currently available on the market as Alzheimer's disease medications. Despite their effectiveness in preventing further destructive progress of this disease, they are not used to cure the disease.
- Some compounds are aimed at improving the general state of neurons and maintaining aged cells in good function. For example, some drugs such as NGF or estrogen act as neuroprotecting agents to delay neurodegeneration, and other drugs such as antioxidants decrease cell damage caused by oxidation of cells resulting from normal aging.
- Alzheimer's disease becomes serious as the amyloid beta peptide is accumulated in the neuritic space. It is thought that the progress of Alzheimer's disease can be delayed by reducing the accumulation of amyloid beta. In addition, amyloid precursor protein (APP) is considered to play a role in combination with proteinases in cells, such as α-, β- and γ-secretases. However, because the process of amyloid beta formation has not been fully elucidated scientifically, it is not yet possible to control the formation of amyloid beta.
- It is not certain how the accumulation of amyloid beta acts on neuronal signaling. Abnormally cleaved APP induces amyloid beta generation, and plaque formation is induced by the accumulation of amyloid beta in the neuritic space. Thus, various factors involved in this cleavage reaction (e.g., inflammation reaction, etc.) increase the phosphorylation of tau protein, and also increase the accumulation of paired helical filaments (PHF) in combination with NFT, resulting in damage to the nerve. All these factors induce dysfunction of the nerve and, ultimately, accelerates the progress of Alzheimer's disease to dementia.
- ELAVL2, or ELAVL-like neuron-specific
RNA binding protein 2, is a type of nELAVL2. nELAVL2 is an RNA-binding protein expressed specifically in the brain and is known to be associated with neurodegenerative diseases. As a result of conducting high-throughput RNA sequencing using brain tissue after post-mortem of patients with Alzheimer's disease, it was found out that ELAVL2 was expressed with low levels. - In this regard, U.S. Pat. No. 5,532,219 discloses a composition for treating Alzheimer's disease containing 4,4′-diaminodiphenylsulfone, etc., U.S. Pat. No. 5,506,097 discloses a composition for treating Alzheimer's disease containing para-amidinophenylmethanesulfonyl fluoride or ebelactone A, and U.S. Pat. No. 6,136,861 discloses a composition for treating Alzheimer's disease containing bicyclo[2.2.1]heptane.
- Recently, the development of therapeutic agents using a microRNA inhibitor is being attempted. WO 2013/045652 (Apr. 4, 2013) discloses a treatment of epilepsy using a miR-134 inhibitor, and WO 2015/025995 (Feb. 26, 2015) discloses treatment of epilepsy using a miR-203 inhibitor. In addition, European Patent Registration No. 2436784 (Sep. 11, 2013) discloses diagnosis and treatment of colon cancer using miR-203.
- Although the development of therapeutic methods to reduce the effect of Alzheimer's disease is carried out actively, temporary improvement of symptoms is the current strategy. In conclusion, the current treatment of Alzheimer's disease is just focused on improvement of symptoms instead of slowing or reversing the progress of the disease. Despite the biological knowledge about the disease, clinical application is still not successful.
- Thus, the inventors of the present disclosure have made efforts to develop an agent for preventing or treating brain diseases including Alzheimer's disease. As a result, they have confirmed that the inhibition of miR-485-3p expression or the inhibition of interaction between miR-485-3p and ELAVL2 leads to inhibition of Aβ42 production, inhibition of APP expression or inhibition of tau protein phosphorylation, thereby being useful in treating brain diseases, and have completed the present disclosure.
- The information described in the Background section is only to enhance the understanding of the background of the present disclosure, and the information forming the prior art already known to those having ordinary skill in the art to which the present disclosure belongs may not be included.
- The present disclosure is directed to providing a pharmaceutical composition for preventing or treating a brain disease using a microRNA. The present disclosure is also directed to providing a method for screening an agent for preventing or treating a brain disease by measuring the expression level of a microRNA.
- In order to achieve the above-described objects, the present disclosure provides a pharmaceutical composition for preventing or treating a brain disease, which contains a miR-485-3p inhibitor.
- The present disclosure also provides a method for preventing or treating a brain disease, which includes a step of administering a pharmaceutically effective amount of a miR-485-3p inhibitor.
- The present disclosure also provides a use of a miR-485-3p inhibitor for preventing or treating a brain disease.
- The present disclosure also provides a use of a miR-485-3p inhibitor for preparing a medication for preventing or treating a brain disease.
- The present disclosure also provides a method for screening an agent for preventing or treating a brain disease, which includes: (A) a step of treating a cell expressing miR-485-3p with a candidate substance and measuring the expression level of miR-485-3p; and (B) a step of screening the candidate substance as an agent for preventing or treating a brain disease if the expression level of miR-485-3p measured in the step (A) is decreased as compared to a control group not treated with the candidate substance.
-
FIG. 1 summarizes a procedure of cDNA synthesis and detection. -
FIGS. 2A-2B show a miRNA expression pattern analysis result (volcano plot) for a patient group as compared to a normal group (FIG. 2A ), and a miRNA expression pattern analysis result (scatter plot) for a patient group as compared to a normal group (FIG. 2B ). -
FIG. 3 compares the expression of miR-485-3p in the hippocampus and the cortex. -
FIG. 4 shows a list of the 3′-untranslated region (UTR) mRNAs of ELAVL2. -
FIGS. 5A-5B shows a comparative quantitative analysis result of Aβ 42 in the cerebral cortex of 5×FAD (FIG. 5A ), and a comparative quantitative analysis result of Aβ 42 in the hippocampus (FIG. 5B ). -
FIG. 6 shows a result of comparing the expression of ELAVL2 in the cerebral cortex and the hippocampus of 5×FAD. -
FIGS. 7A and 7B show results of comparing the expression of ELAVL2 (FIG. 7A ) and Aβ (FIG. 7B ) in hippocampal primary cells depending on transfection with antagomir (AM)-485-3p. -
FIG. 8A-8B show an photograph of a mouse brain (FIG. 8A ) and an imaging analysis of drug delivery after intranasal administration of Cy3-AM-485-3p (FIG. 8B ). -
FIGS. 9A-9B show comparative quantitative analysis results of ELAVL2 (FIG. 9A ) and Aβ (FIG. 9B ) for 5×FAD intranasally treated with AM-485-3p. -
FIGS. 10A-10B show a results of comparing the expression of APP (FIGS. 10A-10B ), tau (FIG. 10B ), and p-tau (FIG. 10B ) in HeLa cells depending on AM-485-3p transfection. -
FIGS. 11A-11B show results of comparing the cognitive function of 5×FAD intranasally treated with AM-485-3p. - Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the skilled experts in the art to which the present disclosure belongs. In general, the nomenclature used herein is known well and commonly used in the art.
- In a specific example of the present disclosure, it was confirmed that the expression of miR-485-3p is increased in Alzheimer's patients and that the expression level of ELAVL2 can be recovered and the production of Aβ 42 can be decreased through an oligonucleotide inhibiting the expression or activity of miR-485-3p, thereby improving behavioral disorder and decline in cognitive function, which are the main symptoms of Alzheimer's disease.
- Accordingly, in an aspect, the present disclosure relates to a pharmaceutical composition for preventing or treating a brain disease, which contains a miR-485-3p inhibitor.
- In the present disclosure, the ‘miR’ or ‘microRNA (miRNA)’ refers to a non-coding RNA consisting of 21-23 nucleotides, which is known to be involved in post-transcriptional regulation of gene expression by suppressing the translation of target RNA or promoting degradation thereof.
- In the present disclosure, the mature sequence of the miRNA can be obtained from the miRNA database (http://www.mirbase.org). As of Aug. 13, 2012, 25,141 mature miRNAs derived from 193 species are listed in the miRNA database (19th edition, miRBase).
- In general, following transcription into a precursor called a pre-miRNA, which has a hairpin structure and is about 70-80 nt (nucleotides) in length, a mature form of miRNA is produced as the pre-miRNA is cleaved by the RNAse III enzyme Dicer. The miRNA forms a ribonucleoprotein complex called a miRNP and cleaves a target gene or inhibits its translation through complementary binding to the target site. 30% or more of human miRNAs exist in the form of a cluster.
- In the present disclosure, the miR-485-3p may be expressed in the brain, particularly in the hippocampus and the cortex, although not being limited thereto. By binding to the 3′-untranslated region of ELAVL2 mRNA which encodes ELAVL2 (ELAV-like RNA binding protein 2), it inhibits its expression, thereby lowering the concentration of the ELAVL2 protein in the brain.
