JP6721189B2 - vWFに結合するDNAアプタマー - Google Patents
vWFに結合するDNAアプタマー Download PDFInfo
- Publication number
- JP6721189B2 JP6721189B2 JP2017547793A JP2017547793A JP6721189B2 JP 6721189 B2 JP6721189 B2 JP 6721189B2 JP 2017547793 A JP2017547793 A JP 2017547793A JP 2017547793 A JP2017547793 A JP 2017547793A JP 6721189 B2 JP6721189 B2 JP 6721189B2
- Authority
- JP
- Japan
- Prior art keywords
- dna aptamer
- nucleic acid
- base sequence
- dna
- seq
- 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.)
- Active
Links
- 108091008102 DNA aptamers Proteins 0.000 title claims description 191
- 108010047303 von Willebrand Factor Proteins 0.000 title claims description 104
- 102100036537 von Willebrand factor Human genes 0.000 title claims description 104
- 239000002773 nucleotide Substances 0.000 claims description 79
- 125000003729 nucleotide group Chemical group 0.000 claims description 78
- 150000007523 nucleic acids Chemical group 0.000 claims description 73
- 108020004707 nucleic acids Proteins 0.000 claims description 68
- 102000039446 nucleic acids Human genes 0.000 claims description 68
- 230000027455 binding Effects 0.000 claims description 62
- 238000009739 binding Methods 0.000 claims description 60
- 238000000034 method Methods 0.000 claims description 58
- -1 7-(2-thienyl)-3H-imidazo[4,5-b]pyridine-3-yl Chemical group 0.000 claims description 33
- 239000008194 pharmaceutical composition Substances 0.000 claims description 26
- 201000010099 disease Diseases 0.000 claims description 23
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 23
- 230000000295 complement effect Effects 0.000 claims description 17
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 201000010849 intracranial embolism Diseases 0.000 claims description 8
- 208000007536 Thrombosis Diseases 0.000 claims description 6
- 206010008088 Cerebral artery embolism Diseases 0.000 claims description 4
- 208000034841 Thrombotic Microangiopathies Diseases 0.000 claims description 4
- 201000007023 Thrombotic Thrombocytopenic Purpura Diseases 0.000 claims description 4
- 206010000891 acute myocardial infarction Diseases 0.000 claims description 4
- 208000006170 carotid stenosis Diseases 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 4
- 238000002331 protein detection Methods 0.000 claims description 4
- 239000002585 base Substances 0.000 description 142
- 108020004414 DNA Proteins 0.000 description 48
- 239000000523 sample Substances 0.000 description 30
- 239000000243 solution Substances 0.000 description 29
- 238000004458 analytical method Methods 0.000 description 24
- 239000000499 gel Substances 0.000 description 19
- 150000001413 amino acids Chemical class 0.000 description 16
- 239000012634 fragment Substances 0.000 description 15
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 11
- 239000013076 target substance Substances 0.000 description 11
- 238000003556 assay Methods 0.000 description 10
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 10
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 239000002552 dosage form Substances 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 238000010494 dissociation reaction Methods 0.000 description 8
- 230000005593 dissociations Effects 0.000 description 8
- 108091008104 nucleic acid aptamers Proteins 0.000 description 8
- 230000001293 nucleolytic effect Effects 0.000 description 8