- In the present disclosure, the sequence of miR-485-3p may be derived from a mammal, for example, a human, mouse or rat. In an exemplary embodiment of the present disclosure, the sequence of miR-485-3p is derived from a human, and includes not only a mature sequence [5′-GUCAUACACGGCUCUCCUCUCU-3′ (SEQ ID NO 1)] but also a precursor sequence [5′-ACUUGGAGAGAGGCUGGCCGUGAUGAAUUCGAUUCAUCAAAGCGAGUCAUAC ACGGCUCUCCUCUCUUUUAGU-3′ (SEQ ID NO 2)].
- In the present disclosure, the miR-485-3p inhibitor may inhibit the expression of miR-485-3p. Alternatively, it may inhibit the interaction between miR-485-3p and the 3′-UTR of ELAVL2 (ELAV-like neuron-specific RNA binding protein 2).
- In the present disclosure, the miR-485-3p inhibitor may inhibit or interfere with the action or function of miR-485-3p in cells. The inhibition of miR-485-3p includes direct inhibition of binding of miR-485-3p to its target, e.g., an mRNA molecule encoding the ELAVL2 protein. Also, direct inhibition of the function of miR-485-3p using a small molecule inhibitor, an antibody or an antibody fragment, or indirect regulation using an inhibitor or a small interfering RNA molecule is included.
- In the present disclosure, the miR-485-3p inhibitor may be a nucleic acid molecule binding to all or a part of the base sequence of
SEQ ID NO 1 orSEQ ID NO 2. - In the present disclosure, the nucleic acid molecule binding to a part of the base sequence of
SEQ ID NO 1 orSEQ ID NO 2 may be 7-50 nt (nucleotides), specifically 10-40 nt, more specifically 15-30 nt, further more specifically 15-25 nt, particularly 16-19 nt, in length, although not being limited thereto. - In the present disclosure, the nucleic acid molecule may bind to the 1st or 2nd through the 7th or 8th base sequence of
SEQ ID NO 1. - In the present disclosure, the nucleic acid molecule may be selected from a group consisting of DNA, RNA, an antagomir (antisense oligonucleotide of miRNA), siRNA, shRNA and an oligonucleotide.
- In an exemplary embodiment of the present disclosure, the activity of the precursor sequence (SEQ ID NO 2) and the mature sequence (SEQ ID NO 1) is inhibited directly or indirectly for the interference with or inhibition of the activity of miR-485-3p. Also, the inhibition of the activity of miR-485-3p includes lowering its cellular level by inhibiting the transcription of miR-485-3p and/or the binding of miR-485-3p to its target mRNA.
- In the present disclosure, the miR-485-3p inhibitor includes any substance capable of inhibiting the expression and/or activity of miR-485-3p. The substance includes a low-molecular-weight compound, an antagomir, an antisense molecule, a small hairpin RNA (shRNA) molecule, a small interfering RNA (siRNA) molecule, a seed target LNA (locked nucleic acid) oligonucleotide, a decoy oligonucleotide, an aptamer, a ribozyme, or an antibody that recognizes a DNA:RNA hybrid, although not being limited thereto.
- In the present disclosure, the miR-485-3p inhibitor may be an antisense oligonucleotide which can inhibit the activity of miR-485-3p by complementarily binding to all or a part of the precursor and/or mature sequence, particularly the seed sequence. The ‘seed sequence’ is a sequence which is very important in recognition of the target molecule of miRNA and is conserved in a variety of species (Krenz, M. et al., J. Am. Coll. Cardiol. 44: 2390-2397 (2004); H. Kiriazis, et al., Annu. Rev. Physiol. 62: 321 (2000)). Because miRNA binds to its target via the sequence seed, the translation, etc. of the target mRNA may be inhibited effectively by inhibiting the interaction between the seed sequence and the target.
- In In the present disclosure, the nucleic acid molecule may be an antisense oligonucleotide containing a sequence all or a part of which is complementary to the base sequence of
SEQ ID NO 1. The antisense oligonucleotide may be represented by a base sequence selected from a group consisting ofSEQ ID NO 3 to SEQ ID NO 7. - In the present disclosure, the antisense oligonucleotide may include a sequence all or a part of which is complementary to the 1st or 2nd through the 7th or 8th base sequence of the base sequence of
SEQ ID NO 1, although not being limited thereto. The antisense oligonucleotide may be represented by a base sequence selected from a group consisting of 5′-GUGUAUGAC-3′ (SEQ ID NO 3), 5′-UGUAUGAC-3′ (SEQ ID NO 4), 5′-GUGUAUGA-3′ (SEQ ID NO 5), 5′-UGUAUGA-3′ (SEQ ID NO 6) or 5′-AGAGAGGAGAGCCGUGUAUGAC-3′ (SEQ ID NO 7). - The antisense oligonucleotide includes a nucleic acid-based molecule having a sequence complementary to all or a part of a target miRNA, particularly the seed sequence of the miRNA, and thus capable of forming a duplex with the miRNA. Thus, the antisense oligonucleotide may be referred to as a complementary nucleic acid-based inhibitor.
- In addition, the antisense oligonucleotide includes a variety of molecules, for example, a ribonucleic acid (RNA), a deoxyribonucleic acid (DNA), an antagomir, a 2′-O-modified oligonucleotide, a phosphorothioate-backbone deoxyribonucleotide, a phosphorothioate-backbone ribonucleotide, a PNA (peptide nucleic acid) oligonucleotide or an LNA (locked nucleic acid) oligonucleotide. Specifically, it may be a ribonucleic acid.
- The ribonucleic acid includes a double-stranded small hairpin RNA (shRNA) molecule, a small interfering RNA (siRNA) molecule and a ribozyme.
- The LNA has a locked conformation due to further modification between the 2 ‘ and 4’ carbon of the ribose moiety of the oligonucleotide and, thus, ensures thermal stability.
- The PNA (peptide nucleic acid) contains a peptide-based backbone instead of a sugar-phosphate backbone.
- The 2′-O-modified oligonucleotide is specifically a 2′-O-alkyl oligonucleotide, more specifically a 2′-O—C1-3 alkyl oligonucleotide, and most specifically a 2′-O-methyl oligonucleotide.
- The antisense oligonucleotide includes an antisense oligonucleotide in a narrow sense, an antagomir and an inhibitory RNA molecule.
- The antagomir is a chemically modified single-stranded oligonucleotide and is used to silence an endogenous microRNA. The antagomir contains a sequence that is not complementary at the Argonaute 2 (Ago2) cleavage site, or inhibits cleavage of Ago2 such that the base is modified with, for example, a 2-′methoxy group, a 3′-cholesterol group or a phosphorothioate. There is a complementary sequence to the target sequence.
- In the present disclosure, the antagomir has a sequence which is at least partially or completely complementary to miR-485-3p. The antagomir may include one or more modification (e.g., 2′-O-methyl-sugar modification or 3′-cholesterol modification). Alternatively, the antagomir may contain one or more phosphorothioate linkage and have a phosphorothioate backbone at least in part.
- In the present disclosure, the appropriate length of the antagomir for inhibiting the expression of miR-485-3p is 7-50 nt (nucleotides), specifically 10-40 nt, more specifically 15-30 nt, more specifically 15-25 nt, more specifically 16-19 nt, although not being limited thereto.
- The term ‘complementary’ as used the present disclosure means that the antisense oligonucleotide is sufficiently complementary to the miR-485-3p target under predetermined hybridization conditions or annealing conditions, specifically under physiological conditions, such that it can selectively hybridize to the target, and encompasses both partially or substantially complementary and completely (perfectly) complementary. Specifically, it means being completely complementary. Substantially complementary means that, although not completely complementary, it has complementarity sufficient to bind to the target sequence and exert an effect according to the present disclosure, i.e., interference with the activity of miR-485-3p.
- The ‘nucleic acid’ includes an oligonucleotide, a DNA, an RNA, a polynucleotide, and analogs and derivatives thereof. For example, a PNA or a mixture thereof is included. In addition, the nucleic acid may be single- or double-stranded and can encode molecules including an mRNA, a microRNA, a siRNA, a polypeptides, etc.
- In the present disclosure, the antisense oligonucleotide may include one or more modification selected from: 1) modification to a LNA (locked nucleic acid) or PNA (peptide nucleic acid) form; 2) substitution of the —OH group at the 2′ carbon of a nucleotide with —CH3 (methyl); and 3) modification of a nucleotide bond to phosphorothioate.
- One or more nucleotide constituting the antisense oligonucleotide may be a LNA or a PNA. The sugar of at least one nucleotide constituting the same may be 2′-O-methylated or methoxylated, or one or more phosphothioate may be contained in the backbone, although not being limited thereto.
- In the present disclosure, the miR-485-3p inhibitor may have one or more of the following features: 1) recovery of the expression level of ELAVL2; 2) inhibition of the production of amyloid beta 42 (Aβ42); 3) inhibition of the expression of amyloid precursor protein (APP); and 4) inhibition of the phosphorylation of tau protein.
- In an example of the present disclosure, it was confirmed that the miR-485-3p inhibitor has the features of recovery of the expression level of ELAVL2, inhibition of the production of Aβ42, inhibition of the expression of APP and inhibition of the phosphorylation of tau protein using 5×FAD mouse, which is an animal model of Alzheimer disease which exhibits severe accumulation of intraneuronal Aβ42 from about 6 weeks due to overexpression of mutant forms of APP and PSEN1.
- It is known that the decreased expression level of ELAVL2 is associated with the onset of Alzheimer's disease, autism spectrum disorder, mental retardation and amyotrophic lateral sclerosis. Especially, it is known that the level of the ELAVL2 protein is decreased by substances inducing excitotoxicity such as kainic acid, NMDA, quisulate, AMPA, glutamate, etc., resulting in neuronal cell death and disturbance of brain function, causing a number of brain diseases such as seizure, stroke, Parkinson's disease, spinal cord injury, etc. (Kaminska, B. et al., Acta Biochim Pol. 44: 781-789). Therefore, the recovery of the ELAVL2 protein through the inhibition of the activity of miR-485-3p can be used in the treatment of various brain diseases such as Alzheimer's disease, autism spectrum disorder, mental retardation, amyotrophic lateral sclerosis, seizure, stroke, Parkinson's disease, spinal cord injury, etc.
- In the present disclosure, the brain disease may be selected from a group consisting of Alzheimer's disease, autism spectrum disorder, mental retardation, amyotrophic lateral sclerosis, seizure, stroke, Parkinson's disease and spinal cord injury, although not being limited thereto.
- In the present disclosure, the pharmaceutical composition may further contain, in addition to the miR-485-3p inhibitor, one or more active ingredient exhibiting the same, similar or synergistic function for the treatment of related diseases or a compound which maintains/increases the solubility and/or absorbency of the miR-485-3p inhibitor or the active ingredient. And, optionally, it may further contain an immunomodulator and/or a chemotherapeutic agent.
- The pharmaceutical composition may further contain one or more pharmaceutically acceptable diluent, carrier and/or adjuvant in addition to the above-mentioned active ingredient. As the pharmaceutically acceptable carrier, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and a mixture of one or more of these components may be used. If necessary, other common additives such as an antioxidant, a buffer, a bacteriostatic agent, etc. may be added.
- In addition, it can be formulated into an injectable formulation such as an aqueous solution, a suspension, an emulsion, etc., a pill, a capsule, a granule or a tablet by additionally adding a diluent, a dispersant, a surfactant, a binder and a lubricant, and it can be used by binding a target organ-specific antibody or other ligand with the carrier.
- Furthermore, it can be suitably formulated depending on the particular disease or ingredient by using appropriate methods in the art or using the methods disclosed in the Remington's literature (Remington's Pharmaceutical Science (newest edition), Mack Publishing Company, Easton Pa.). For example, it can be formulated into one of a suspension, a liposomal formulation, an emulsion, a tablet, a capsule, a gel, a syrup or a suppository.
- The pharmaceutical composition may be prepared into a suspension using an aqueous, nonaqueous or mixed medium. An aqueous suspension may further contain a material increasing the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol and/or dextran.
- In the present disclosure, the pharmaceutical composition may be formulated into a formulation for intranasal administration, intravenous administration, subcutaneous injection, intrathecal injection, inhalation administration or oral administration.
- The administration method of the pharmaceutical composition according to the present disclosure is not particularly limited and any known administration method of inhibitors may be applied. Depending on purposes, parenteral administration (e.g., intranasal, intravenous, subcutaneous, intraperitoneal or topical administration) or oral administration may be employed. Specifically, administration by intranasal injection may be selected to achieve a quick therapeutic effect.
- The pharmaceutical composition may be delivered via various routes, e.g., via infusion, bolus injection, transdermal or transmucosal administration (via buccal, anal or intestinal mucosa), or systemic or topical administration.
- In the present disclosure, the pharmaceutical composition may be delivered to the brain. Specifically, the pharmaceutical composition may be introduced to the central or peripheral nerves via an appropriate route. The appropriate route includes intraventricular or intrathecal administration. The administration may be achieved using a catheter connected to a reservoir. Also, the pharmaceutical composition may be formulated as an aerosol and may be administered to the lungs using an inhaler or a nebulizer. However, the appropriate route is not limited as long as the effect of the present disclosure is achieved, and includes intravenous administration, subcutaneous injection, intrathecal injection, inhalation administration or oral administration.
- In the present disclosure, the pharmaceutical composition can be prepared into a variety of unit dosage forms. Such forms include a nasal drop, a nasal spray, a nasal gel, a nasal ointment and a nasal powder, although not being limited thereto.
- In an exemplary example of the present disclosure, the composition could be administered intranasally. The effect of the pharmaceutical composition can be enhanced when it is administered intranasally because it is delivered to the brain through the olfactory pathway. The nasal cavity refers to a space in the nose, which is divided into left and right fossae by the nasal septum, and the intranasal administration refers to delivery of the composition of the present disclosure to any tissue of the nasal epithelium. For the intranasal administration, an intranasally acceptable carrier may be contained. The carrier refers to one or more solid or liquid filler, diluent or encapsulating material which is suitable for administration to any portion of the nasal epithelium of a mammal, specifically human. Typically, the carrier may be a liquid, a solution, a suspension, a gel, an ointment, a lotion, or a combination thereof. Specifically, the carrier may be a pharmaceutically acceptable aqueous carrier.
- In addition, the carrier may contain a delivery-enhancing agent. An intranasal delivery-enhancing agent may include an aggregation-inhibiting agent, a dosage-changing agent, a pH control agent, a degradative enzyme-inhibiting agent, a mucolytic or mucus-clearing agent, a ciliostatic agent, a membrane penetration-enhancing agent, a surfactant, a bile salt, a phospholipid or fatty acid additive, a mixed micelle, a liposome or carrier, an alcohol, an enamine, a nitric oxide-donating compound, a long-chain amphiphilic molecule, a small hydrophobic penetration enhancer, a sodium or salicylic acid derivative, a glycerol ester of acetoacetic acid, a cyclodextrin or beta-cyclodextrin derivative, a medium-chain fatty acid, a chelating agent, an amino acid or a salt thereof, a N-acetylamino acid or a salt thereof, a degradative enzyme for a selected membrane component, a fatty acid synthesis inhibitor, a cholesterol synthesis inhibitor, a nitric oxide-stimulating material, a modulatory agent of epithelial junction physiology such as chitosan or a chitosan derivative, a vasodilator, a selective transport-enhancing agent, etc. In order to enhance intranasal mucosal delivery, a stabilizing delivery vehicle, carrier, support, complex-forming species, etc. which allows effective combination, association, storage and encapsulation of the composition of the present disclosure and stabilizes the active ingredient may be contained.
- In the present disclosure, the pharmaceutical composition may be administered in a pharmaceutically or therapeutically effective amount. The pharmaceutically or therapeutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and an effective dose level will depend on factors including the type and severity of the disease, the activity of a drug, sensitivity to the drug, the time of administration, the route of administration, the rate of excretion, the duration of the treatment, and drugs used together, and other factors well known in the medical field.
- In addition, the pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or multiply. It is important that the pharmaceutical composition is administered in such an amount that the maximum effect can be obtained with a minimum amount without side effects considering all of the above-mentioned factors, which can be easily determined by those skilled in the art.
- The dosage may vary depending on the patient's body weight, age, sex, health condition and diet, administration time, administration method, excretion rate, the severity of the disease, etc., and a proper dosage may also vary depending on the amount of the drug accumulated in the patient's body and/or the specific efficacy of the polynucleotide used. In general, it can be calculated on the basis of EC50 measured as effective from an in-vivo animal model and in vitro. For example, it may be from 0.01 μg to 1 g per 1 kg of body weight, and may be administered once to several times per unit period in a daily, weekly, monthly, or annual unit period. Also, it can be administered continuously for a long period of time using an infusion pump. The number of repeated administrations is determined in consideration of the time during which the drug remains in the body, the drug concentration in the body, and the like. Even after treatment according to the course of disease treatment, the pharmaceutical composition can be continuously administered to prevent the recurrence of the disease.