- 239000013641 positive control Substances 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 7
- 239000011324 bead Substances 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 229910052751 metal Inorganic materials 0.000 description 7
- 239000002184 metal Substances 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 239000002202 Polyethylene glycol Substances 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 229920002472 Starch Polymers 0.000 description 6
- 235000014113 dietary fatty acids Nutrition 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 239000000194 fatty acid Substances 0.000 description 6
- 229930195729 fatty acid Natural products 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 235000002639 sodium chloride Nutrition 0.000 description 6
- 239000008107 starch Substances 0.000 description 6
- 235000019698 starch Nutrition 0.000 description 6
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 5
- 229930024421 Adenine Natural products 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 229960000643 adenine Drugs 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 229940104302 cytosine Drugs 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 235000000346 sugar Nutrition 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 239000010931 gold Substances 0.000 description 4
- 229910052737 gold Inorganic materials 0.000 description 4
- 230000003902 lesion Effects 0.000 description 4
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 4
- 229920000151 polyglycol Polymers 0.000 description 4
- 239000010695 polyglycol Substances 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000003826 tablet Substances 0.000 description 4
- 239000010409 thin film Substances 0.000 description 4
- 229940113082 thymine Drugs 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WNWHHMBRJJOGFJ-UHFFFAOYSA-N 16-methylheptadecan-1-ol Chemical class CC(C)CCCCCCCCCCCCCCCO WNWHHMBRJJOGFJ-UHFFFAOYSA-N 0.000 description 3
- 108091023037 Aptamer Proteins 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 206010064571 Gene mutation Diseases 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 239000000796 flavoring agent Substances 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 229940014259 gelatin Drugs 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000012146 running buffer Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- 238000007910 systemic administration Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 3
- 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 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 2
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 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 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 230000004568 DNA-binding Effects 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000004909 Moisturizer Substances 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 206010037549 Purpura Diseases 0.000 description 2
- 241001672981 Purpura Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 208000027276 Von Willebrand disease Diseases 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 239000003114 blood coagulation factor Substances 0.000 description 2
- 229940019700 blood coagulation factors Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- 239000007884 disintegrant Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 239000003889 eye drop Substances 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 2