- In the present disclosure, the active ingredient of the pharmaceutical composition, e.g., the antisense oligonucleotide, can be used in the composition as it is or in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salt refers to a salt that retains the desired biological activity of the oligonucleotide according to the present disclosure and exhibits minimal undesired toxicological effect. The salt includes, for example, a base addition salt formed with a metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, etc., or a salt formed with a cation derived from ammonia, N,N-dibenzylethylenediamine, D-glucosamine, tetraethylammonium or ethylenediamine, although not being limited thereto.
- In the present disclosure, the antisense oligonucleotide, which is the active ingredient of the pharmaceutical composition, may be negatively charged due to the characteristic of the nucleotide. The cellular uptake of the antisense oligonucleotide may be reduced due to the lipophilic nature of cell membranes. The hindered uptake due to polarity can be avoided by using the prodrug approach described in Crooke, R. M. (1998) in Crooke, S. T. Antisense research and Application. Springer-Verlag, Berlin, Germany, vol. 131, pp. 103-140.
- The term ‘improvement’, ‘treatment’, or ‘alleviation’ as used in the present disclosure means any action to change favorably or improve the symptoms of related diseases by administering the composition. Those of ordinary skill in the art to which the present disclosure belongs will know the exact criteria of diseases by referring to the data presented, for example, by the Korean Academy of Medical Sciences and will be able to judge the degree of improvement, progress and treatment.
- The term “prevention” used in the present disclosure means any action to inhibit or delay the onset of related diseases. It will be apparent to those skilled in the art that the related diseases can be prevented if the pharmaceutical composition according to the present disclosure is administered when or before early symptoms appear.
- In an example of the present disclosure, it was confirmed that the expression of miR-485-3p is increased in Alzheimer's patients and that behavioral disorder and decline in cognitive function, which are the main symptoms of Alzheimer's disease, can be improved by an oligonucleotide which inhibits the expression or activity of miR-485-3p.
- Accordingly, in another aspect, the present disclosure relates to a method for preventing or treating a brain disease, which includes a step of administering a pharmaceutically effective amount of a miR-485-3p inhibitor.
- In the present disclosure, the method for preventing or treating a brain disease inhibits the activity of miR-485-3p in the cells or tissues, particularly in the brain cells or brain tissues, of a subject.
- In another aspect, the present disclosure relates to a use of a miR-485-3p inhibitor for preventing or treating a brain disease.
- In another aspect, the present disclosure relates to a use of a miR-485-3p inhibitor for preparing a medication for preventing or treating a brain disease.
- Reference can be made to the above description regarding the miR-485-3p inhibitor, the regulation or inhibition of the activity of miR-485-3p, administration method, diseases that can be treated, etc.
- In another aspect, the present disclosure relates to a method for screening an agent for preventing or treating a brain disease, which includes: (A) a step of treating a cell expressing miR-485-3p with a candidate substance and measuring the expression level of miR-485-3p; and (B) a step of screening the candidate substance as an agent for preventing or treating a brain disease if the expression level of miR-485-3p measured in the step (A) is decreased as compared to a control group not treated with the candidate substance.
- In the present disclosure, the activity of miR-485-3p may be determined by analyzing the interaction between miR-485-3p and the 3′-UTR of ELAVL2 (ELAV-like neuron-specific RNA binding protein 2).
- In the present disclosure, the brain disease may be selected from a group consisting of Alzheimer's disease, autism spectrum disorder, mental retardation, amyotrophic lateral sclerosis, seizure, stroke, Parkinson's disease and spinal cord injury.
- In the screening method of the present disclosure, after contacting a cell expressing miR-485-3p with candidate substances, the change in the expression level of miR-485-3p may be compared with that before the contacting or with a control group cell not in contact with the test substances and the substance which shows change, particularly decrease, in the expression level may be selected as an agent for preventing or treating a brain disease.
- The expression level of miR-485-3p may be measured by performing a known method such as northern blot, RT-PCR, a hybridization method using a microarray, etc.
- In the present disclosure, the miR-485-3p is provided in the form of a cell expressing the same, and the activity is determined by analyzing the interaction between miR-485-3p and the 3′-UTR of its target ELAVL2 protein. For example, after contacting a cell expressing the miR-485-3p according to the present disclosure with candidate substances, the change in the expression level of miR-485-3p may be compared with that before the contacting or with a control group cell not in contact with the test substances and the substance which shows change, particularly decrease, in the expression level may be selected as an agent for preventing or treating a brain disease.
- In the present disclosure, the type of the cell and the amount and kind of the candidate substance used in the screening method will vary depending on the particular test method and candidate substance used, and those skilled in the art will be able to select the suitable type, amount and/or condition of the cell. Based on the test result, the substance which leads to decreased activity of miR-485-3p in the presence of the test substance as compared to the control group not in contact with the test substance is selected as a therapeutic agent. The decrease means decrease by about 99% or less, decrease by about 95% or less, decrease by about 90% or less, decrease by about 85% or less, decrease by about 80% or less, decrease by about 75% or less, decrease by about 70% or less, decrease by about 65% or less, decrease by about 60% or less, decrease by about 55% or less, decrease by about 50% or less, decrease by about 45% or less, decrease by about 40% or less, decrease by about 30% or less, or decrease by about 20% or less, as compared to the control group, although not being limited thereto.
- The RNA-RNA interaction used in the screening method according to the present disclosure may be detected by a method known in the art, for example, RNA walk (Lusting et al., Nucleic Acids Res. 2010; 38 (1): e5) or yeast two-hybrid system (Piganeau et al, RNA 2006; 12: 177-184, and RNA: A Laboratory Manual (Cold Spring Harbor Laboratory Press 2011)).
- The candidate substance means a substance which is expected to inhibit the activity of miR-485-3p, and includes a low-molecular-weight compound, a high-molecular-weight compound, a mixture of compounds (e.g., a natural extract or a cell or tissue culture), a biomedicine (e.g., a protein, an antibody, a peptide, DNA, RNA, an antisense oligonucleotide, RNAi, an aptamer, RNAzyme and DNAzyme), a sugar and a lipid, although not being limited thereto. The candidate substance can be a polypeptide having two or more amino acid residues, for example, 6, 10, 12, 20 or fewer, or more than 20, e.g., 50, amino acid residues. The candidate substance may be obtained from a library of synthetic or natural compounds, and a method for obtaining a library of such compounds is known in the art. The libraries of synthetic compounds are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and the libraries of natural compounds are commercially available from Pan Laboratories (USA) and MycoSearch (USA). The test substance may be obtained by a variety of combinatorial library methods known in the art, for example, a biological library, a spatially addressable parallel solid-phase or solution-phase library, a synthetic library requiring deconvolution, a “one-bead/one-compound” library, and a synthetic library using affinity chromatography selection. Method for the synthesis of molecular libraries are disclosed in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al.,
Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, or the like. - In the present disclosure, a low-molecular-weight compound exhibiting a therapeutic effect may be used for the screening purpose of a drug which treats a brain. For example, a compound with a molecular weight of about 1000 Da, e.g., 400 Da, 600 Da or 800 Da, may be used. Depending on purposes, these compounds can form a part of a compound library, and the number of compounds that make up the library can also vary from dozens to millions. The compound library may contain peptides, peptoids, other cyclic or linear oligomeric compounds, template-based low-molecular-weight compounds, e.g., benzodiazepines, hydantoins, biaryls, carbocycles and polycyclic compounds (e.g., naphthalene, phenothiazine, acridine, steroid, etc.), carbohydrates, amino acid derivatives, dihydropyridines, benzhydryls and heterocycles (e.g. triazine, indole, thiazolidine, etc.), although not being limited thereto.
- Also, biologics may be used for the screening. The biologics refers to use of a cell or a biomolecule, and the biomolecule refers to a protein, a nucleic acid, a carbohydrate, a lipid or a material produced in vivo or in vitro using a cellular system. The biomolecule may be provided either alone or in combination with other biomolecules or cells. For example, the biomolecule includes polynucleotides, peptides, antibodies or other proteins or biological organic materials found in the plasma.
- Hereinafter, the present disclosure will be described in detail through examples. However, the following examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples.
- (1) Patients and Sample Preparation
- Table 1 shows the characteristics of the patients used in the study. About 3 mL of blood was collected in blood tubes (Becton Dickinson, Germany) containing sodium citrate (3.2% w/v) from 4 patients diagnosed with Alzheimer's dementia by physicians. Four healthy adults of corresponding ages (±4 years) were included as a control group.
-
TABLE 1 Sex and age of normal group and patient group Group Sample No. Sex Age Normal group N1 Female 78 Normal group N2 Male 72 Normal group N3 Female 74 Normal group N4 Male 79 Patient Group S1 Female 72 Patient Group S2 Female 82 Patient Group S3 Female 84 Patient Group S4 Male 75 - The blood was centrifuged for 10 minutes at 3,500 rpm to separate plasma and then stored at −80° C. until RNA extraction. miRNA was extracted using the miRNAeasy Serum/Plasma kit (Qiagen, USA) according to the manufacturer's recommendations. The concentration and purity of the extracted RNA were analyzed using Bioanalyzer 2100 (Agilent, USA). Eight groups including a normal group satisfied the quality criteria and were used in the study.
- (2) Microarray Screening
- Table 2 shows a list of genes used in microarray assay. The mature sequence of each miRNA is available from the miRNA database (http://www.mirbase.org). The extracted RNA was screened using a miRNA array containing 84 different miRNAs known to be associated with human neurological development and the progress of neurological disease.
-
TABLE 2 List of genes used in miRNA qPCR array assay No. Mature miRNA 1ist 1 hsa-let-7b-5p 2 hsa-let-7c-5p 3 hsa-let-7d-5p 4 hsa-let-7e-5p 5 hsa-let-7i-5p 6 hsa-miR-101-3p 7 hsa-miR-105-5p 8 hsa miR 106b 5p 9 hsa-miR-107 10 hsa-miR-124-3p 11 hsa-miR-125b-5p 12 hsa-miR-126-5p 13 hsa-miR-128-3p 14 hsa-miR-130a-3p 15 hsa miR 132 3p 16 hsa-miR-133b 17 hsa-miR-134-5p 18 hsa-miR-135b-5p 19 hsa-miR-138-5p 20 hsa-miR-139-5p 21 hsa-miR-140-5p 22 hsa-miR-146a-5p 23 hsa-miR-146b-5p 24 hsa-miR-148b-3p 25 hsa-miR-151a-3p 26 hsa-miR-152-3p 27 hsa-miR-15a-5p 28 hsa-miR-15b-5p 29 hsa-miR-181a-5p 30 hsa-miR-181d-5p 31 hsa-miR-191-5p 32 hsa-miR-193b-3p 33 hsa-miR-195-5p 34 hsa miR 19b 3p 35 hsa miR 203a 3p 36 hsa-miR-20a-5p 37 hsa-miR-212-3p 38 hsa miR 22 3p 39 hsa miR 24 3p 40 hsa-miR-26b-5p 41 hsa-miR-27a-3p 42 hsa miR 28 5p 43 hsa-miR-298 44 hsa-miR-29a-3p 45 hsa-miR-29b-3p 46 hsa-miR-29c-3p 47 hsa-miR-302a-5p 48 hsa-miR-302b-5p 49 hsa-miR-30d-5p 50 hsa miR 320a 51 hsa-miR-328-3p 52 hsa-miR-337-3p 53 hsa-miR-338-3p 54 hsa-miR-339-5p 55 hsa-miR-342-3p 56 hsa-miR-346 57 hsa miR 34a 5p 58 hsa-miR-376b-3p 59 hsa-miR-381-3p 60 hsa-miR-409-3p 61 hsa-miR-431-5p 62 hsa-miR-432-5p 63 hsa-miR-433-3p 64 hsa-miR-455-5p 65 hsa-miR-484 66 hsa-miR-485-3p 67 hsa-miR-485-5p 68 hsa-miR-487a-3p 69 hsa-miR-488-3p 70 hsa-miR-489-3p 71 hsa-miR-499a-5p 72 hsa-miR-509-3p 73 hsa-miR-511-5p 74 hsa-miR-512-3p 75 hsa-miR-518b 76 hsa miR 539 5p 77 hsa miR 652 3p 78 hsa-miR-7-5p 79 hsa-miR-9-5p 80 hsa miR 9 3p 81 hsa miR 92a 3p 82 hsa-miR-93-5p 83 hsa-miR-95-3p 84 hsa miR 98 5p -
FIG. 1 summarizes the procedure of cDNA synthesis and detection. The quantitative PCR assay method can be summarized as follows. A mature miRNA is generally a 22-nt, non-coding RNA and is responsible for post-transcriptional regulation. Polyadenylation of mature miRNA was induced by poly(A) polymerase, and cDNA was synthesized using oligo-dT primers. The oligo-dT primer enables the amplification of the mature miRNA during the real-time PCR process because it has a 3′ degenerate anchor and a universal tag sequence at the 5′ end. The mature miRNA was quantified during the real-time PCR process using the miScript SYBR Green PCR kit (Qiagen). - (3) Analysis of miRNA Expression Pattern Through Volcano Plot
-
FIG. 2A shows a miRNA expression pattern analysis result (volcano plot) for the patient group as compared to the normal group, andFIG. 2B shows a miRNA expression pattern analysis result (scatter plot) for the patient group as compared to the normal group. The expression pattern of 84 miRNAs was analyzed as compared to the normal group. - The x axis represents fold-change and the y axis represents −log 10 of the p value. The horizontal black line shows where the p value is 0.05 or smaller. As a result of the volcano plot analysis, it was confirmed that the expression of hsa-miR-105-5p, hsa-miR-98-5p, hsa-miR-15a-5p, hsa-miR-134-5p, hsa-miR-409-3p, hsa-miR-19b-3p, hsa-miR-92a-3p, hsa-miR-28-5p, hsa-miR-30d-5p, hsa-miR-212-3p, hsa-miR-93-5p, hsa-miR-342-3p, hsa-miR-381-3p, hsa-miR-431-5p, hsa-miR-130a-3p, hsa-miR-146b-5p, hsa-miR-29a-3p, hsa-miR-132-3p, hsa-miR-376b-3p, hsa-miR-22-3p, hsa-miR-509-3p, hsa-miR-139-5p, hsa-miR-499a-5p, hsa-miR-203a-3p, hsa-miR-95-3p, hsa-miR-128-3p, hsa-miR-487a-3p, hsa-miR-485-3p, hsa-miR-195-5p, hsa-miR-433-3p, hsa-miR-133b, hsa-miR-191-5p, hsa-miR-489-3p, hsa-miR-432-5p, hsa-miR-29c-3p, hsa-miR-485-5p, hsa-miR-652-3p, hsa-miR-126-5p, hsa-miR-328-3p, hsa-let-7b-5p, hsa-miR-539-5p, hsa-miR-106b-5p, hsa-miR-101-3p, hsa-miR-302a-5p, hsa-miR-484, hsa-miR-518b, hsa-miR-148b-3p, hsa-miR-181d-5p, hsa-miR-7-5p, hsa-miR-512-3p, hsa-miR-151a-3p, hsa-miR-15b-5p, hsa-let-7e-5p, hsa-miR-135b-5p, hsa-miR-181a-5p, hsa-miR-138-5p, hsa-miR-34a-5p, hsa-miR-346, hsa-miR-511-5p, hsa-miR-485-3p, hsa-miR-485-5p, hsa-miR-487a-3p, hsa-miR-489-3p, hsa-miR-499a-5p, hsa-miR-509-3p, hsa-miR-511-5p, hsa-miR-512-3p, hsa-miR-518b, hsa-miR-539-5p, hsa-miR-652-3p, hsa-miR-7-5p, hsa-miR-92a-3p, hsa-miR-93-5p hsa-miR-95-3p and hsa-miR-98-5p was increased in the patient group. However, the regulation of miRNA was not statistically significant except for hsa-miR-485-3p. The expression of hsa-485-3p was significantly increased as compared to the normal group, with a p value of 0.00439. Therefore, hsa-miR-485-3p can be used as a marker for treatment of Alzheimer's disease.
- Table 3 shows the base sequence of has-miR-485-3p. Based on the above result, a functional study was conducted to elucidate the physiological functions of has-miR-485-3p on cells by synthesizing the sequence.
-
TABLE 3 Base sequence of hsa-miR485-3p Gene Sequence (5′ → 3′) SEQ ID NO hsa-miR485- 3p GUCAUACACGGCUCUCCUCUCU 1 - (1) Research Methods
- The 5×FAD transgenic mouse is an animal model of Alzheimer disease obtained by overexpressing mutant forms of APP and PSEN1, which exhibits severe accumulation of intraneuronal Aβ42 from about 6 weeks.