- 229920000609 methyl cellulose Polymers 0.000 description 2
- 239000001923 methylcellulose Substances 0.000 description 2
- 235000010981 methylcellulose Nutrition 0.000 description 2
- 230000001333 moisturizer Effects 0.000 description 2
- PSZYNBSKGUBXEH-UHFFFAOYSA-N naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1 PSZYNBSKGUBXEH-UHFFFAOYSA-N 0.000 description 2
- 150000002790 naphthalenes Chemical class 0.000 description 2
- 239000007923 nasal drop Substances 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001732 thrombotic effect Effects 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- 208000012137 von Willebrand disease (hereditary or acquired) Diseases 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- WHOZNOZYMBRCBL-OUKQBFOZSA-N (2E)-2-Tetradecenal Chemical compound CCCCCCCCCCC\C=C\C=O WHOZNOZYMBRCBL-OUKQBFOZSA-N 0.000 description 1
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 1
- KSTJOICDZAFYTD-UHFFFAOYSA-N 2-amino-1h-imidazo[1,2-a][1,3,5]triazin-4-one Chemical compound O=C1N=C(N)N=C2NC=CN21 KSTJOICDZAFYTD-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- OTDJAMXESTUWLO-UUOKFMHZSA-N 2-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-oxolanyl]-3H-purine-6-thione Chemical compound C12=NC(N)=NC(S)=C2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OTDJAMXESTUWLO-UUOKFMHZSA-N 0.000 description 1
- FTBBGQKRYUTLMP-UHFFFAOYSA-N 2-nitro-1h-pyrrole Chemical compound [O-][N+](=O)C1=CC=CN1 FTBBGQKRYUTLMP-UHFFFAOYSA-N 0.000 description 1
- FOGYNLXERPKEGN-UHFFFAOYSA-N 3-(2-hydroxy-3-methoxyphenyl)-2-[2-methoxy-4-(3-sulfopropyl)phenoxy]propane-1-sulfonic acid Chemical compound COC1=CC=CC(CC(CS(O)(=O)=O)OC=2C(=CC(CCCS(O)(=O)=O)=CC=2)OC)=C1O FOGYNLXERPKEGN-UHFFFAOYSA-N 0.000 description 1
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 1
- NLLCDONDZDHLCI-UHFFFAOYSA-N 6-amino-5-hydroxy-1h-pyrimidin-2-one Chemical compound NC=1NC(=O)N=CC=1O NLLCDONDZDHLCI-UHFFFAOYSA-N 0.000 description 1
- JFGLOZOVAGYLKU-UHFFFAOYSA-N 6-amino-5-nitro-1h-pyridin-2-one Chemical compound NC=1NC(=O)C=CC=1[N+]([O-])=O JFGLOZOVAGYLKU-UHFFFAOYSA-N 0.000 description 1
- ITWHUNFGCBDAQH-UHFFFAOYSA-N 6-methyl-2h-isoquinoline-1-thione Chemical compound C1=CNC(=S)C=2C1=CC(C)=CC=2 ITWHUNFGCBDAQH-UHFFFAOYSA-N 0.000 description 1
- CKOMXBHMKXXTNW-UHFFFAOYSA-N 6-methyladenine Chemical compound CNC1=NC=NC2=C1N=CN2 CKOMXBHMKXXTNW-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 206010007687 Carotid artery stenosis Diseases 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-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- DCXXMTOCNZCJGO-UHFFFAOYSA-N Glycerol trioctadecanoate Natural products CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 239000004705 High-molecular-weight polyethylene Substances 0.000 description 1
- 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 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- IGFHQQFPSIBGKE-UHFFFAOYSA-N Nonylphenol Natural products CCCCCCCCCC1=CC=C(O)C=C1 IGFHQQFPSIBGKE-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- CJVZJAHFWKCJIJ-UHFFFAOYSA-N OC1=CC=C(C=C1)C(C)C=1C(NC(NC=1)=O)=O Chemical compound OC1=CC=C(C=C1)C(C)C=1C(NC(NC=1)=O)=O CJVZJAHFWKCJIJ-UHFFFAOYSA-N 0.000 description 1