- Given the results of Example 1, RT-qPCR was performed to confirm the expression of miR-485-3p in the dementia animal model. 5×FAD transgenic mice and wild-type (WT) mice were deeply anesthetized and sacrificed by decapitation. After excising the brain immediately, the hippocampus and cerebral cortex were dissected from the remaining brain structure. Total miRNA was isolated from the hippocampus using the PAXgene Tissue miRNA kit (Qiagen, USA) according to the manufacturer's instructions. cDNA was synthesized using the miScript II RT kit (Qiagen, USA), and qPCR was performed using the mmu_miR-485-3p miScript Primer Assay kit and the miScript SYBR Green PCR kit. The miRNA level was quantified by normalizing to snoRNA202 (control mouse).
- (2) Research Results
-
FIG. 3 compares the expression of miR-485-3p in the hippocampus and the cortex. RT-PCR was conducted to investigate the expression pattern of miR-485-3p in the hippocampus and the cerebral cortex of 5×FAD. The result showed that the expression of miR-485-3p was increased in the hippocampus of 5×FAD as compared to WT. This, together with the results of Example 1, shows that the expression of miR-485-3p is increased in Alzheimer's dementia. Therefore, the neuronal target mRNA or protein that may be affected by miR-485-3p was investigated. - In order to analyze the base sequence and target location of hsa-miR-485-3p, it was confirmed using a target prediction software (miRDB) that the 3′-untranslated region (UTR) of human-derived ELAVL2 is the target of hsa-miR-485-3p. It was confirmed that the identified seed sequence was conserved also in mmu-miR-485-3p and the 3′-untranslated region of mouse-derived ELAVL2.
-
FIG. 4 shows a list of the 3′-untranslated region (UTR) mRNAs of ELAVL2, and shows thetarget 3′-untranslated region (UTR) mRNAs of miR485-3p. The 5′ seed sequence of miR-485-3p (ELAVL2) is shown in blue color. Table 4 shows the base sequence and target location of mmu-miR485-3p. It was confirmed using a target prediction software (miRDB) that the 3′-untranslated region (UTR) of human-derived ELAVL2 is the target of mmu-miR-485-3p. - (1) Research Methods
- Given the results of Example 3, the expression of Aβ 42 and ELAVL2 in the hippocampus and the cerebral cortex of 5×FAD was investigated. After sacrificing an anesthetized mouse by decapitation, the brain was extracted immediately. After preparing a homogenate of the brain (hippocampus and cerebral cortex), western blot was conducted using anti-ELAVL2 antibody (Abcam, USA). The immunoreactive protein was visualized with a chemiluminescence reagent (GE Healthcare, UK) and was measured and quantified using a chemiluminescence analyzer (Fusion SL). Aβ 42 in the hippocampus and the cerebral cortex was quantified by using the mouse/rat amyloid beta (1-42) ELISA kit (IBL) according to the manufacturer's instructions.
- (2) Research Results
- 1) Confirmation of Aβ 42 Expression in Hippocampus and Cerebral Cortex
-
FIGS. 5A-5B show results of quantitatively comparing the expression of Aβ42 in the cerebral cortex (FIG. 5A ) and the hippocampus (FIG. 5B ) of 5×FAD. It was confirmed that Aβ42 was significantly increased as compared towild-typ (WT) both in the cerebral cortex and in the hippocampus. - 2) Confirmation of ELAVL2 Expression in Hippocampus and Cerebral Cortex
-
FIG. 6 shows the expression of ELAVL2 in the cerebral cortex and hippocampus of 5×FAD. ELAVL2, an ELAV-like RNA-binding protein, is known as a protein that regulates neural functions such as neuronal excitation or synaptic transmission, which are directly associated with cognitive and behavioral functions. FromFIG. 6 , it was confirmed that the expression of ELAVL2 in the hippocampus and cerebral cortex of 5×FAD was decreased as compared to WT. This suggests that the dementia induced in 5×FAD is associated with the decline in cognitive and behavioral functions caused by decreased ELAVL2. - (1) Research Methods
- Primary cells derived from the tissues of the hippocampus and the cerebral cortex excised from the embryo of 5×FAD were cultured. The methods for cell preparation and culture followed the previous research (Seibenhener, M. L & Woonten M. W, Isolation and culture of Hippocampal Neurons from Prenatal Mice, Jove, 2012). 50 nM of miR-485-3p duplex (or scrambled miRNA duplex; Bioneer, Daejon, South Korea) and 50 nM of antagomir (AM) 485-3p were transfected into primary cells in vitro using Lipofectamine 2000. A cell homogenate was obtained 48 hours after the transfection, which was subjected to western blot using ELAVL2 antibody (Abcam, UK). The immunoreactive protein was visualized with a chemiluminescence reagent (GE Healthcare, UK) and was measured and quantified using a chemiluminescence analyzer (Fusion SL). The amyloid beta 42 protein was measured by using the mouse/rat amyloid beta (1-42) ELISA kit (IBL) according to the manufacturer's instructions.
- (2) Research Results
-
FIGS. 7A-7B show the results of comparing the expression of ELAVL2 and AP depending on transfection of hippocampal primary cells with AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)). - It was confirmed that ELAVL2 was expressed in the hippocampal primary cells of 5×FAD, and the expression of ELAVL2 was increased in the cells transfected with antagomir (AM)-485-3p as compared to the control (
FIG. 7 A). This means that miR-485-3p inhibits the expression of ElAVL2 in the cells treated with the antagomir. Because ELAVL2 is an important factor affecting cognitive function by being involved in excitation of neurons, the development of a drug or a composition that increases ELAVL2, such as a miR-485-3p inhibitor, can be a key strategy in preventing or treating Alzheimer's disease. - Also, it was confirmed that the expression of Aβ 42 was decreased in the cells transfected with AM-485-3p ((2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9);
FIG. 7B ). This means that miR-485-3p affects the production of Aβ 42 and suggests that the development of a drug or a composition capable of inhibiting miR-485-3p can relieve the pathological symptoms of Alzheimer's dementia by inhibiting the accumulation of Aβ. - (1) Research Methods
- The inhibition of miR-485-3p was induced by intranasally administering a sequence-specific antagomir. The intranasal administration of the antagomir was carried out according to a method targeting the brain without anesthetizing the mouse (Leah R. T., et al. (2013) Intranasal Administration of CNS Therapeutics to Awake Mice. J Vis Exp. 2013; (74): 4440). After immobilizing the accustomed mouse for intranasal inhalation (intranasal grip) and positioning so that the abdomen faces upward, a pipette was positioned in front of one nasal cavity. 6 μL was inhaled dropwise twice using the pipette (1 drop=3 μL). After maintaining the position for 15 seconds, intranasal inhalation into the right nasal cavity was conducted in the same manner. The same procedure was repeated 2 minutes later. A total of 24 μL was inhaled (AM485 (2′-O-methylated)-5′-gagaggagagccguguaugacu-3′ (SEQ ID NO 9); 5 nmol in 24 μL of distilled water treated with 0.1% v/v diethylpyrocarbonate; Bioneer, Korea). A vehicle of the same volume was administered to a control mouse. 12 weeks after the nasal administration, the anesthetized mouse was sacrificed by decapitation and the brain was excised immediately. After fixing the sagittally sectioned brain tissue, the tissue was treated with DAPI to stain DNA. The stained sample was imaged using a confocal laser scanning microscope (LSM510).
- (2) Research Results
- As a result of intranasally administering the AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)) fluorescence-labeled with Cy3, it was confirmed that the target neurons were stained with DAPI (
FIG. 8B ). - (1) Research Methods
- The intranasal administration of AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)) was conducted as described in Example 6 (Lee, S. T. et al. (2012) miR-206 regulates brain-derived neurotrophic factor in Alzheimer disease model. Ann Neurol, 72, 269-277). For intranasal administration of the antagomir, an anesthetized mouse was placed in supine position with its head flat on the surface. AM-485 (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9); 5 nmol in 24 μL of distilled water treated with 0.1% v/v diethylpyrocarbonate; Bioneer, Korea) was administered with a pipette while alternating nares every 2 minutes, with 4 μL per each administration (6 times in total). A vehicle of the same volume was administered to a control mouse. On day 7 after the intranasal administration, the anesthetized mouse was sacrificed by decapitation and the brain was excised immediately. After preparing a homogenate of the brain (hippocampus and cerebral cortex), western blot was conducted using ELAVL2 antibody (Abcam, USA). The immunoreactive protein was visualized with a chemiluminescence reagent (GE Healthcare, UK) and was measured and quantified using a chemiluminescence analyzer (Fusion SL). Aβ 42 was measured by using the mouse/rat amyloid beta (1-42) ELISA kit (IBL) according to the manufacturer's instructions.
- (2) Research Results
-
FIGS. 9A-9B show the comparative quantitative analysis of ELAVL2 and Aβ in 5×FAD intranasally treated with the AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)). Because it was confirmed that the treatment of a mouse primary cell line with AM-485-3p induces change in ELAVL2 and Aβ (Example 5), the effect of AM-485-3p in vivo was investigated by intranasally treating 5×FAD with AM-485-3p. The AM-485-3p group showed increased expression of ELAVL2 as compared to the control group (FIG. 9 A). This suggests that the expression of ELAVL2 is decreased as the expression of miR-485-3p is increased, and that the decreased level of ELAVL2 can be increased by treating with a miR-485-3p inhibitor such as AM-485-3p. - In addition, since it was confirmed in the animal model that the treatment with AM-485-3p affects the inhibition of Aβ42 production (
FIG. 9 B), it can be seen that treatment with the related inhibitor or drug can relieve the pathological symptoms of Alzheimer's dementia. - (1) Research Methods
- HeLa cells in which AβPPsw was expressed stably were transfected with 5-500 or 50 nM of miR-485-3p duplex (or scrambled miRNA duplex; Bioneer, Daej on, South Korea) and 50 nM of antagomir (AM)-485-3p in vitro using Lipofectamine 2000. A cell homogenate was obtained 48 hours after the transfection, which was subjected to western blot using APP antibody (Cell Signaling, USA), Tau (Thermofisher Scientific) and p-Tau (Thermofisher Scientific). The immunoreactive protein was visualized with a chemiluminescence reagent (GE Healthcare, UK) and was measured and quantified using a chemiluminescence analyzer (Fusion SL).
- (2) Research Results
- The expression of APP and pattern of Tau and p-Tau in the HeLa cells depending on the treatment with AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)) was compared (
FIGS. 10A-10B ). It was confirmed that the expression of APP is decreased in the cells transfected with AM-485-3p in a concentration-dependent manner as compared to the control group. In addition, it was confirmed that the HeLa cell treated with 50 nM of AM-485-3p showed decreased phosphorylation of tau protein, which is known as another cause of Alzheimer's disease. This suggests that the development of a drug such as a miR-485-3p inhibitor or a composition thereof can be a key strategy in preventing or treating Alzheimer's disease by inhibiting the precursor of amyloid beta and the phosphorylation of tau protein, which are known as main causes of Alzheimer's disease, at the same time. -
-
- (1) Research Methods
- Y-maze and passive avoidance tests were carried out to investigate whether the intranasal treatment of AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)) improved the cognitive function of 5×FAD.
- 1) Y-Maze Test
- A Y-maze test apparatus is composed of Y-shaped maze prepared with black acrylic plates (10 cm wide, 41 cm long, 25 cm high). The maze is arranged with an angle of 120°. After dividing each maze into A, B and C zones, the experimental animals were placed carefully in each zone and allowed to move freely for 8 minutes. Spontaneous alternation (%) was evaluated by measuring the number and sequence of entries into each maze. The entrance into the three different zones in sequence was given one point (actual alternation, e.g., A-B—C, B—C-A, C-A-B, etc.). No point was given to discontinuous entrance. The spontaneous alternation (%) was calculated by the following formula.
-
% Spontaneous alteration=total number of alternation/(total number of entries−2)×100 - 2) Passive Avoidance Test
- The passive avoidance test is a widely used method for measuring the working memory ability of rodents. A passive avoidance test apparatus is a shuttle box divided into two chambers, one equipped with a light bulb to create a bright environment that the test animals dislike, and the other with light blocked to create an environment which is comfort for the animals. After two hours of stress application, the passive avoidance response was tested (training test). Aluminum grids were placed on the floor of the dark chamber at regular intervals so as to apply electric shocks to the sole of the animals. The experimental animals tend to enter the dark chamber. After keeping the animal in the bright chamber and then allowing to enter the dark chamber, electric shock (5 V, 0.5 mA, 10 sec) was applied so that it could remember it. 24 hours later, the time (latency time) lapsed until the entry into the dark chamber was measured up to 90 seconds without applying electric shock (
retention tests - (2) Research Results
-
FIGS. 11A-11B show the results of comparing the cognitive function of the 5×FAD intranasally treated with AM-485-3p (2′-O-methylated-5′-GAGAGGAGAGCCGUGUAUGACU-3′ (SEQ ID NO 9)). As a result, both the spontaneous alteration and the latency time were decreased in the 5×FAD and the control mouse as compared to WT. Because the typical symptoms of Alzheimer's dementia are behavior disorder and memory decline, the behavior disorder of 5×FAD seems to be due to the excessive accumulation and pathology of Aβ. However, the group intranasally treated with AM-485-3p showed significant increase in both the spontaneous alteration (FIG. 11 A) and the latency time (FIG. 11 B) as compared to 5×FAD. It means that the treatment with AM-485-3p can improve the main symptoms of Alzheimer's by relieving the pathological symptoms such as behavioral disorder and memory decline caused by the production of Aβ42 facilitated by miR-485-3p. Therefore, the preparation of a drug that regulates miR-485-3p or a composition thereof can be a new strategy to improve the main symptoms of Alzheimer's dementia, i.e., behavioral disorder and cognitive function. - Two groups were compared by the Student's t-test, and three or more groups were compared by the Krushall-Wallis test. When the P value obtained from the Krushall-Wallis test was <0.05, two groups were tested post-hoc by the Mann-Whitney U test. P value of 0.05 or smaller for the two-tailed test was considered statistically significant.
- According to the present disclosure, a composition for treating a brain disease, which contains a miR-485-3p inhibitor, can restore the ELAVL2 protein unlike the exiting therapeutic agents for Alzheimer's disease, which are limited only to alleviating symptoms by inducing decreased expression of amyloid beta 42. Therefore, it can fundamentally treat various diseases caused by decreased expression of ELAVL2, such as Alzheimer's disease, autism spectrum disorder, mental retardation, amyotrophic lateral sclerosis, etc. Accordingly, the present disclosure is useful for treating brain diseases including Alzheimer's disease fundamentally.
- While the specific embodiments of the present disclosure have been described in detail above, those skilled of ordinary skill in the art will appreciate that the specific embodiments are merely specific exemplary embodiments and the scope of the present disclosure is not limited by them. It is to be understood that the substantial scope of the disclosure is defined by the appended claims and their equivalents.