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004721 Polyphenylene oxide Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 229940124532 absorption promoter Drugs 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
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000002156 adsorbate Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 229940023476 agar Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 125000002877 alkyl aryl group Chemical group 0.000 description 1
- 150000008055 alkyl aryl sulfonates Chemical class 0.000 description 1
- 125000005037 alkyl phenyl group Chemical group 0.000 description 1
- 150000008051 alkyl sulfates Chemical class 0.000 description 1
- 229940045714 alkyl sulfonate alkylating agent Drugs 0.000 description 1
- 150000008052 alkyl sulfonates Chemical class 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
- 235000011132 calcium sulphate Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 229940097362 cyclodextrins Drugs 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- LQZZUXJYWNFBMV-UHFFFAOYSA-N dodecan-1-ol Chemical compound CCCCCCCCCCCCO LQZZUXJYWNFBMV-UHFFFAOYSA-N 0.000 description 1
- 239000008298 dragée Substances 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000002662 enteric coated tablet Substances 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 239000007941 film coated tablet Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000696 magnetic material Substances 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 229960002900 methylcellulose Drugs 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 229940100662 nasal drops Drugs 0.000 description 1
- SNQQPOLDUKLAAF-UHFFFAOYSA-N nonylphenol Chemical compound CCCCCCCCCC1=CC=CC=C1O SNQQPOLDUKLAAF-UHFFFAOYSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- 229920002114 octoxynol-9 Polymers 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 229940044654 phenolsulfonic acid Drugs 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920000193 polymethacrylate Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 229920002102 polyvinyl toluene Polymers 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 229940116317 potato starch Drugs 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229940100486 rice starch Drugs 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 238000010583 slow cooling Methods 0.000 description 1
- 235000015424 sodium Nutrition 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000007940 sugar coated tablet Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000003582 thrombocytopenic effect Effects 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 229940078499 tricalcium phosphate Drugs 0.000 description 1
- 235000019731 tricalcium phosphate Nutrition 0.000 description 1
- 229910000391 tricalcium phosphate Inorganic materials 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 229940100445 wheat starch Drugs 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
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- 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
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
- C12N2310/16—Aptamers
-
- 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
- C12N2310/33—Chemical structure of the base
- C12N2310/333—Modified A
-
- 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
- C12N2310/50—Physical structure
- C12N2310/53—Physical structure partially self-complementary or closed