Claims (22)
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/443,700 US10844380B1 (en) | 2019-06-17 | 2019-06-17 | Uses for prevention or treatment of brain diseases using microrna |
US17/039,075 US11542503B2 (en) | 2019-06-17 | 2020-09-30 | Uses for prevention or treatment of brain diseases using microRNA |
US18/149,251 US20230340477A1 (en) | 2019-06-17 | 2023-01-03 | Uses for prevention or treatment of brain diseases using microrna |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/443,700 US10844380B1 (en) | 2019-06-17 | 2019-06-17 | Uses for prevention or treatment of brain diseases using microrna |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/039,075 Continuation US11542503B2 (en) | 2019-06-17 | 2020-09-30 | Uses for prevention or treatment of brain diseases using microRNA |
Publications (2)
Publication Number | Publication Date |
---|---|
US10844380B1 US10844380B1 (en) | 2020-11-24 |
US20200392489A1 true US20200392489A1 (en) | 2020-12-17 |
Family
ID=73462266
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/443,700 Active US10844380B1 (en) | 2019-06-17 | 2019-06-17 | Uses for prevention or treatment of brain diseases using microrna |
US17/039,075 Active 2039-07-30 US11542503B2 (en) | 2019-06-17 | 2020-09-30 | Uses for prevention or treatment of brain diseases using microRNA |
US18/149,251 Pending US20230340477A1 (en) | 2019-06-17 | 2023-01-03 | Uses for prevention or treatment of brain diseases using microrna |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/039,075 Active 2039-07-30 US11542503B2 (en) | 2019-06-17 | 2020-09-30 | Uses for prevention or treatment of brain diseases using microRNA |
US18/149,251 Pending US20230340477A1 (en) | 2019-06-17 | 2023-01-03 | Uses for prevention or treatment of brain diseases using microrna |
Country Status (1)
Country | Link |
---|---|
US (3) | US10844380B1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021156833A1 (en) * | 2020-02-07 | 2021-08-12 | Biorchestra Co., Ltd. | Use of mirna-485 inhibitors for treating tauopathy |
WO2021156832A1 (en) * | 2020-02-07 | 2021-08-12 | Biorchestra Co., Ltd. | Use of mirna-485 inhibitors for treating amyotrophic lateral sclerosis (als) |
WO2022168007A1 (en) * | 2021-02-05 | 2022-08-11 | Biorchestra Co., Ltd. | Use of mirna-485 inhibitors for treating diseases or disorders associated with abnormal nlrp3 expression |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11198908B2 (en) * | 2019-06-17 | 2021-12-14 | Biorchestra Co., Ltd. | Method for diagnosis of Alzheimer's disease using microRNA |
US10844380B1 (en) | 2019-06-17 | 2020-11-24 | Biorchestra Co., Ltd. | Uses for prevention or treatment of brain diseases using microrna |
KR20240022540A (en) * | 2021-06-14 | 2024-02-20 | 주식회사 바이오오케스트라 | Use of MIRNA-485 inhibitors to treat inflammasome-related diseases or disorders |
WO2023089506A1 (en) * | 2021-11-17 | 2023-05-25 | Biorchestra Co., Ltd. | Amplification-free methods for measuring mirna |
Family Cites Families (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5506097A (en) | 1990-08-24 | 1996-04-09 | President And Fellows Of Harvard College | Method for inhibiting β-protein enzymatic activity |
US5532219A (en) | 1991-04-23 | 1996-07-02 | The University Of British Columbia | Dapsone and promin for the treatment of dementia |
PT1064256E (en) | 1998-03-17 | 2004-10-29 | Pfizer Prod Inc | BICYCLE [2.2.1] HEPTANS AND RELATED COMPOUNDS |
PL2302055T3 (en) | 2004-11-12 | 2015-02-27 | Asuragen Inc | Methods and compositions involving miRNA and miRNA inhibitor molecules |
CN101535331B (en) | 2005-04-29 | 2013-01-30 | 洛克菲勒大学 | Human micrornas and methods for inhibiting same |
EP2388328A1 (en) | 2006-01-27 | 2011-11-23 | Isis Pharmaceuticals, Inc. | Oligomeric compounds and compositions for the use in modulation of micrornas |
EP2455494B1 (en) | 2006-07-13 | 2013-12-18 | The Ohio State University Research Foundation | Micro-RNA-based methods and compositions for the diagnosis and treatment of colon related diseases |
JP5653899B2 (en) | 2008-03-17 | 2015-01-14 | ボード・オブ・リージエンツ,ザ・ユニバーシテイ・オブ・テキサス・システム | Identification of microRNAs involved in the maintenance and regeneration of neuromuscular synapses |
KR101235256B1 (en) | 2010-09-13 | 2013-02-21 | 서울대학교산학협력단 | Treatment of Neurodegenerative Diseases by Targeting a miRNA |
CA2850258A1 (en) | 2011-09-28 | 2013-04-04 | Royal College Of Surgeons In Ireland | Inhibition of microrna-134 for the treatment of seizure-related disorders and neurologic injuries |
KR101566586B1 (en) | 2013-08-21 | 2015-11-16 | 서울대학교산학협력단 | Composition and method for preventing or treating epilepsy or diseases related to seizure comprising agent targeting microRNA-203 |
KR101615424B1 (en) | 2014-02-13 | 2016-04-25 | 서울대학교산학협력단 | Composition and method for treating neurodegenerative diseases targeting miRNA |
WO2018139819A1 (en) * | 2017-01-26 | 2018-08-02 | 주식회사 바이오오케스트라 | Uses for prevention or treatment of brain diseases using microrna |
WO2018139759A1 (en) | 2017-01-26 | 2018-08-02 | 주식회사 바이오오케스트라 | Method for diagnosis of alzheimer's disease using microrna |
US11198908B2 (en) | 2019-06-17 | 2021-12-14 | Biorchestra Co., Ltd. | Method for diagnosis of Alzheimer's disease using microRNA |
KR20220024160A (en) | 2019-06-17 | 2022-03-03 | 주식회사 바이오오케스트라 | Compositions and methods for preparing an animal model of Alzheimer's disease using microRNA |
US10844380B1 (en) | 2019-06-17 | 2020-11-24 | Biorchestra Co., Ltd. | Uses for prevention or treatment of brain diseases using microrna |
EP3990028A4 (en) | 2019-06-26 | 2023-08-09 | Biorchestra Co., Ltd. | Micellar nanoparticles and uses thereof |
-
2019
- 2019-06-17 US US16/443,700 patent/US10844380B1/en active Active
-
2020
- 2020-09-30 US US17/039,075 patent/US11542503B2/en active Active
-
2023
- 2023-01-03 US US18/149,251 patent/US20230340477A1/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021156833A1 (en) * | 2020-02-07 | 2021-08-12 | Biorchestra Co., Ltd. | Use of mirna-485 inhibitors for treating tauopathy |
WO2021156832A1 (en) * | 2020-02-07 | 2021-08-12 | Biorchestra Co., Ltd. | Use of mirna-485 inhibitors for treating amyotrophic lateral sclerosis (als) |
WO2022168007A1 (en) * | 2021-02-05 | 2022-08-11 | Biorchestra Co., Ltd. | Use of mirna-485 inhibitors for treating diseases or disorders associated with abnormal nlrp3 expression |
Also Published As
Publication number | Publication date |
---|---|
US20210123051A1 (en) | 2021-04-29 |
US10844380B1 (en) | 2020-11-24 |
US11542503B2 (en) | 2023-01-03 |
US20230340477A1 (en) | 2023-10-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11542503B2 (en) | Uses for prevention or treatment of brain diseases using microRNA | |
Gupta et al. | miRNAs in Alzheimer disease–a therapeutic perspective | |
WO2018139819A1 (en) | Uses for prevention or treatment of brain diseases using microrna | |
US11198908B2 (en) | Method for diagnosis of Alzheimer's disease using microRNA | |
JP2022101541A (en) | Agent for removing senescent cell | |
Guo et al. | Up-regulation of miR-122 protects against neuronal cell death in ischemic stroke through the heat shock protein 70-dependent NF-κB pathway by targeting FOXO3 | |
EP3394259B1 (en) | Compositions and methods for decreasing tau expression | |
KR20220104677A (en) | Compositions and methods for modulating splicing and protein expression | |
KR102105016B1 (en) | Method for Diagnosing Alzheimer's disease Using microRNA-485-3p | |
EP3575401A1 (en) | Methods of treating or preventing an aging-related disease | |
KR101566586B1 (en) | Composition and method for preventing or treating epilepsy or diseases related to seizure comprising agent targeting microRNA-203 | |
KR101615424B1 (en) | Composition and method for treating neurodegenerative diseases targeting miRNA | |
US20230242909A1 (en) | Methods and compositions for neuroprotection | |
US20170246200A1 (en) | Microrna-132/212 for the treatment of neurodegenerative disorders | |
KR102304878B1 (en) | Method for Treating Alzheimer's Disease Using microRNA-485-3p Inhibitor | |
AU2019204230A1 (en) | Uses for prevention or treatment of brain diseases using microRNA | |
CA3046767A1 (en) | Uses for prevention or treatment of brain diseases using microrna | |
KR102074407B1 (en) | Method for Diagnosing Alzheimer's disease Using microRNA-485-3p | |
CA3046766A1 (en) | Method for diagnosis of alzheimer's disease using microrna | |
US20120108648A1 (en) | Mitochondrial function-improving agent | |
KR101722861B1 (en) | Composition and method for enhancing differentiation of neuronal precursor cell comprising miRNA-193a | |
KR101904795B1 (en) | The detection method of miRNA-206, the method of providing information for diagnosis and prognostic analysis of mood disorders, the composition targeting miRNA-206 | |
US9951331B2 (en) | Targeting microRNA-26a/b for the treatment of neurodegenerative disease | |
AU2019204231A1 (en) | Method for diagnosis of Alzheimer's disease using microRNA | |
KR101611071B1 (en) | Composition and method for treating or preventing multiple system atrophy targeting miRNA202 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: BIG.); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO SMALL (ORIGINAL EVENT CODE: SMAL); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
AS | Assignment |
Owner name: BIORCHESTRA CO., LTD., KOREA, REPUBLIC OF Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:RYU, JIN-HYEOB;CHO, HYUN-JEONG;REEL/FRAME:053694/0796 Effective date: 20191210 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 4TH YR, SMALL ENTITY (ORIGINAL EVENT CODE: M2551); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY Year of fee payment: 4 |