- C12N2310/531—Stem-loop; Hairpin
-
- 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
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/745—Assays involving non-enzymic blood coagulation factors
- G01N2333/755—Factors VIII, e.g. factor VIII C [AHF], factor VIII Ag [VWF]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Plant Pathology (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Epidemiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
Description
(1)以下の(i)又は(ii)の塩基配列を含む、vWFタンパク質に結合するDNAアプタマー:
(i)配列番号13〜16、19及び20のいずれかに示される塩基配列、又は
(ii)(i)に示される塩基配列において、7-(2-thienyl)-3H-imidazo[4,5-b]pyridine-3-yl以外の位置において1又は数個のヌクレオチドが付加、欠失、及び/又は置換された塩基配列。
(2)(i)の塩基配列が、配列番号13、14、19又は20に示される配列である、(1)に記載のDNAアプタマー。
(3)1〜5個のGC対を塩基配列の末端に含む、(1)又は(2)に記載のDNAアプタマー。
(4)塩基配列の3'末端にミニヘアピン構造をさらに含み、
前記ミニヘアピン構造が、
5'末端側から3'末端側に向かって順番に連結された以下の(A)〜(C)の核酸領域:
(A)2〜5個の任意のヌクレオチドからなる第1核酸領域、
(B)GNA又はGNNA(ここで、各Nは、独立に、G、T、A若しくはCのいずれかである)の塩基配列からなる第2核酸領域、及び
(C)第1核酸領域に相補的な塩基配列からなる第3核酸領域
からなり、
かつ、第1核酸領域及び第3核酸領域が互いに塩基対合したステム部分と第2核酸領域からなるループ部分によって構成される、
(1)〜(3)のいずれかに記載のDNAアプタマー。
(5)以下の(i)又は(ii)の塩基配列を含む、vWFタンパク質に結合するDNAアプタマー:
(i)配列番号18若しくは21に示される塩基配列、又は
(ii)(i)に示される塩基配列において、7-(2-thienyl)-3H-imidazo[4,5-b]pyridine-3-yl以外の位置において1又は数個のヌクレオチドが付加、欠失、及び/又は置換された塩基配列。
(6)(1)〜(5)のいずれかに記載の塩基配列からなる、vWFタンパク質に結合するDNAアプタマー。
(7)以下の(I)又は(II)の塩基配列を含む、vWFタンパク質に結合するDNAアプタマー:
(I)配列番号1〜4、9及び11のいずれかに示される塩基配列、又は
(II)(I)に示される塩基配列において、7-(2-thienyl)-3H-imidazo[4,5-b]pyridine-3-yl以外の位置において1又は数個のヌクレオチドが付加、欠失、及び/又は置換された塩基配列。
(8)(I)の塩基配列が、配列番号1又は11に示される配列である、(7)に記載のDNAアプタマー。
(9)1〜5個のGC対を塩基配列の末端に含む、(7)又は(8)に記載のDNAアプタマー。
(10)塩基配列の3'末端にミニヘアピン構造をさらに含み、
前記ミニヘアピン構造が、
5'末端側から3'末端側に向かって順番に連結された以下の(A)〜(C)の核酸領域:
(A)2〜5個の任意のヌクレオチドからなる第1核酸領域、
(B)GNA又はGNNA(ここで、各Nは、独立に、G、T、A若しくはCのいずれかである)の塩基配列からなる第2核酸領域、及び
(C)第1核酸領域に相補的な塩基配列からなる第3核酸領域
からなり、
かつ、第1核酸領域及び第3核酸領域が互いに塩基対合したステム部分と第2核酸領域からなるループ部分によって構成される、
(7)〜(9)のいずれかに記載のDNAアプタマー。
(11)以下の(I)又は(II)の塩基配列を含む、vWFタンパク質に結合するDNAアプタマー:
(I)配列番号12に示される塩基配列、又は
(II)(I)に示される塩基配列において、7-(2-thienyl)-3H-imidazo[4,5-b]pyridine-3-yl以外の位置において1又は数個のヌクレオチドが付加、欠失、及び/又は置換された塩基配列。
(12)(7)〜(11)のいずれかに記載の塩基配列からなる、vWFタンパク質に結合するDNAアプタマー。
(13)(1)〜(12)のいずれかに記載のDNAアプタマーを含むvWFタンパク質検出剤。
(14)(1)〜(12)のいずれかに記載のDNAアプタマーを含むvWFタンパク質検出用キット。
(15)(1)〜(12)のいずれかに記載のDNAアプタマーを含む医薬組成物。
(16)血栓症、血栓性血小板減少性紫斑病、頭蓋内塞栓、脳塞栓症、頸動脈狭窄、血栓性微小血管症、及び急性心筋梗塞症からなる群から選択される疾患の治療及び/又は予防のための、(15)に記載の医薬組成物。
(17)被験体から得られたサンプルを、(1)〜(12)のいずれかに記載のDNAアプタマーと接触させる工程、及び
前記サンプルと前記DNAアプタマーの結合に基づいてvWFタンパク質を検出する工程、
を含む、vWFタンパク質を検出する方法。
本明細書で使用する一般的な用語の定義について以下で説明する。
一態様において、本発明は、以下の(i)又は(ii)の塩基配列を含む、vWFに結合するDNAアプタマーに関する:
(i)配列番号13〜16、19及び20のいずれか、好ましくは配列番号13、14、19及び20のいずれかに示される塩基配列、又は
(ii)(i)に示される塩基配列において、Ds以外の位置において1又は数個のヌクレオチドが付加、欠失、及び/又は置換された塩基配列。
(I)配列番号1〜4、9及び11のいずれか、好ましくは配列番号1又は11に示される塩基配列、又は
(II)(I)に示される塩基配列において、Ds以外の位置において1又は数個のヌクレオチドが付加、欠失、及び/又は置換された塩基配列。
一態様において、本発明は、本発明のDNAアプタマーを含む医薬組成物に関する。本発明の医薬組成物は、本発明のDNAアプタマーが有するvWFへの結合能を失わない範囲において、他の薬剤を一以上含有することもできる。
一態様において、本発明は本発明のDNAアプタマー又は上記医薬組成物を被験体に投与することを含む、疾患の治療及び/又は予防方法に関する。
一態様において、本発明は、本発明のDNAアプタマーを含むvWF検出剤に関する。本発明のvWF検出剤は、本発明のDNAアプタマーのvWFへの結合能を利用して、in vivo又はin vitroにおいてvWFを検出するための薬剤である。例えば、DNAアプタマーをあらかじめ蛍光試薬等で標識し、これを投与することによって、生体内でvWFの発現強度を決定し、またその局在を調べることができる。これによって、上記vWF関連疾患の診断を補助することが可能となり得る。本発明のDNAアプタマーは、イメージング及び組織染色等において有用である。
一態様において、本発明は、vWFを検出する方法に関する。本方法は、被験体から得られたサンプルを、本発明のDNAアプタマーと接触させる工程、及びサンプルとDNAアプタマーの結合に基づいてvWFを検出する工程を含む。本方法によって、上記vWF関連疾患の診断を補助することが可能となり得る。
WO2013/073602に記載のプレデターミン法に従って人工塩基Dsを含むDNAライブラリーの調製を行った。プレデターミン法で用いるライブラリーは、ランダムな塩基配列中の特定の固定された位置に人工塩基Dsを含むようにデザインされたものである。手短には、DNA断片(分子種総数は300 pmol、約2×1014分子)を1ラウンド目のライブラリーとして用い、標的タンパク質であるvWF A1ドメイン(U-Protein社、V003)を混合した後、磁気ビーズを用いて標的タンパク質に結合するDNAを選別及び単離し、さらにポリアクリルアミドゲル電気泳動を行いDNA-vWF A1ドメイン複合体を切り出すことにより選別及び単離後、PCR増幅を行い、計8ラウンドのセレクション操作を行った。各セレクションの条件は表1に示した。8ラウンドのセレクション終了後、配列の解析を行い、人工塩基Dsを含むDNAアプタマーの配列を得た。
人工塩基Dsの位置を正確に同定するため、以下の操作を行い、正確な配列決定を行った。
配列同定した人工塩基Dsを3箇所に含むDNAアプタマーのvWF A1ドメインタンパクに対する結合を調べるために、プライマー領域を切り詰めた40-mer及び38-merのDNAアプタマーを調製した。調製した各DNAアプタマーの名称及び配列を表2に、セレクションで得られた塩基配列等から予測される二次構造を図1に示す。
得られたDNAアプタマーの結合能をGEヘルスケア社のBiacoreT200を用いた表面プラズモン共鳴(SPR)によって測定した。解析に用いた各種DNAアプタマーの配列を上記表2に、予測される二次構造を図3に示す。vWF1-DsDsDs(配列番号1)を元に、三つのDsをAに置換したvWF1-AAA(配列番号8)、ステム領域のAT対の一部をGC対に置換したvWF1-DsDsDs-GC(配列番号11)、vWF1-DsDsDs-GC の3'末端にミニヘアピンを付加したvWF1-DsDsDs-mhGC(配列番号12)を調製した。また、陽性対照として、ARC1172(配列番号10)を用いた。
各種DNAアプタマー(vWF1-DsDsDs、vWF1-DsDsDs-GC、vWF1-DsDsDs-GCmh、vWF1-AAA最終濃度:2 μM)の熱安定性(Tm値)を測定した。DNAアプタマーの吸光度変化を、紫外可視分光光度計 UV-2450(島津)により測定し、その一次微分から融解温度であるTm値を算出した。結果を図5に示す。vWF1-DsDsDsはTm = 65.7℃、vWF1-DsDsDs-GCはTm = 73.0℃、vWF1-DsDsDs-mhGCはTm = 74.7℃と、ステム部分のGC対の増加及びミニヘアピンDNAの付加により熱安定性が向上していることが示された。特に、vWF1-DsDsDs-GC及びvWF1-DsDsDs-mhGCは、既存のDNAアプタマーであるARC1172と比較してもTm値が10℃以上高く、熱安定性がARC1172より顕著に優れていた。一方で、DsをAに置換したvWF1-AAAはTm = 63.0℃と僅かに低下しており、Ds自体が熱安定性に関与していることが示された。
WO2013/073602に記載のランダムライブラリー法に従って、人工塩基Dsを含むDNAライブラリーの調製を行った。ランダムライブラリー法で用いるライブラリーは、ランダムな塩基配列中のランダムな位置に所定の確率で人工塩基Dsを含むようにデザインされたものである。セレクション方法は実施例1の方法に従って行った。手短には、DNA断片(分子種総数は300 pmol、すなわち約2×1014分子)を1ラウンド目のライブラリーとして用い、標的タンパク質であるvWF A1ドメイン(U-Protein社、V003)を混合した後、磁気ビーズを用いて標的タンパク質に結合するDNAを選別及び単離し、さらにポリアクリルアミドゲル電気泳動によってDNA-vWF A1ドメイン複合体を切り出すことにより選別及び単離後、PCR増幅し、計7ラウンドのセレクション操作を行った。各セレクションラウンドの条件は表3に示した。7ラウンドのセレクション終了後、配列の解析を行い、人工塩基Dsを含むDNAアプタマー配列を得た。
人工塩基Dsの位置を正確に同定するため、以下の操作を行い、正確な配列決定を行った。
配列同定した人工塩基Dsを3箇所に含むDNAアプタマーのvWF A1ドメインタンパクに対する結合を調べるために、プライマー領域を切り詰めたDNAアプタマーを作製し、ゲルシフトアッセイによって結合解析を行った。本実施例に使用したDNAアプタマーの配列を表4に、予想される二次構造を図6に示した。
得られたDNAアプタマーの結合能をGEヘルスケア社のBiacoreT200を用いた表面プラズモン共鳴(SPR)により測定した。解析に用いたDNAアプタマーの配列は、上記表4に示し、セレクションで得られた塩基配列等から予測される二次構造を図6に示した。
各DNAアプタマーのヒト血清中に含まれる核酸分解酵素に対する安定性を調べた。各DNAアプタマー(vWF2-DsDsDs、vWF2-DsDsDs-mhGC、vWF2-DsDsDs-2mhGC、vWF2-AAA、ARC1172、最終濃度2 μM)を、ヒト血清濃度が96%となるように混合し、この溶液を37℃でインキュベートした。0時間後、1時間後、6時間後、24時間後、48時間後、及び72時間後に混合溶液から10 μLを分取し、110μLの1×TBE、10M Urea溶液と混合して分解反応を止めた。反応後のサンプルを変性15%ポリアクリルアミドゲル電気泳動で分離した後、ゲルをSYBR GOLD(Thermo Fisher Scientific)で染色して1本鎖核酸を検出した。ヒト血清中の核酸分解酵素による分解産物のバンドパターンをバイオイメージャーLAS-4000(富士フィルム)で解析した。
各DNAアプタマー(vWF2-DsDsDs、vWF2-DsADs、vWF2-DsAA、vWF2-AADs、vWF2-AAA、vWF2-DsDsDs-mhGC、vWF2-DsDsDs-2mhGC、最終濃度2 μM)の熱安定性(Tm値)を測定した。DNAアプタマーの吸光度変化を、紫外可視分光光度計 UV-2450(島津)により測定し、その一次微分から融解温度であるTm値を算出した。
Claims (17)
- 以下の(i)又は(ii)の塩基配列を含む、vWFタンパク質に結合するDNAアプタマー: (i)配列番号13〜16、19及び20のいずれかに示される塩基配列、又は
(ii)(i)に示される塩基配列において、7-(2-thienyl)-3H-imidazo[4,5-b]pyridine-3-yl以外の位置において1又は数個のヌクレオチドが付加、欠失、及び/又は置換された塩基配列。 - (i)の塩基配列が、配列番号13、14、19又は20に示される配列である、請求項1に記載のDNAアプタマー。
- 1〜5個のGC対を塩基配列の末端に含む、請求項1又は2に記載のDNAアプタマー。
- 塩基配列の3'末端にミニヘアピン構造をさらに含み、
前記ミニヘアピン構造が、
5'末端側から3'末端側に向かって順番に連結された以下の(A)〜(C)の核酸領域:
(A)2〜5個の任意のヌクレオチドからなる第1核酸領域、
(B)GNA又はGNNA(ここで、各Nは、独立に、G、T、A若しくはCのいずれかである)の塩基配列からなる第2核酸領域、及び
(C)第1核酸領域に相補的な塩基配列からなる第3核酸領域
からなり、
かつ、第1核酸領域及び第3核酸領域が互いに塩基対合したステム部分と第2核酸領域からなるループ部分によって構成される、
請求項1〜3のいずれか一項に記載のDNAアプタマー。 - 以下の(i)又は(ii)の塩基配列を含む、vWFタンパク質に結合するDNAアプタマー: (i)配列番号18若しくは21に示される塩基配列、又は
(ii)(i)に示される塩基配列において、7-(2-thienyl)-3H-imidazo[4,5-b]pyridine-3-yl以外の位置において1又は数個のヌクレオチドが付加、欠失、及び/又は置換された塩基配列。 - 請求項1〜5のいずれか一項に記載の塩基配列からなる、vWFタンパク質に結合するDNAアプタマー。
- 以下の(I)又は(II)の塩基配列を含む、vWFタンパク質に結合するDNAアプタマー: (I)配列番号1〜4、9及び11のいずれかに示される塩基配列、又は
(II)(I)に示される塩基配列において、7-(2-thienyl)-3H-imidazo[4,5-b]pyridine-3-yl以外の位置において1又は数個のヌクレオチドが付加、欠失、及び/又は置換された塩基配列。 - (I)の塩基配列が、配列番号1又は11に示される配列である、請求項7に記載のDNAアプタマー。
- 1〜5個のGC対を塩基配列の末端に含む、請求項7又は8に記載のDNAアプタマー。
- 塩基配列の3'末端にミニヘアピン構造をさらに含み、
前記ミニヘアピン構造が、
5'末端側から3'末端側に向かって順番に連結された以下の(A)〜(C)の核酸領域:
(A)2〜5個の任意のヌクレオチドからなる第1核酸領域、
(B)GNA又はGNNA(ここで、各Nは、独立に、G、T、A若しくはCのいずれかである)の塩基配列からなる第2核酸領域、及び
(C)第1核酸領域に相補的な塩基配列からなる第3核酸領域
からなり、
かつ、第1核酸領域及び第3核酸領域が互いに塩基対合したステム部分と第2核酸領域からなるループ部分によって構成される、
請求項7〜9のいずれか一項に記載のDNAアプタマー。 - 以下の(I)又は(II)の塩基配列を含む、vWFタンパク質に結合するDNAアプタマー: (I)配列番号12に示される塩基配列、又は
(II)(I)に示される塩基配列において、7-(2-thienyl)-3H-imidazo[4,5-b]pyridine-3-yl以外の位置において1又は数個のヌクレオチドが付加、欠失、及び/又は置換された塩基配列。 - 請求項7〜11のいずれか一項に記載の塩基配列からなる、vWFタンパク質に結合するDNAアプタマー。
- 請求項1〜12のいずれか一項に記載のDNAアプタマーを含むvWFタンパク質検出剤。
- 請求項1〜12のいずれか一項に記載のDNAアプタマーを含むvWFタンパク質検出用キット。
- 請求項1〜12のいずれか一項に記載のDNAアプタマーを含む医薬組成物。
- 血栓症、血栓性血小板減少性紫斑病、頭蓋内塞栓、脳塞栓症、頸動脈狭窄、血栓性微小血管症、及び急性心筋梗塞症からなる群から選択される疾患の治療及び/又は予防のための、請求項15に記載の医薬組成物。
- 被験体から得られたサンプルを、請求項1〜12のいずれか一項に記載のDNAアプタマーと接触させる工程、及び
前記サンプルと前記DNAアプタマーの結合に基づいてvWFタンパク質を検出する工程、
を含む、vWFタンパク質を検出する方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2015214848 | 2015-10-30 | ||
JP2015214848 | 2015-10-30 | ||
PCT/JP2016/081518 WO2017073536A1 (ja) | 2015-10-30 | 2016-10-25 | vWFに結合するDNAアプタマー |
Publications (2)
Publication Number | Publication Date |
---|---|
JPWO2017073536A1 JPWO2017073536A1 (ja) | 2018-08-16 |
JP6721189B2 true JP6721189B2 (ja) | 2020-07-08 |
Family
ID=58630544
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2017547793A Active JP6721189B2 (ja) | 2015-10-30 | 2016-10-25 | vWFに結合するDNAアプタマー |
Country Status (11)
Country | Link |
---|---|
US (1) | US10760083B2 (ja) |
EP (1) | EP3369820B1 (ja) |
JP (1) | JP6721189B2 (ja) |
KR (2) | KR20180072722A (ja) |
CN (1) | CN108350461B (ja) |
AU (1) | AU2016346270A1 (ja) |
CA (1) | CA3003576A1 (ja) |
ES (1) | ES2903041T3 (ja) |
HK (1) | HK1253955A1 (ja) |
SG (1) | SG11201803631YA (ja) |
WO (1) | WO2017073536A1 (ja) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3266871B1 (en) * | 2015-03-06 | 2021-04-28 | TAGCyx Biotechnologies Inc. | Method for stabilizing dna aptamers |
EP3624801A4 (en) | 2017-05-19 | 2021-02-24 | Band Therapeutics, LLC | COMPOSITIONS AND METHODS FOR THE TREATMENT OF COMPLICATIONS AND DISORDERS RELATED TO VON WILLEBRAND FACTOR |
US20220315926A1 (en) * | 2019-06-21 | 2022-10-06 | Agency For Science, Technology And Research | An Aptamer for Dengue Virus and Related Methods and Products |
US10788488B1 (en) * | 2019-11-12 | 2020-09-29 | Alfaisal University | Full-length and truncated anti-coagulant Dabigatran etexilate specific DNA aptamers for electrochemical and fluorescence sensing applications |
US20230038761A1 (en) * | 2020-02-04 | 2023-02-09 | Band Therapeutics, Llc | Regulation of von willebrand factor (vwf) |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101300361A (zh) * | 2004-09-07 | 2008-11-05 | 阿切埃米克斯有限公司 | 关于血管性血友病因子的适体及其作为血栓形成疾病治疗剂的应用 |
CA2579374A1 (en) * | 2004-09-07 | 2006-03-30 | Archemix Corp. | Aptamers to von willebrand factor and their use as thrombotic disease therapeutics |
JP5649018B2 (ja) * | 2005-08-04 | 2015-01-07 | タグシクス・バイオ株式会社 | 新規人工塩基対及びその利用 |
CN101365791B (zh) * | 2005-12-09 | 2012-07-25 | 独立行政法人理化学研究所 | 核酸的复制方法及新型人工碱基对 |
WO2008150495A2 (en) * | 2007-06-01 | 2008-12-11 | Archemix Corp. | Vwf aptamer formulations and methods for use |
WO2010091396A2 (en) * | 2009-02-09 | 2010-08-12 | Archemix Corp. | Aptamers to von willerbrand factor and their use as thrombotic, hematologic and cardiovascular disease therapeutics |
SG11201402402SA (en) * | 2011-11-18 | 2014-09-26 | Tagcyx Biotechnologies | Nucleic acid fragment binding to target protein |
EP3266871B1 (en) * | 2015-03-06 | 2021-04-28 | TAGCyx Biotechnologies Inc. | Method for stabilizing dna aptamers |
-
2016
- 2016-10-25 EP EP16859766.4A patent/EP3369820B1/en active Active
- 2016-10-25 CA CA3003576A patent/CA3003576A1/en active Pending
- 2016-10-25 KR KR1020187013314A patent/KR20180072722A/ko not_active IP Right Cessation
- 2016-10-25 WO PCT/JP2016/081518 patent/WO2017073536A1/ja active Application Filing
- 2016-10-25 US US15/771,588 patent/US10760083B2/en active Active
- 2016-10-25 KR KR1020237014013A patent/KR102640903B1/ko active IP Right Grant
- 2016-10-25 ES ES16859766T patent/ES2903041T3/es active Active
- 2016-10-25 AU AU2016346270A patent/AU2016346270A1/en not_active Abandoned
- 2016-10-25 JP JP2017547793A patent/JP6721189B2/ja active Active
- 2016-10-25 CN CN201680063729.0A patent/CN108350461B/zh active Active
- 2016-10-25 SG SG11201803631YA patent/SG11201803631YA/en unknown
-
2018
- 2018-10-12 HK HK18113106.4A patent/HK1253955A1/zh unknown
Also Published As
Publication number | Publication date |
---|---|
KR20180072722A (ko) | 2018-06-29 |
ES2903041T3 (es) | 2022-03-30 |
CN108350461A (zh) | 2018-07-31 |
EP3369820A1 (en) | 2018-09-05 |
CA3003576A1 (en) | 2017-05-04 |
CN108350461B (zh) | 2021-10-19 |
HK1253955A1 (zh) | 2019-07-05 |
EP3369820B1 (en) | 2021-12-08 |
WO2017073536A1 (ja) | 2017-05-04 |
SG11201803631YA (en) | 2018-05-30 |
US10760083B2 (en) | 2020-09-01 |
EP3369820A4 (en) | 2019-06-19 |
AU2016346270A1 (en) | 2018-05-24 |
US20180305695A1 (en) | 2018-10-25 |
KR102640903B1 (ko) | 2024-02-27 |
JPWO2017073536A1 (ja) | 2018-08-16 |
KR20230061569A (ko) | 2023-05-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6721189B2 (ja) | vWFに結合するDNAアプタマー | |
US9857362B2 (en) | Method for preparing nucleic acid aptamer | |
Fernández et al. | TLR4-binding DNA aptamers show a protective effect against acute stroke in animal models | |
JP6544750B2 (ja) | Dnaアプタマーの安定化法 | |
JP7391074B2 (ja) | 癌細胞に結合するdnaアプタマー | |
US20030232400A1 (en) | Methods of screening subjects for expression of soluble receptors of vascular endothelial growth factor (VEGF) for use in managing treatment and determining prognostic outcome | |
US20180066263A1 (en) | Nucleic acid aptamers binding to vascular endothelial growth factor receptors | |
Kohn et al. | Terminal alkyne-modified DNA Aptamers with enhanced protein binding affinities | |
JP2007043917A (ja) | 腫瘍成長因子β受容体III型に結合する核酸リガンド | |
WO2020163974A1 (zh) | 针对骨硬化蛋白的适体的诊断用途 | |
KR101385783B1 (ko) | 간암 진단 마커 및 치료제로서의 dbc1 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20190626 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20200512 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20200611 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6721189 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |