KR20230134446A - Composition for preventing or treating coronavirus infection - Google Patents
Composition for preventing or treating coronavirus infection Download PDFInfo
- Publication number
- KR20230134446A KR20230134446A KR1020230033179A KR20230033179A KR20230134446A KR 20230134446 A KR20230134446 A KR 20230134446A KR 1020230033179 A KR1020230033179 A KR 1020230033179A KR 20230033179 A KR20230033179 A KR 20230033179A KR 20230134446 A KR20230134446 A KR 20230134446A
- Authority
- KR
- South Korea
- Prior art keywords
- composition
- cells
- heparin
- hlf
- preventing
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 39
- 208000001528 Coronaviridae Infections Diseases 0.000 title description 2
- HSHNITRMYYLLCV-UHFFFAOYSA-N 4-methylumbelliferone Chemical compound C1=C(O)C=CC2=C1OC(=O)C=C2C HSHNITRMYYLLCV-UHFFFAOYSA-N 0.000 claims abstract description 43
- 229920000669 heparin Polymers 0.000 claims abstract description 37
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 34
- 229960002897 heparin Drugs 0.000 claims abstract description 32
- 230000000694 effects Effects 0.000 claims abstract description 23
- 229940079593 drug Drugs 0.000 claims abstract description 21
- 239000003814 drug Substances 0.000 claims abstract description 21
- 208000009341 RNA Virus Infections Diseases 0.000 claims abstract description 18
- 239000004480 active ingredient Substances 0.000 claims abstract description 8
- 241001678559 COVID-19 virus Species 0.000 claims description 21
- 229920002674 hyaluronan Polymers 0.000 claims description 19
- 229940099552 hyaluronan Drugs 0.000 claims description 19
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-MNSSHETKSA-N 0.000 claims description 19
- 208000025721 COVID-19 Diseases 0.000 claims description 18
- 101710198474 Spike protein Proteins 0.000 claims description 16
- 229940096437 Protein S Drugs 0.000 claims description 15
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims description 7
- 238000003786 synthesis reaction Methods 0.000 claims description 7
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 208000037847 SARS-CoV-2-infection Diseases 0.000 claims description 4
- 238000005516 engineering process Methods 0.000 abstract description 7
- 238000009511 drug repositioning Methods 0.000 abstract description 5
- 239000002547 new drug Substances 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 72
- 230000014509 gene expression Effects 0.000 description 41
- 238000011282 treatment Methods 0.000 description 27
- 238000012360 testing method Methods 0.000 description 26
- 230000008859 change Effects 0.000 description 21
- 239000002953 phosphate buffered saline Substances 0.000 description 19
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 18
- 102100035765 Angiotensin-converting enzyme 2 Human genes 0.000 description 17
- 102000003918 Hyaluronan Synthases Human genes 0.000 description 17
- 108090000320 Hyaluronan Synthases Proteins 0.000 description 15
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 13
- 241000700605 Viruses Species 0.000 description 13
- 241000713666 Lentivirus Species 0.000 description 12
- 239000002609 medium Substances 0.000 description 11
- 210000002889 endothelial cell Anatomy 0.000 description 10
- 239000012091 fetal bovine serum Substances 0.000 description 9
- 208000015181 infectious disease Diseases 0.000 description 9
- 210000004072 lung Anatomy 0.000 description 9
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 8
- 101150027313 Has2 gene Proteins 0.000 description 8
- 210000002919 epithelial cell Anatomy 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 101150017002 CD44 gene Proteins 0.000 description 6
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 5
- 230000000840 anti-viral effect Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 238000003501 co-culture Methods 0.000 description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 5
- 210000002950 fibroblast Anatomy 0.000 description 5
- 230000003472 neutralizing effect Effects 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 230000002265 prevention Effects 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 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 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 241000315672 SARS coronavirus Species 0.000 description 4
- 101150082427 Tlr4 gene Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000012881 co-culture medium Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 102100032912 CD44 antigen Human genes 0.000 description 3
- 241000711573 Coronaviridae Species 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 101150063416 add gene Proteins 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 210000003501 vero cell Anatomy 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 2
- 101150090724 3 gene Proteins 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
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 101710128038 Hyaluronan synthase Proteins 0.000 description 2
- 229930182816 L-glutamine Natural products 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
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 229920002873 Polyethylenimine Polymers 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 229930006000 Sucrose Natural products 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
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 2
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 108010067390 Viral Proteins Proteins 0.000 description 2
- 101150054399 ace2 gene Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000003246 corticosteroid Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 239000000645 desinfectant Substances 0.000 description 2
- 229960003957 dexamethasone Drugs 0.000 description 2
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000010185 immunofluorescence analysis Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000003055 low molecular weight heparin Substances 0.000 description 2
- 229940127215 low-molecular weight heparin Drugs 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 238000004519 manufacturing process Methods 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
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 229940056360 penicillin g Drugs 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 229960002385 streptomycin sulfate Drugs 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000454 talc Substances 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 235000012222 talc Nutrition 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- KZDCMKVLEYCGQX-UDPGNSCCSA-N 2-(diethylamino)ethyl 4-aminobenzoate;(2s,5r,6r)-3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid;hydrate Chemical compound O.CCN(CC)CCOC(=O)C1=CC=C(N)C=C1.N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 KZDCMKVLEYCGQX-UDPGNSCCSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- CFNMUZCFSDMZPQ-GHXNOFRVSA-N 7-[(z)-3-methyl-4-(4-methyl-5-oxo-2h-furan-2-yl)but-2-enoxy]chromen-2-one Chemical compound C=1C=C2C=CC(=O)OC2=CC=1OC/C=C(/C)CC1OC(=O)C(C)=C1 CFNMUZCFSDMZPQ-GHXNOFRVSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 235000006491 Acacia senegal Nutrition 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 description 1
- 101100272788 Arabidopsis thaliana BSL3 gene Proteins 0.000 description 1
- 238000007808 Cell invasion assay Methods 0.000 description 1
- PTHCMJGKKRQCBF-UHFFFAOYSA-N Cellulose, microcrystalline Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC)C(CO)O1 PTHCMJGKKRQCBF-UHFFFAOYSA-N 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010061994 Coronavirus Spike Glycoprotein Proteins 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
- 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
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 101150089672 HAS3 gene Proteins 0.000 description 1
- 102000004989 Hepsin Human genes 0.000 description 1
- 108090001101 Hepsin Proteins 0.000 description 1
- 101000962530 Homo sapiens Hyaluronidase-1 Proteins 0.000 description 1
- 101000962526 Homo sapiens Hyaluronidase-2 Proteins 0.000 description 1
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 description 1
- 101800000120 Host translation inhibitor nsp1 Proteins 0.000 description 1
- 101001057752 Human cytomegalovirus (strain AD169) Uncharacterized protein IRL4 Proteins 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 102100039283 Hyaluronidase-1 Human genes 0.000 description 1
- 102100039285 Hyaluronidase-2 Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 101800000512 Non-structural protein 1 Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 108091005634 SARS-CoV-2 receptor-binding domains Proteins 0.000 description 1
- 238000010818 SYBR green PCR Master Mix Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000000424 bronchial epithelial cell Anatomy 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960001334 corticosteroids Drugs 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 210000001985 kidney epithelial cell Anatomy 0.000 description 1
- 230000023404 leukocyte cell-cell adhesion Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 231100000516 lung damage Toxicity 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229940041678 oral spray Drugs 0.000 description 1
- 239000000668 oral spray Substances 0.000 description 1
- 230000004768 organ dysfunction Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- RWWYLEGWBNMMLJ-MEUHYHILSA-N remdesivir Drugs C([C@@H]1[C@H]([C@@H](O)[C@@](C#N)(O1)C=1N2N=CN=C(N)C2=CC=1)O)OP(=O)(N[C@@H](C)C(=O)OCC(CC)CC)OC1=CC=CC=C1 RWWYLEGWBNMMLJ-MEUHYHILSA-N 0.000 description 1
- RWWYLEGWBNMMLJ-YSOARWBDSA-N remdesivir Chemical compound NC1=NC=NN2C1=CC=C2[C@]1([C@@H]([C@@H]([C@H](O1)CO[P@](=O)(OC1=CC=CC=C1)N[C@H](C(=O)OCC(CC)CC)C)O)O)C#N RWWYLEGWBNMMLJ-YSOARWBDSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 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 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 230000007502 viral entry Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
-
- 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
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/727—Heparin; Heparan
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Dermatology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
본 발명은 RNA 바이러스 감염 예방 또는 치료용 조성물에 관한 것으로, 더욱 구체적으로는 4-메틸움벨리페론(4-Methylumbelliferon) 및 헤파린(Heparin)을 유효성분으로 포함하는 것을 특징으로 한다. 본 발명에 의하면, 약물 재창출 기술을 이용하여 이미 약효가 입증된 약물의 다른 용도를 도출한 것으로, 새로운 약물에 비하여 부작용이 현저히 낮고, RNA 바이러스 감염의 예방 또는 치료 효과가 우수하다는 장점이 있다. The present invention relates to a composition for preventing or treating RNA virus infection, and more specifically, to a composition comprising 4-Methylumbelliferon and heparin as active ingredients. According to the present invention, a different use for a drug whose efficacy has already been proven is derived using drug repositioning technology, and has the advantage of significantly lower side effects compared to new drugs and excellent effectiveness in preventing or treating RNA virus infections.
Description
본 발명은 RNA 바이러스 감염 예방 또는 치료용 조성물에 관한 것으로, 더욱 구체적으로는 SARS-CoV-2의 스파이크 단백질에 결합능을 가지면서도 하이알루로난(hyaluronan)의 합성을 억제하는 활성을 가져, RNA 바이러스의 감염을 예방 또는 치료하는 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating RNA virus infection, and more specifically, has the ability to bind to the spike protein of SARS-CoV-2 and has the activity of inhibiting the synthesis of hyaluronan, thereby preventing RNA virus infection. It relates to a composition for preventing or treating infections.
코로나바이러스 감염증-19(coronavirus disease 2019, COVID-19)는 SARS-CoV-2(Severe acute respiratory syndrome coronavirus 2) 바이러스의 감염에 의해 발병하는 새로운 호흡기 감염질환으로, 2019년 12월 중국 우한지역에서 처음 발생한 뒤 전 세계적으로 확산되고 있으며, 국내 확진자가 급속도로 증가하고 있다.Coronavirus disease 2019 (COVID-19) is a new respiratory infectious disease caused by infection with the SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) virus. It was first reported in Wuhan, China in December 2019. Since its outbreak, it has spread globally, and the number of confirmed cases in Korea is rapidly increasing.
COVID-19 호흡기 감염의 치료를 위해 현재 많은 약품들이 테스트되고 있지만, 미국 식품의약국(FDA)이 승인한 항바이러스제는 Remdesivir 단 한 가지뿐이었다. 이밖에 항염증 치료제 중 하나인 코르티코스테로이드 덱사메타손에 대한 장기기능장애와 염증으로 인한 폐손상을 치료하거나 예방하는 연구를 진행하고 있다. 일부 연구에서는 인공호흡기를 사용하는 사람의 경우 사망 위험이 약 30%, 보조 산소가 필요한 사람의 경우 약 20% 감소한다는 것이 입증되었다. 그러나 덱사메타손과 다른 코르티코스테로이드는 덜 심각한 COVID-19 감염에 투여되면 매우 해로울 수 있다는 우려가 있다. Many drugs are currently being tested to treat COVID-19 respiratory infections, but only one antiviral drug has been approved by the U.S. Food and Drug Administration (FDA), Remdesivir. In addition, research is being conducted on the corticosteroid dexamethasone, one of the anti-inflammatory treatments, to treat or prevent lung damage caused by organ dysfunction and inflammation. Some studies have shown that the risk of death is reduced by about 30% for people on ventilators and by about 20% for people who require supplemental oxygen. However, there are concerns that dexamethasone and other corticosteroids could be very harmful when administered to less severe COVID-19 infections.
SARS-CoV-2의 세포로의 진입은 바이러스 외피에 있고, 외관상으로 '코로나'와 같이 외부로 돌출 된 1273개 아미노산으로 구성된 스파이크 (S) 바이러스 단백질이 안지오텐신 전환 효소 2 (ACE2) 수용체와 결합하여 이루어진다. ACE2는 3가지 코로나 바이러스 균주인 SARS-CoV, NL63 및 SARSCoV-2의 세포 진입을 매개하며, 특히, SARS-CoV 및 SARS-CoV2는 아미노산 서열에서 76% 동일성을 공유함으로써, ACE2와의 결합에 대한 이들 바이러스의 경향을 설명한다. 바이러스 진입 과정의 첫 번째 단계는 바이러스 단백질 단위 S1의 N- 말단 부분이 ACE2 수용체의 포켓에 결합하는 것이다. 바이러스 진입에 가장 중요한 것으로 여겨지는 두 번째 단계는 S1과 S2 단위 사이의 단백질 절단으로, Hepsin / TMPRSS 서브 패밀리의 구성원인 수용체 막횡 단백질 세린 2(TMPRSS2)에 의해 매개되는 것으로 알려져 있다(Polo V et al, European Journal of Internal Medicine, 2020).SARS-CoV-2 enters cells through the viral envelope, and the spike (S) viral protein, which consists of 1,273 amino acids that protrudes outwards like a 'corona' in appearance, binds to the angiotensin-converting enzyme 2 (ACE2) receptor. It comes true. ACE2 mediates cellular entry of three coronavirus strains, SARS-CoV, NL63, and SARSCoV-2. In particular, SARS-CoV and SARS-CoV2 share 76% identity in their amino acid sequences, suggesting their binding to ACE2. Describe the trends of viruses. The first step in the viral entry process is the binding of the N-terminal portion of viral protein unit S1 to the pocket of the ACE2 receptor. The second step, believed to be most important for virus entry, is protein cleavage between the S1 and S2 units, which is known to be mediated by the receptor transmembrane protein serine 2 (TMPRSS2), a member of the Hepsin/TMPRSS subfamily (Polo V et al , European Journal of Internal Medicine, 2020).
최근 보고에 의하면 히알루로난(HA) 수치와 COVID-19 증상 사이의 상관관계가 있는 것으로 보고되고 있다. 즉, 건강한 사람들보다 COVID-19 환자는 히알루로난이 20배 이상 높고, 중증 환자의 히알루로난 수치가 경증 환자의 히알루로난 수치보다 유의하게 높으며, COVID-19 환자의 폐 조직에서도 다량의 히알루로난이 검출됨이 밝혀졌다. Recent reports have shown a correlation between hyaluronan (HA) levels and COVID-19 symptoms. In other words, hyaluronan is more than 20 times higher in COVID-19 patients than in healthy people, the hyaluronan level in severe patients is significantly higher than that in mild patients, and the lung tissue of COVID-19 patients also contains a large amount of hyaluronan. It was revealed that Ronan had been detected.
따라서 COVID-19 환자의 HA 억제는 코로나바이러스 감염에 의한 질병의 중중도 진행을 멈출 수 있음이 확인된 것이다.Therefore, it has been confirmed that HA inhibition in COVID-19 patients can stop the severe progression of the disease caused by coronavirus infection.
그러나 이러한 HA 억제제가 COVID-19를 치료하거나 예방하는 데 얼마나 효과적일 수 있는지는 테스트되지 않았고 알려지지 않았다. However, it is untested and unknown how effective these HA inhibitors may be in treating or preventing COVID-19.
한편, 약물 재창출은 이미 시판 중이거나 임상 단계에서 안정성 이외의 이유로 실패한 약물을 대상으로 새로운 적응증을 규명하는 신약 개발 전략 중의 하나이다. 약물 재창출은 동일한 약물 타겟을 통해 새로운 적응증에 효과를 확인하거나, 신규 약물 타겟을 찾아내 새로운 적응증에 효과가 있음을 입증하는 것이다. 약물 재창출은 약물 후보물질 발굴에 소요되는 비용과 시간을 획기적으로 줄일 수 있고, 이미 임상적으로 안전성이 확보된 후보 물질을 대상으로 하기 때문에 약물의 안전성 문제로 임상 중간 단계에서 실패하는 문제를 해결할 수 있는 장점이 있다.Meanwhile, drug repositioning is one of the new drug development strategies that identifies new indications for drugs that are already on the market or have failed in the clinical stage for reasons other than safety. Drug repositioning involves confirming effectiveness for new indications through the same drug target, or discovering new drug targets and proving that they are effective for new indications. Drug re-creation can dramatically reduce the cost and time required to discover drug candidates, and because it targets candidates whose safety has already been clinically confirmed, it can solve the problem of drug failure in the mid-clinical stage due to safety issues. There are advantages to this.
따라서, 본 발명자는 코로나바이러스감염증-19를 치료하기 위한 약물을 개발하기 위해 예의 노력한 결과, 약물 재창출을 통해 SARS-CoV-2의 스파이크 단백질에 결합능을 가지며, HA를 억제하여 뛰어난 항 SARS-CoV-2 효과를 나타내는 물질을 도출하였다. Accordingly, the present inventor has made diligent efforts to develop a drug to treat COVID-19, and as a result, the drug has been reinvented to have the ability to bind to the spike protein of SARS-CoV-2 and to inhibit HA, thereby providing excellent anti-SARS-CoV A substance showing a -2 effect was derived.
따라서, 본 발명의 목적은 약물 재창출 기술을 이용하여 이미 약효가 입증된 약물의 다른 RNA 바이러스, 특히 코로나바이러스감염증-19의 예방 또는 치료용 용도를 도출함으로써, 부작용이 현저히 낮은 RNA 바이러스 감염 예방 또는 치료용 조성물을 제공하는 데 있다. Therefore, the purpose of the present invention is to use drug repositioning technology to derive a use for the prevention or treatment of other RNA viruses, especially COVID-19, of drugs whose efficacy has already been proven, thereby preventing or treating RNA virus infections with significantly lower side effects. The object is to provide a therapeutic composition.
상기한 목적을 달성하기 위한 본 발명의 RNA 바이러스 감염 예방 또는 치료용 조성물은, 4-메틸움벨리페론(4-Methylumbelliferon) 및 헤파린(Heparin)을 유효성분으로 포함하는 것을 특징으로 한다.The composition for preventing or treating RNA virus infection of the present invention for achieving the above object is characterized by containing 4-Methylumbelliferon and heparin as active ingredients.
상기 조성물은, 하이알루로난(hyaluronan)의 합성을 억제하는 활성을 가져, SARS-CoV-2의 진행을 억제하는 것을 특징으로 한다.The composition has the activity of inhibiting the synthesis of hyaluronan, thereby inhibiting the progression of SARS-CoV-2.
상기 조성물은, SARS-CoV-2의 스파이크 단백질에 결합능을 가지는 것을 특징으로 한다.The composition is characterized by having the ability to bind to the spike protein of SARS-CoV-2.
상기 4-메틸움벨리페론은 50~200microM의 농도로 포함되고, 상기 헤파린은 3.5~5nM의 농도로 포함되는 것을 특징으로 한다. The 4-methylumbelliferone is contained at a concentration of 50 to 200 microM, and the heparin is contained at a concentration of 3.5 to 5 nM.
상기 조성물은, 약학 조성물, 의약외품 조성물 중 1종의 것임을 특징으로 한다.The composition is characterized as being one of a pharmaceutical composition and a quasi-drug composition.
본 발명에 의하면, 약물 재창출 기술을 이용하여 이미 약효가 입증된 약물의 다른 용도를 도출한 것으로, 새로운 약물에 비하여 부작용이 현저히 낮고, RNA 바이러스 감염의 예방 또는 치료용 조성물 효과가 우수하다는 장점이 있다. According to the present invention, a different use of a drug whose efficacy has already been proven was derived using drug repositioning technology, and the advantages are that the side effects are significantly lower than those of new drugs and the composition for preventing or treating RNA virus infection is highly effective. there is.
도 1은 본 발명의 시험예 1의 원리를 나타낸 개략도.
도 2 및 도 3은 본 발명의 시험예 1의 결과를 나타낸 도면.
도 4는 본 발명의 시험예 3에 따른 인간 폐 상피세포 미니 장기에서 헤파린의 항코로나 바이러스 효과를 나타낸 도면.
도 5 및 도 6은 본 발명의 시험예 3에 따른 Heparin의 농도에 따른 항코로나 바이러스 효과를 나타낸 그래프.
도 7은 본 발명의 시험예 3에 따른 Heparin과, Heparin과 4-MU의 농도에 따른 항코로나 바이러스 효과를 나타낸 그래프.
도 8은 본 발명의 시험예 3에 따른 Human Primary Epithelial Cell Models에서 Heparin의 농도에 따른 항바이러스 효과를 나타낸 그래프.
도 9는 본 발명의 시험예 3에 따른 Human Primary Epithelial Cell Models에서 Heparin과 4-MU의 처리에 따른 HAS2/3 유전자 복제의 발현변화를 나타낸 그래프.
도 10은 본 발명의 시험예 3에 따른 각 Cell Models에서 4-MU의 처리에 따른 HAS2 유전자의 발현변화를 나타낸 그래프.
도 11은 본 발명의 시험예 3에 따른 각 Cell Models에서 4-MU의 처리에 따른 HAS3 유전자의 발현변화를 나타낸 그래프.
도 12는 본 발명의 시험예 3에 따른 각 Cell Models에서 4-MU의 처리에 따른 Hy1 유전자의 발현변화를 나타낸 그래프.
도 13은 본 발명의 시험예 3에 따른 각 Cell Models에서 4-MU의 처리에 따른 Hy2 유전자의 발현변화를 나타낸 그래프.
도 14는 본 발명의 시험예 3에 따른 각 Cell Models에서 4-MU의 처리에 따른 CD44 유전자의 발현변화를 나타낸 그래프.
도 15는 본 발명의 시험예 3에 따른 HLF cell Models에서 4-MU의 처리 농도에 따른 has2유전자의 발현변화를 나타낸 그래프이고,
도 16은 본 발명의 시험예 3에 따른 HLF cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 Has2 유전자의 발현변화를 나타낸 그래프.
도 17은 본 발명의 시험예 3에 따른 HLF cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 CD44 유전자의 발현변화를 나타낸 그래프.
도 18은 본 발명의 시험예 3에 따른 HLF cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 TLR4 유전자의 발현변화를 나타낸 그래프
도 19는 본 발명의 시험예 3에 따른 HEC cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 Has2 유전자의 발현변화를 나타낸 그래프
도 20은 본 발명의 시험예 3에 따른 HEC cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 CD44 유전자의 발현변화를 나타낸 그래프.
도 21은 본 발명의 시험예 3에 따른 HEC cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 TLR4 유전자의 발현변화를 나타낸 그래프.1 is a schematic diagram showing the principle of Test Example 1 of the present invention.
Figures 2 and 3 are diagrams showing the results of Test Example 1 of the present invention.
Figure 4 is a diagram showing the anti-coronavirus effect of heparin on human lung epithelial cell mini-organs according to Test Example 3 of the present invention.
Figures 5 and 6 are graphs showing the anti-coronavirus effect according to the concentration of heparin according to Test Example 3 of the present invention.
Figure 7 is a graph showing the anti-coronavirus effect according to the concentration of heparin, heparin, and 4-MU according to Test Example 3 of the present invention.
Figure 8 is a graph showing the antiviral effect according to the concentration of heparin in Human Primary Epithelial Cell Models according to Test Example 3 of the present invention.
Figure 9 is a graph showing the expression change of HAS2/3 gene replication according to treatment with heparin and 4-MU in Human Primary Epithelial Cell Models according to Test Example 3 of the present invention.
Figure 10 is a graph showing the expression change of the HAS2 gene according to treatment with 4-MU in each Cell Model according to Test Example 3 of the present invention.
Figure 11 is a graph showing the expression change of the HAS3 gene according to treatment with 4-MU in each Cell Model according to Test Example 3 of the present invention.
Figure 12 is a graph showing the expression change of the Hy1 gene according to treatment with 4-MU in each Cell Model according to Test Example 3 of the present invention.
Figure 13 is a graph showing the expression change of the Hy2 gene according to treatment with 4-MU in each Cell Model according to Test Example 3 of the present invention.
Figure 14 is a graph showing the change in expression of the CD44 gene according to treatment with 4-MU in each Cell Model according to Test Example 3 of the present invention.
Figure 15 is a graph showing the change in expression of the has2 gene according to the treatment concentration of 4-MU in HLF cell models according to Test Example 3 of the present invention.
Figure 16 is a graph showing the expression change of the Has2 gene after treatment of TGF&TNF in cells treated with 4-MU in HLF cell models according to Test Example 3 of the present invention.
Figure 17 is a graph showing the change in expression of the CD44 gene after treatment of TGF&TNF in cells treated with 4-MU in HLF cell models according to Test Example 3 of the present invention.
Figure 18 is a graph showing the change in expression of the TLR4 gene after treating cells treated with 4-MU with TGF&TNF in HLF cell models according to Test Example 3 of the present invention.
Figure 19 is a graph showing the change in expression of the Has2 gene after treating cells treated with 4-MU with TGF&TNF in HEC cell models according to Test Example 3 of the present invention.
Figure 20 is a graph showing the change in expression of the CD44 gene after treatment of TGF&TNF in cells treated with 4-MU in HEC cell models according to Test Example 3 of the present invention.
Figure 21 is a graph showing the change in expression of the TLR4 gene after treatment of TGF&TNF in cells treated with 4-MU in HEC cell models according to Test Example 3 of the present invention.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명에서 사용된 용어 "예방"은 조성물에 의해 SARS-CoV-2 감염을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.The term “prevention” as used in the present invention refers to any action that inhibits or delays the onset of SARS-CoV-2 infection by a composition.
본 발명에서 사용된 용어 "치료"는 조성물에 의해 SARS-CoV-2 감염의 증세가 호전되거나 이롭게 변경하는 모든 행위를 의미한다.The term “treatment” used in the present invention refers to any action that improves or beneficially changes the symptoms of SARS-CoV-2 infection by the composition.
본 발명에서는 기존에 승인된 약물을 이용하여 SARS-CoV-2의 스파이크 단백질에 결합능이 우수하면서도, HA의 합성을 억제하여 SARS-CoV-2에 대해 높은 항바이러스 효과를 나타낸다는 데 특징이 있다.The present invention uses a previously approved drug that has excellent binding ability to the spike protein of SARS-CoV-2, and is characterized by showing a high antiviral effect against SARS-CoV-2 by inhibiting the synthesis of HA.
본 발명의 RNA 바이러스 감염의 예방 또는 치료용 조성물은, 4-메틸움벨리페론(4-Methylumbelliferon) 및 헤파린(Heparin)을 유효성분으로 포함하는 것을 특징으로 한다.The composition for preventing or treating RNA virus infection of the present invention is characterized by containing 4-Methylumbelliferon and heparin as active ingredients.
먼저, 4-메틸움벨리페론(4-Methylumbelliferon; 4-MU)은 coumarin 유도체이자 HA 합성효소의 억제제로, 4-MU를 이용하면 SARS-CoV-2에 의해 촉진되는 HA의 합성을 효과적으로 억제함으로써, RNA 바이러스 감염, 특히 코로나-19의 진행을 억제하여 중증도 진행을 예방할 수 있다.First, 4-Methylumbelliferon (4-MU) is a coumarin derivative and an inhibitor of HA synthase. Using 4-MU effectively inhibits the synthesis of HA promoted by SARS-CoV-2. , it can inhibit the progression of RNA virus infections, especially COVID-19, and prevent the progression of severity.
이러한 4-MU는 생물학적 안전성이 매우 우수함이 밝혀진 물질로, RNA 바이러스 감염 치료에 안정적으로 사용할 수 있다.This 4-MU is a substance that has been shown to have excellent biological safety and can be stably used to treat RNA virus infections.
상기 4-MU는 조성물 전체에 대하여 50~200microM의 농도로 포함됨이 바람직한데, 상기한 농도를 벗어날 경우 그 효과가 미미하거나, 안정성이 좋지 못하기 때문이다.The 4-MU is preferably included in a concentration of 50 to 200 microM relative to the entire composition, because if the concentration exceeds the above concentration, the effect is minimal or the stability is poor.
또한, 헤파린(Heparin)은 항응고제로서, 항응고성을 가지는 글리코스아미노글리칸으로 일컬어지는 비이온성 직쇄 모노폴리사카라이드 등의 비균질 군이다. 이러한 헤파린은 크게 미분획 헤파린(Unfractionated heparin)과 저분자량 헤파린(Low molecular weight heparin)으로 구분하는데, 통상 헤파린이라고 부르는 약물은 미분획 헤파린으로 분자량이 3,000~30,000 정도의 이종다당류의 혼합물이며, 헤파린을 화학적 또는 효소적으로 중합체를 잘라서 소분자화한 것이 저분자량 헤파으로 평균 분자량은 4,000~5,000 정도이다.Additionally, heparin is an anticoagulant and is a heterogeneous group of nonionic linear monopolysaccharides called glycosaminoglycans with anticoagulant properties. Heparin is largely divided into unfractionated heparin and low molecular weight heparin. The drug commonly called heparin is unfractionated heparin, which is a mixture of heteropolysaccharides with a molecular weight of about 3,000 to 30,000. Low-molecular-weight Hepa is made by cutting the polymer chemically or enzymatically into small molecules, and its average molecular weight is about 4,000 to 5,000.
상기 헤파린은 SARS-CoV-2의 스파이크 단백질에 결합능을 가지는 바, 이를 통해 SARS-CoV-2의 스파이크 단백질이 무력화되어 더 이상 감염력을 가지지 않도록 해준다.The heparin has the ability to bind to the spike protein of SARS-CoV-2, thereby neutralizing the spike protein of SARS-CoV-2 so that it is no longer infectious.
이러한 헤파린 역시 이미 항응고제로 범용 사용되고 있는바, RNA 바이러스 감염 치료에 안정적으로 사용할 수 있다.Heparin is already widely used as an anticoagulant and can be reliably used to treat RNA virus infections.
상기 헤파린은 조성물 전체에 대하여 3.5~5nM의 농도로 포함되는 것이 바람직한데, 상기한 농도를 벗어날 경우 그 효과가 미미하거나, 안정성이 좋지 못하기 때문이다.The heparin is preferably included at a concentration of 3.5 to 5 nM relative to the entire composition, because if the concentration exceeds the above concentration, the effect is minimal or the stability is poor.
즉, 본 발명은 상기 4-MU 및 헤파린의 조합으로 인해, HA의 합성을 억제하는 활성을 가져, SARS-CoV-2 감염의 진행을 억제하며, SARS-CoV-2의 스파이크 단백질에 결합능을 가져, SARS-CoV-2의 스파이크 단백질을 무력화함으로써, RNA 바이러스 감염, 특히 COVID-19를 효과적으로 예방 또는 치료하는 것인바, 상기 4-MU 및 헤파린은 뛰어난 시너지 효과를 나타낸다. That is, the present invention has the activity of inhibiting the synthesis of HA due to the combination of 4-MU and heparin, inhibits the progression of SARS-CoV-2 infection, and has the ability to bind to the spike protein of SARS-CoV-2. , By neutralizing the spike protein of SARS-CoV-2, RNA virus infections, especially COVID-19, are effectively prevented or treated, and the 4-MU and heparin show an excellent synergistic effect.
이러한 본 발명의 조성물은, 약학 조성물, 의약외품 조성물 중 1종의 것임이 바람직하다.The composition of the present invention is preferably one of a pharmaceutical composition and a quasi-drug composition.
본 발명의 약학 조성물은, 투여를 위하여, 상기 기재한 성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.For administration, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier, excipient, or diluent in addition to the ingredients described above. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Examples include polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
본 발명의 약학적 조성물은 각각 통상의 방법에 따라 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용할 수 있다. 상세하게는 제형화할 경우 통상 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 이러한 고형 제제는 상기 유효성분 외에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 비경구 투여를 위한 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제 및 과제를 포함한다.The pharmaceutical composition of the present invention can be formulated and used in the form of topical preparations, suppositories, or sterile injectable solutions according to conventional methods. In detail, it can be prepared using commonly used diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants. Such solid preparations may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., in addition to the above active ingredients. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and preparations.
상기 약학 조성물은 증상 정도에 따라 투여 방법이 결정되는데, 통상적으로는 국소 투여 방식이 바람직하다. 또한, 상기 약학 조성물의 유효성분의 유효량은 질환의 예방 또는 치료 요구되는 양을 의미한다. 따라서, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효 성분 및 다른 성분의 종류 및 함량, 제형의 종류 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. The method of administration of the pharmaceutical composition is determined depending on the severity of symptoms, and topical administration is usually preferable. In addition, the effective amount of the active ingredient of the pharmaceutical composition refers to the amount required for the prevention or treatment of disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form and the patient's age, weight, general health condition, gender and diet, administration time, administration route and composition. It can be adjusted depending on a variety of factors, including secretion rate, duration of treatment, and concurrent medications.
아울러, 본 발명의 의약외품 조성물은, 다른 의약외품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있으며 이에 제한되는 것은 아니다. 유효성분의 혼합양은 사용 목적(예방, 건강 또는 개선)에 따라 적합하게 결정될 수 있다. 본 발명의 의약외품 조성물의 종류에는 제한은 없으나 바람직하게는 소독 청결제, 가그린, 구강 스프레이, 소독제, 공기 스프레이 등으로 사용될 수 있으며, 그 형태가 제한되지 않는다.In addition, the quasi-drug composition of the present invention can be used together with other quasi-drug ingredients and can be used appropriately according to conventional methods, but is not limited thereto. The amount of active ingredients mixed can be appropriately determined depending on the purpose of use (prevention, health, or improvement). There is no limitation on the type of the quasi-drug composition of the present invention, but it can preferably be used as a disinfectant cleaner, gargreen, oral spray, disinfectant, air spray, etc., and its form is not limited.
이하, 실시예를 통해 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail through examples.
(실시예 1)(Example 1)
4-MU 200μM, 저분자량 헤파린 4nM 및 정제수로 되는 조성물을 제조하였다(Soluble ACE2). 그리고 이를 동결건조하여 냉장보관하였다.A composition consisting of 200 μM of 4-MU, 4 nM of low molecular weight heparin, and purified water was prepared (Soluble ACE2). It was then freeze-dried and stored in the refrigerator.
(시험예 1)(Test Example 1)
본 발명의 조성물과 SARS-CoV-2의 스파이크 단백질의 결합을 검증하기 위하여 도 1과 같이 리포터 사람 신장 세포주를 이용한 결합 억제능 테스트를 수행하였다.To verify the binding of the composition of the present invention to the spike protein of SARS-CoV-2, a binding inhibition test was performed using a reporter human kidney cell line as shown in Figure 1.
HEK293T는 고포도당 DMEM(Dulbecco's modified Eagle's medium; Gibco)에 10% 우태아 혈청(fetal bovine serum; Hyclone)을 첨가한 배지에서 배양하였다.HEK293T was cultured in high-glucose DMEM (Dulbecco's modified Eagle's medium; Gibco) supplemented with 10% fetal bovine serum (Hyclone).
대상 바이러스는 SARS-CoV-2와 렌티바이러스를 조합하여 인위적인 코로나 바이러스 스파이크 단백질을 표면에 가지면서 녹색형광 단백질을 발현하도록 만든 유사 렌티바이러스를 준비하였다. 이러한 유사 렌티바이러스는 종래 공지된 방법에 의하여 제조, 배양하였다.The target virus was prepared by combining SARS-CoV-2 and lentivirus to prepare a similar lentivirus that had an artificial coronavirus spike protein on its surface and expressed a green fluorescent protein. These similar lentiviruses were manufactured and cultured by conventionally known methods.
유사 렌티바이러스 배양액을 HEK293T 및 Ace2 수용체가 과발현된 HEK293T에 1시간 동안 반응시켰다. 그 결과를 도 2에 나타내었다. Similar lentivirus cultures were reacted with HEK293T and HEK293T overexpressing the Ace2 receptor for 1 hour. The results are shown in Figure 2.
또한, 유사 렌티바이러스 배양액에 미리 식염수, 백신접종자의 혈청 또는 Soluble ACE2을 200μM의 농도로 미리 처리한 후, ACE2 과발현 HEK293T에 1시간 동안 반응시켰다. 그 결과를 도 3에 나타내었다. In addition, the similar lentivirus culture was pre-treated with saline solution, vaccinee's serum, or Soluble ACE2 at a concentration of 200 μM, and then reacted with ACE2-overexpressing HEK293T for 1 hour. The results are shown in Figure 3.
상기 유사 렌티바이러스는 Ace2 수용체가 과발현된 HEK293T에 감염되어 해당 세포주에서 녹색 형광을 발현하게 한다. 그러나 이러한 유사 렌티바이러스에 코로나 바이러스 중화 항체를 미리 처리한 후 동일한 실험을 수행하면 중화항체에 의해 유사 렌티바이러스 표면 스파이크 단백질이 무력화되어 더 이상 감염력을 가지지 않게 된다. 같은 원리로 Soluble ACE2가 스파이크 단백질에 결합능을 가지는 경우, 유사 렌티바이러스의 감염력이 약해지므로, HEK293T 세포주에서의 녹색 형광 정도에 따라 Soluble ACE2의 스파이크 단백질에 대한 결합능을 테스트 할 수 있다.The similar lentivirus infects HEK293T, which overexpresses the Ace2 receptor, and causes the cell line to express green fluorescence. However, if the same experiment is performed after pre-treating these similar lentiviruses with coronavirus neutralizing antibodies, the similar lentivirus surface spike proteins will be neutralized by the neutralizing antibodies and will no longer be infectious. In the same principle, if Soluble ACE2 has the ability to bind to the spike protein, the infectivity of similar lentiviruses is weakened, so the binding ability of Soluble ACE2 to the spike protein can be tested according to the level of green fluorescence in the HEK293T cell line.
도 2와 같이 일반적인 HEK293T는 코로나 바이러스 스파이크 유사 렌티바이러스에 거의 감염되지 않지만, ACE2가 과발현된 HEK293T는 감염 정도가 증가됨을 확인할 수 있었다.As shown in Figure 2, it was confirmed that typical HEK293T is rarely infected with coronavirus spike-like lentivirus, but HEK293T with overexpressed ACE2 has an increased degree of infection.
도 3에서와 같이, 대조군으로서 식염수를 미리 처리한 경우 비해 백신 접종자 혈청을 전처리한 경우 녹색형광이 완전히 제거된 것으로 미루어 보아 혈청 내에 중화항체가 존재하여 유사 렌티바이러스의 감염력을 차단하는 것을 알 수 있으며, Soluble ACE2의 경우 혈청 처리군에 비해서는 많지만 식염수 처리군에 비해서는 녹색형광이 현저히 감소함을 미루어 Soluble ACE2가 스파이크 단백질에 결합하여 바이러스를 무력화함을 확인할 수 있었다.As shown in Figure 3, green fluorescence was completely removed when the vaccine recipient's serum was pretreated compared to when the saline solution was pretreated as a control, indicating that neutralizing antibodies were present in the serum, blocking the infectivity of similar lentiviruses. , In the case of Soluble ACE2, green fluorescence was greater than that of the serum-treated group, but significantly decreased compared to the saline-treated group, confirming that Soluble ACE2 binds to the spike protein and neutralizes the virus.
(시험예 2)(Test Example 2)
인간 정상 내피 세포(NEC) 및 폐 섬유아세포(HLF)는 ACTT에서 구입하여 모든 공배양 시험에 사용되었다. 두 셀라인은 모두 5000cll/㎠의 동일한 밀도로 시딩되었으며, 10% FBS-DMEM and Endothelial Cell Medium(ECM)의 배지를 사용하였다. 배지는 48h 간격으로 교체하였다.Human normal endothelial cells (NEC) and lung fibroblasts (HLF) were purchased from ACTT and used for all co-culture tests. Both cell lines were seeded at the same density of 5000cll/㎠, and 10% FBS-DMEM and Endothelial Cell Medium (ECM) were used. The medium was changed every 48 h.
실험 0일차 부터는 공배양 배지(1:1 DMEM and ECM MEdia)를 사용하였으며, 배지는 24h 간격으로 교체하면서 96h 배양하였다.From day 0 of the experiment, co-culture medium (1:1 DMEM and ECM MEdia) was used, and the culture was cultured for 96 h, changing the medium at 24-h intervals.
NEC(normal Endothelial cell)/HLF(Human lung fibroblast) 및 SPEC(Spike protein exposed Endothelial cell)/HLF, HLF 배양물 내 HA 생성효소인 HAS2의 상대적 mRNA 발현을 테스트하였다. The relative mRNA expression of HAS2, an HA synthase, was tested in normal endothelial cell (NEC)/human lung fibroblast (HLF) and spike protein exposed endothelial cell (SPEC)/HLF and HLF cultures.
그 결과 NEC/HLF 대비 SPEC/HLF에서 HAS2의 mRNA 발현은 약 2.6배 증가함을 확인하였다. 또한, HLF 단독 배양에 비해 NEC/HLF 및 SPEC/HLF에서 각각 약 4.0배 및 5.8배 증가하였다. As a result, it was confirmed that the mRNA expression of HAS2 increased approximately 2.6-fold in SPEC/HLF compared to NEC/HLF. In addition, compared to HLF culture alone, NEC/HLF and SPEC/HLF increased by about 4.0-fold and 5.8-fold, respectively.
아울러, HAS2의 발현도 NEC/HLF에 비해 SPEC/HLF가 2.2배 더 증가하였다. 또한, SPEC/HLF은 HLF 단독 배양에 비해 3.1배 더 증가하였다.In addition, the expression of HAS2 also increased 2.2 times more in SPEC/HLF than in NEC/HLF. Additionally, SPEC/HLF increased 3.1 times compared to HLF culture alone.
이를 통해 SPEC/HLF의 공동 배양군의 HAS2 및 HAS3의 발현도는 HLF의 단독 배양군에 비해 그 발현이 유의미하게 높음을 확인하였는바, 이는 COVID 감염 이후 HA 합성이 활성화되고 증가했음을 의미하는 것이다. Through this, it was confirmed that the expression levels of HAS2 and HAS3 in the SPEC/HLF co-culture group were significantly higher than those in the HLF alone culture group, which means that HA synthesis was activated and increased after COVID infection.
그리고 상기한 배양군에 Soluble ACE2을 200μM로 처리하고 72시간 후, 다시 HAS2 및 HAS3의 mRNA 발현을 측정한 결과, SPEC/HLF에서 HAS2의 발현이 60% 하향 조절되었고, NEC/HLF에서 HAS3가 85% 하향 조절됨을 확인하였다.And 72 hours after treating the above-mentioned culture group with 200 μM of Soluble ACE2, the mRNA expression of HAS2 and HAS3 was measured again. As a result, the expression of HAS2 was down-regulated by 60% in SPEC/HLF, and HAS3 was downregulated by 85% in NEC/HLF. It was confirmed that the % was down-regulated.
또한, HA 염색을 통해 HLF, NEC/HLF, SPEC/HLF, Soluble ACE2-NEC/HLF, Soluble ACE2-SPEC/HLF에서의 HA 발현량을 비교하였다. In addition, HA expression levels in HLF, NEC/HLF, SPEC/HLF, Soluble ACE2-NEC/HLF, and Soluble ACE2-SPEC/HLF were compared through HA staining.
SPEC/HLF의 염색 비율은 NEC/HLF에 비해 더 큰 면적을 보였으며(15.3±1.1% vs 2.1±0.3%), Soluble ACE2-NEC/HLF, Soluble ACE2-SPEC/HLF에서는 모두 HA의 발현이 증가하지 않았으며, 오히려 그 면적이 다소 감소하는 경향을 보였음을 확인하였다.The staining ratio of SPEC/HLF showed a larger area compared to NEC/HLF (15.3±1.1% vs 2.1±0.3%), and HA expression increased in both Soluble ACE2-NEC/HLF and Soluble ACE2-SPEC/HLF. It was confirmed that this was not the case, and rather, the area showed a tendency to decrease somewhat.
(시험예 3)(Test Example 3)
Heparin, 4-MU, Heparin-4-MU의 처리에 따른 항바이러스 효과 및 HAS2 HAS3, Hy1, Hy2, CD44, TLR4 유전자의 발현변화를 테스트하였다.Antiviral effects and changes in expression of HAS2 HAS3, Hy1, Hy2, CD44, and TLR4 genes were tested according to treatment with heparin, 4-MU, and Heparin-4-MU.
테스트 방법은 하기와 같았다.The test method was as follows.
Maintenance and coculture of Human normal Endothelial cells and lung fibroblastsMaintenance and coculture of Human normal Endothelial cells and lung fibroblasts
인간 정상 내피 세포(NEC) 및 폐 섬유아세포(HLF)는 ACTT에서 구입하여 모든 공배양 시험에 사용되었다. 두 셀라인은 모두 6-well 배양접시에 5000cll/㎠의 동일한 밀도로 시딩되었으며, 10% FBS-DMEM and Endothelial Cell Medium(ECM)의 배지를 사용하여 유지하였다. 배양액 교환은 48시간 간격이었다.Human normal endothelial cells (NEC) and lung fibroblasts (HLF) were purchased from ACTT and used for all co-culture tests. Both cell lines were seeded at the same density of 5000 cll/cm2 in 6-well culture dishes and maintained using 10% FBS-DMEM and Endothelial Cell Medium (ECM). Culture medium exchange was every 48 hours.
실험 0일부터 시작하여 HLF 배양액을 공동 배양액(1:1, DMEM 및 ECM 미디어)로 교체하였고, 실험 0일에 트랜스배양접시을 옮겨 HLF를 트랜스배양접시 플레이트의 바닥에 놓았다. 배양된 NEC가 들어 있는 트랜스배양접시를 HLF가 들어 있는 웰(챔버)에 넣고 하프앤하프 배지를 유지했으며, 공동 배양 배지 교환은 매일하였다. 96시간의 공동 배양 후, NEC 트랜스배양접시를 제거하고 mRNA 발현, HA 함량 분석 또는 백혈구 부착 연구를 위해 HLF 샘플을 분리하였다. 부착 연구에 사용된 HLF 및 NEC/HLF 공동 배양은 DMEM 및 ECM(1:1) 배지로 유지되었다.Starting from experimental day 0, the HLF culture medium was replaced with co-culture medium (1:1, DMEM and ECM media), and on experimental day 0, the transculture dish was transferred and HLF was placed on the bottom of the transculture dish plate. The transculture dish containing the cultured NEC was placed in a well (chamber) containing HLF and maintained on half-and-half medium, and the co-culture medium was exchanged daily. After 96 hours of co-culture, NEC transculture dishes were removed and HLF samples were isolated for mRNA expression, HA content analysis, or leukocyte adhesion studies. HLF and NEC/HLF co-cultures used for attachment studies were maintained with DMEM and ECM (1:1) medium.
Cell-to-Cell TransmissionCell-to-Cell Transmission
렌티바이러스 벡터 시스템에서, 안티센스 리포터 유전자 Gluc의 발현은 HIV-1 게놈의 센스 방향으로 배향된 인트론에 의해 방해를 받아 Gluc 생산이 바이러스 생산 세포가 아닌 감염된 표적 세포에서만 일어나게 된다. 바이러스 생산자와 표적 세포를 공동 배양함으로써, 렌티바이러스 위형을 생성하는 공여체 세포(예: 293T)와 표적 세포(예: 293T/ACE2) 사이의 공동 배양 배지의 Gluc 활성을 측정하여 세포 간의 감염 정도를 정하였다. 구체적으로, 293T 세포를 6웰 플레이트에 시딩하고, 1.4μg NL4.3-inGluc와 SARS-CoV 또는 SARS-CoV-2 스파이크를 인코딩하는 플라스미드 0.7μg으로 형질 감염 시키고, 다음날 형질 감염된 293T 공여자 세포를 PBS(phosphate-buffered saline)/5mM EDTA(ethylenediaminetetraacetic acid)로 충분히 세척하여 EDTA를 제거한 후, 표적 세포(293T/ACE2, Caco-2, Calu-3, NCI-H520 또는 PBMC)를 24-웰 플레이트에서 24~72시간 동안 1:1 비율로 처리하였다. 그리고 필요에 따라 억제제 또는 혈청을 추가하였다. 상청액을 덜어내고 Gluc의 활성을 측정하였다.In the lentiviral vector system, expression of the antisense reporter gene Gluc is hindered by an intron oriented in the sense direction of the HIV-1 genome, such that Gluc production occurs only in infected target cells and not in virus-producing cells. By co-culturing virus producers and target cells, the extent of infection between cells is determined by measuring the Gluc activity in the co-culture medium between donor cells producing lentiviral pseudotypes (e.g. 293T) and target cells (e.g. 293T/ACE2). did. Specifically, 293T cells were seeded in 6-well plates, transfected with 1.4 μg NL4.3-inGluc and 0.7 μg of plasmid encoding SARS-CoV or SARS-CoV-2 spike, and the next day, transfected 293T donor cells were incubated with PBS. After washing thoroughly with (phosphate-buffered saline)/5mM EDTA (ethylenediaminetetraacetic acid) to remove EDTA, target cells (293T/ACE2, Caco-2, Calu-3, NCI-H520 or PBMC) were cultured in a 24-well plate for 24 hours. Treated at a 1:1 ratio for ~72 hours. And inhibitors or serum were added as needed. The supernatant was removed and the Gluc activity was measured.
실제 SARS-CoV-2 WT 및 VOC의 세포 간 전파를 조사하기 위해 기증자 Vero-ACE2 세포를 MOI 0.2(WT) 또는 0.02(VOC)로 20시간 동안 감염시키고, 동일한 세포 배양 백신 혈청의 존재 또는 없는 조건 하에서 추가 6시간 동안 Vero-mTomato-Red 세포의 수, 다른 목적을 위해 Vero-ACE2-TMPRSS2-mTomato 세포는 WT SARS-CoV-2(USA-WA1/2020, MOI=0.01)로 18시간 동안 감염시킨 후, Vero-ACE2-TMPRSS2-mTomato 세포를 공동 배양하였다. 그런 다음 세포를 3.7% 포름알데히드로 1시간 동안 고정하고, PBS로 3회 세척한 후, BSL3 실험실에서 꺼내고, 고정된 세포를 항-SARS-CoV-2 뉴클레오캡시드 및 항-마우스-FITC와 함께 배양하고 flow cytometry 분석을 실시하였다.To investigate cell-to-cell transmission of real-world SARS-CoV-2 WT and VOC, donor Vero-ACE2 cells were infected at an MOI of 0.2 (WT) or 0.02 (VOC) for 20 h, in the presence or absence of identical cell culture vaccine sera. Number of Vero-mTomato-Red cells for an additional 6 h under Afterwards, Vero-ACE2-TMPRSS2-mTomato cells were co-cultured. The cells were then fixed with 3.7% formaldehyde for 1 hour, washed three times with PBS, taken out in a BSL3 laboratory, and the fixed cells were incubated with anti-SARS-CoV-2 nucleocapsid and anti-mouse-FITC. Culture was performed and flow cytometry analysis was performed.
Vero Cell Culture and AssaysVero Cell Culture and Assays
아프리카 녹색 원숭이 Vero 신장 상피 세포는 ECACC에서 구입했고 DMEM(Gibco, 영국)배양액에서 50~75% 컨플루언스(세포밀도)로 유지하고, 10%(v/v) 태아 소 혈청(SLS, 영국), 20mM L -글루타민(Gibco, 영국), 100 U mL -1 페니실린-G(Gibco, 영국) 및 100 U mL -1 스트렙토마이신 설페이트(Gibco, 영국), 37℃에서 5% CO2의 조건에서 세포들을 배양하였다.African green monkey Vero kidney epithelial cells were purchased from ECACC and maintained at 50–75% confluence (cell density) in DMEM (Gibco, UK) culture with 10% (v/v) fetal bovine serum (SLS, UK). , 20mM L -glutamine (Gibco, UK), 100 U mL -1 penicillin-G (Gibco, UK) and 100 U mL -1 streptomycin sulfate (Gibco, UK) at 37°C and 5% CO2. Cultured.
세포 침윤 검정 테스트를 위해, Vero 세포를 24-웰 플레이트에서 웰당 2.5 ×105개의 세포로 플레이팅하고, 10%(v/v) 소 태아 혈청(완전 배지)이 보충된 EMEM(Sigma-Aldrich, Italy)으로 배양하였다. 24시간 후, 감염 1시간 전에 세포를 300μL의 완전 배지에서 나트륨 헤파린(6.25~200μg mL-1; Celsus Laboratories, Cincinnati, United States)과 함께 배양한 다음 50개의 플라크 형성을 포함하는 바이러스 용액과 함께 배양하였다. Italy/UniSR1/2020 분리주(GISAID 가입 ID: EPI_ISL_413489) 또는 SARS-CoV HSR-1(EID 2004))의 단위(PFU). 37℃에서 1시간 동안 배양한 후, 상청액을 버리고 1%의 500μL(w/v) methylcellulose (Sigma-Aldrich, Italy) overlay (in complete medium)를 각 well에 첨가하고 3일 후 6% (v/v) formaldehyde:phosphate-buffered saline (PBS)을 사용하여 세포를 고정하였다. 70%(v/v) 메탄올(Sigma-Aldrich, Italy) 중 1%(w/v) 크리스탈 바이올렛(Sigma-Aldrich, Italy)으로 염색한 후, 입체 현미경(SMZ-1500, 니콘)으로 카운트하여 분석하였다.For cell invasion assay testing, Vero cells were plated at 2.5 × 10 cells per well in 24-well plates and cultured in EMEM (Sigma-Aldrich, 2018) supplemented with 10% (v/v) fetal bovine serum (complete medium). Italy). Twenty-four hours later, 1 h before infection, cells were incubated with sodium heparin (6.25–200 μg mL−1; Celsus Laboratories, Cincinnati, United States) in 300 μL of complete medium and then incubated with virus solution containing 50 plaque-forming cells. did. units (PFU) of the Italy/UniSR1/2020 isolate (GISAID accession ID: EPI_ISL_413489) or SARS-CoV HSR-1 (EID 2004). After incubation at 37°C for 1 hour, the supernatant was discarded and 500 μL (w/v) 1% methylcellulose (Sigma-Aldrich, Italy) overlay (in complete medium) was added to each well, and after 3 days, 6% (v/v) was added to each well. v) Cells were fixed using formaldehyde:phosphate-buffered saline (PBS). After staining with 1% (w/v) crystal violet (Sigma-Aldrich, Italy) in 70% (v/v) methanol (Sigma-Aldrich, Italy), counting and analysis using a stereomicroscope (SMZ-1500, Nikon) did.
RBD-결합 검정을 위해, Vero 세포를 10%(v/v) 소 태아 혈청, 20 mM L-글루타민, 100U mL로 보충된 DMEM 100μL에서 웰당 1,000개 세포로 96-웰 세포 배양 플레이트에 플레이팅하였다. -1 페니실린-G 및 100UmL -1 스트렙토마이신 설페이트를 첨가하고 하루밤을 부착시켰다. 배지는 200μL의 1X PBS(CMF-PBS; Ca 2+ 및 Mg 2+ 없음; Lonza, 영국)로 3회 세척하기 전에 제거되었다. 세포를 실온에서 10분 동안 100μL의 10% 중성 완충 포르말린(Thermo Fisher, 영국)으로 고정시켰다. 웰을 CMF-PBS 200μL로 3회 세척한 다음, 100μL CMF-PBS를 각 웰에 첨가하고 플레이트를 사용할 때까지 4℃에서 보관하였다. 결합 분석 전에 웰을 실온에서 1시간 동안 200μL CMF-PBS + 1%(w/v) 소 혈청 알부민(BSA; Sigma-Aldrich, 영국)으로 블록킹하고, 200μL CMF-PBS로 3회 세척하였다. 0.1%(v/v) Tween-20(PBST, Sigma-Aldrich, United Kingdom)으로 200μL의 CMF-PBS로 2회 세척한 후, SARS-CoV-2 S1 RBD(50㎍/㎖-1)(헤파린(100㎍/㎖) 포함 또는 포함되지 않음)를 25㎕ PBST + 0.1%(w/v) BSA와 함께 각 웰에 첨가하고, 1시간 동안 배양하였다. 흔들면서 실온. 배양 후, 웰을 200μL PBST로 3회, 200μL CMF-PBS로 2회 세척한 후 실온에서 1시간 동안 Alexa Fluor 488 항-his 태그 항체(클론 J095G46, Biolegend , 영국) 웰당 0.1%(w/v) BSA가 포함된 25μL PBST에서 1:5,000(v/v). 웰을 200μL PBST로 3회, 200μL CMF-PBS로 2회 세척하였다. 형광 방출은 Tecan Infinite M200Pro 플레이트 판독기(λ ex. = 495 nm, λ em. = 520 nm)를 사용하여 측정되었으며, 결과는 평균 ± 표준 편차(n = 3)로 표시되었다.For RBD-binding assays, Vero cells were plated in 96-well cell culture plates at 1,000 cells per well in 100 μL of DMEM supplemented with 10% (v/v) fetal bovine serum, 20 mM L-glutamine, and 100 U mL. . -1 Penicillin-G and 100 UmL -1 streptomycin sulfate were added and allowed to adhere overnight. The medium was removed before washing three times with 200 μL of 1× PBS (CMF-PBS; free of Ca 2+ and Mg 2+ ; Lonza, UK). Cells were fixed with 100 μL of 10% neutral buffered formalin (Thermo Fisher, UK) for 10 min at room temperature. Wells were washed three times with 200 μL of CMF-PBS, then 100 μL of CMF-PBS was added to each well and the plates were stored at 4°C until use. Before binding analysis, wells were blocked with 200 μL CMF-PBS + 1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, UK) for 1 h at room temperature and washed three times with 200 μL CMF-PBS. After washing twice with 200 μL of CMF-PBS with 0.1% (v/v) Tween-20 (PBST, Sigma-Aldrich, United Kingdom), the SARS-CoV-2 S1 RBD (50 μg/mL-1) (heparin (with or without 100 μg/ml) was added to each well along with 25 μl PBST + 0.1% (w/v) BSA and incubated for 1 hour. Room temperature while shaking. After incubation, the wells were washed three times with 200 μL PBST and twice with 200 μL CMF-PBS and then incubated with Alexa Fluor 488 anti-his tag antibody (clone J095G46, Biolegend, UK) 0.1% (w/v) per well for 1 hour at room temperature. 1:5,000 (v/v) in 25 μL PBST with BSA. Wells were washed three times with 200 μL PBST and twice with 200 μL CMF-PBS. Fluorescence emission was measured using a Tecan Infinite M200Pro plate reader (λ ex. = 495 nm, λ em. = 520 nm), and results are expressed as mean ± standard deviation (n = 3).
Establishment of 293T ACE2 cells, production of lentiviral particles, and transfection of virus to endothelial cellsEstablishment of 293T ACE2 cells, production of lentiviral particles, and transfection of virus to endothelial cells
VSV G-pseudotyped lentivirus를 사용하는 293T-ACE2 세포주는 앞서 ACE2 발현을 확립하고 검증했으며, D10 성장 배지(10% 열 불활성화 FBS, 2mM L-글루타민, 100U/ml가 포함된 DMEM)에서 성장할 수 있음이 확인되었다. 페니실린 및 100 ug/ml 스트렙토마이신, 37℃ 및 5% 이산화탄소에서 렌티바이러스 입자 생성, 준비된 내피세포에 바이러스 입자를 감염시켜 항-ACE2 항체에 의한 ACE2 단백질 발현을 확인하였다.The 293T-ACE2 cell line using VSV G-pseudotyped lentivirus has previously been established and validated for ACE2 expression and can be grown in D10 growth medium (DMEM with 10% heat-inactivated FBS, 2mM L-glutamine, 100U/ml). This has been confirmed. Lentiviral particles were produced in the presence of penicillin and 100 ug/ml streptomycin at 37°C and 5% carbon dioxide, and the prepared endothelial cells were infected with the virus particles to confirm ACE2 protein expression by anti-ACE2 antibody.
Generation of VSV pseudotype particlesGeneration of VSV pseudotype particles
HEK293T 세포(ATCC 번호 CRL3216)를 10% 태아 소 혈청(FBS; Fisher Scientific)이 보충된 Dulbecco modified Eagle 배지(DMEM)(Corning Inc.)에서 37℃, 5% CO2에서 배양하였다. HEK293T 세포(2 × 106)를 100-mm 조직 배양 접시에 플레이팅하고, 플라스미드의 조합으로 약 75% 컨플루언트(세포밀도)일 때 다음 날 형질 감염시켰다. 9μg의 pLV-eGFP는 Addgene에서 구입하였다. 플라스미드 번호 36083; RRID Addgene_36083), 9μg의 psPAX2; 애드진 플라스미드 번호 12260; RRID Addgene_12260), 및 3μg의 pCAGGS-S(SARS-CoV-2)(카탈로그 번호 NR-52310; BEI 리소스) 또는 VSV-G; 애드진 플라스미드 번호 14888; RRID Addgene_14888)을 대조군으로 사용하였고, 폴리에틸렌이민(PEI) 시약(Millipore Sigma 번호 408727)을 제조업체의 프로토콜에 따라 형질감염에 사용하였다. 다음날, GFP 형광으로 표시되는 형광 현미경 하에서 형질감염 효율에 대해 세포를 확인하였다. 24시간 세포 배양 상청액을 수확하여 4℃에 보관하고 더 많은(10ml) 완전 배지(10% FBS 함유 DMEM)를 플레이트에 추가했으며, 48시간 세포 배양 상청액을 수확하고 각 샘플에 대한 24시간 상청액과 결합시켰다. 결합된 상청액을 2,000 x g에서 5분 동안 테이블탑 원심분리기에서 회전시켜 잔류 세포를 펠릿화한 다음, 0.45-μm 주사기 필터를 통과시켰다. 분취액은 -80°C에서 동결되었으며, 12웰 조직 배양 접시에 플레이팅된 새로운 HEK293T 세포를 102 내지 107의 희석 범위로 수확된 바이러스(상청액)로 감염시켰다. 바이러스(pLV-S) 역가는 20~100개의 GFP 양성 세포에 의해 정하였다.HEK293T cells (ATCC number CRL3216) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Corning Inc.) supplemented with 10% fetal bovine serum (FBS; Fisher Scientific) at 37°C and 5% CO 2 . HEK293T cells (2 × 10 6 ) were plated in 100-mm tissue culture dishes and transfected the next day when approximately 75% confluent (cell density) with the combination of plasmids. 9 μg of pLV-eGFP was purchased from Addgene. Plasmid number 36083; RRID Addgene_36083), 9 μg of psPAX2; Addgene plasmid number 12260; RRID Addgene_12260), and 3 μg of pCAGGS-S (SARS-CoV-2) (catalog number NR-52310; BEI Resources) or VSV-G; Addgene plasmid number 14888; RRID Addgene_14888) was used as a control, and polyethyleneimine (PEI) reagent (Millipore Sigma number 408727) was used for transfection according to the manufacturer's protocol. The next day, cells were checked for transfection efficiency under a fluorescence microscope as indicated by GFP fluorescence. The 24-h cell culture supernatant was harvested and stored at 4°C, more (10 ml) complete medium (DMEM with 10% FBS) was added to the plate, and the 48-h cell culture supernatant was harvested and combined with the 24-h supernatant for each sample. I ordered it. The combined supernatant was spun in a tabletop centrifuge at 2,000 × g for 5 min to pellet any remaining cells and then passed through a 0.45-μm syringe filter. Aliquots were frozen at -80°C, and fresh HEK293T cells plated in 12-well tissue culture dishes were infected with the harvested virus (supernatant) at a dilution range of 10 2 to 10 7 . Virus (pLV-S) titer was determined by 20 to 100 GFP-positive cells.
RNA extraction and real-time PCRRNA extraction and real-time PCR
총 RNA는 HEC와 공동 배양된 HLF에서 분리되었으며, 각 실험 조건에서 3개의 웰을 수확하고 풀링하여 제조업체 권장 사항(RNAqueous 키트, Ambion®-Applied Biosystems, Austin, TX)에 따라 RNA를 분리하였다. RNA 농도 및 무결성은 Agilent® 2100 Bioanalyzer 시스템 및 Agilent® RNA 6000 Nano Chips(Agilent® Technologies, Foster City, CA)를 사용하여 결정하였으며, RNA 무결성 번호가 ≥ 8인 RNA 샘플은 SuperScript® VILO cDNA Synthesis Kit(Life Technologies, Grand Island, NY)를 사용하여 역전사 시켰다. HAS1, HAS2, HAS3, HYAL1, HYAL2, CD44, TRL4 및 GAPDH(glyceraldehyde 3-phosphate dehydrogenase)에 대해 검증된 TaqMan® probe(Life Technologies)를 사용하여 정량적 실시간 PCR을 수행했으며, 분석은 TaqMan® Fast Advanced Master Mix 시약 및 Applied Biosystems StepOnePlus™ Real-Time PCR System(Life Technologies)을 사용하여 수행하였다.Total RNA was isolated from HLF co-cultured with HEC, and three wells from each experimental condition were harvested, pooled, and RNA isolated according to manufacturer recommendations (RNAqueous kit, Ambion®-Applied Biosystems, Austin, TX). RNA concentration and integrity were determined using the Agilent® 2100 Bioanalyzer System and Agilent® RNA 6000 Nano Chips (Agilent® Technologies, Foster City, CA), and RNA samples with RNA integrity number ≥ 8 were analyzed using the SuperScript® VILO cDNA Synthesis Kit ( Reverse transcription was performed using Life Technologies, Grand Island, NY). Quantitative real-time PCR was performed using validated TaqMan® probes (Life Technologies) for HAS1, HAS2, HAS3, HYAL1, HYAL2, CD44, TRL4, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the assay was performed using TaqMan® Fast Advanced Master. It was performed using Mix reagent and Applied Biosystems StepOnePlus™ Real-Time PCR System (Life Technologies).
Immunofluorescence analysis Immunofluorescence analysis
CoV2-mut에 감염되지 않았거나 감염된 인간 폐 상피세포를 4℃에서 밤새 PBS에서 4% 파라포름알데히드로 고정하고, 실온에서 15분 동안 0.1% Triton X-100으로 투과시켰다. 이후 항-SARS-CoV-1 nsp1 항체 또는 친화성 정제된 토끼 항-SARS-CoV N 다클론 항체를 1차 항체로 하여 세포를 면역형광 분석한 후, SlowFade Gold Antifade 시약(Invitrogen)을 사용하여 알렉사 플루오르 488-공역 2차 항체(Invitrogen)와 DAPI 역염색을 실시하였다. 세포는 UTMB에서 40X 대물 렌즈가 장착된 Olympus BX53 현미경을 사용하여 검사하고 ImageJ(NIH) 소프트웨어로 처리하였다. 세포를 ibidi 슬라이드에 플레이팅하고, MOI 3에서 '세포 및 바이러스'에 기술된 바와 같이 감염시키거나 감염시키지 않고, 8hpi에서 PBS로 1회 세척하고, 3% 파라포름알데히드에서 20분 동안 고정하고, PBS에서 세척하고, 0.5% 트리톤으로 투과성으로 만들었다. PBS에서 X-100을 2분 동안 처리한 다음 PBS에서 2% FBS로 30분 동안 블록킹하였다. 토끼 항-SARS-CoV-2 혈청을 1:200 희석하여 면역염색을 수행하였다. 세포를 세척하고 항-토끼 FITC 접합 항체(1:200 희석) 및 DAPI(4',6-디아미디노-2-페닐인돌)(1:200 희석)로 표지했으며, ×40 대물렌즈와 Axiocam 506 모노 카메라를 사용하여 Zeiss AxioObserver Z1 광시야 현미경에서 이미징을 수행하였다.Human lung epithelial cells uninfected or infected with CoV2-mut were fixed with 4% paraformaldehyde in PBS overnight at 4°C and permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. Afterwards, the cells were subjected to immunofluorescence analysis using anti-SARS-CoV-1 nsp1 antibody or affinity-purified rabbit anti-SARS-CoV N polyclonal antibody as the primary antibody, and then Alexa stained using SlowFade Gold Antifade reagent (Invitrogen). Counterstaining with fluorine 488-conjugated secondary antibody (Invitrogen) and DAPI was performed. Cells were examined using an Olympus BX53 microscope equipped with a 40X objective at UTMB and processed with ImageJ (NIH) software. Cells were plated on ibidi slides, infected or uninfected as described in ‘Cells and Viruses’ at an MOI of 3, washed once with PBS at 8 hpi, fixed in 3% paraformaldehyde for 20 min; Washed in PBS and permeabilized with 0.5% Triton. X-100 was treated in PBS for 2 minutes and then blocked with 2% FBS in PBS for 30 minutes. Immunostaining was performed with rabbit anti-SARS-CoV-2 serum diluted 1:200. Cells were washed and labeled with anti-rabbit FITC conjugated antibody (1:200 dilution) and DAPI (4',6-diamidino-2-phenylindole) (1:200 dilution) using a ×40 objective and Axiocam 506. Imaging was performed on a Zeiss AxioObserver Z1 wide-field microscope using a monocamera.
Quantification of mRNA expressionQuantification of mRNA expression
RNAqueous™-4PCR 키트(Ambion)를 사용하여 총 RNA를 정제하고, 제조업체의 지침에 따라 SuperScriptTM II RNase H- 역전사 효소 키트(Invitrogen)를 사용하여 cDNA로 역전하였다. 제조자의 프로토콜(Power SYBR Green PCR Master Mix, Applied Biosystems)에 따라 PCR 생성물의 실시간 검출을 가능하게 하는 형광 염료로서 SYBR-green을 갖는 ABI Prism 7500 검출 시스템(Applied Biosystems)을 사용하여 생성된 cDNA에서의 유전자 발현을 검사하였다. 유전자의 상대적인 발현 수준은 샘플의 GAPDH 수준에 대해 결정되다. PCR에 사용한 프라이머는 표 1에 나타내었다.Total RNA was purified using the RNAqueous™-4PCR kit (Ambion) and reversed to cDNA using the SuperScript™ II RNase H-Reverse Transcriptase Kit (Invitrogen) according to the manufacturer's instructions. in cDNA generated using an ABI Prism 7500 detection system (Applied Biosystems) with SYBR-green as a fluorescent dye to enable real-time detection of PCR products according to the manufacturer's protocol (Power SYBR Green PCR Master Mix, Applied Biosystems). Gene expression was examined. The relative expression level of the gene was determined relative to the GAPDH level in the sample. Primers used for PCR are shown in Table 1.
HAS1-RHAS1-F
HAS1-R
5’-GCCGGTCATCCCCAAAAG-3’5'-GGAATAACCTCTTGCAGCAGTTTC-3'
5'-GCCGGTCATCCCCAAAAG-3'
HAS2-RHAS2-F
HAS2-R
5’-ACTTCTCTTTTTCCACCCCATTT-3’5'-TCGCAACACGTAACGCAAT-3'
5'-ACTTCTCTTTTTCCACCCCATTT-3'
HAS3-RHAS3-F
HAS3-R
5’-GCCCGCTCCACGTTGA-35'-AACAAGTACGACTCATGGATTTCCT-3'
5'-GCCCGCTCCACGTTGA-3
HYAL1-RHYAL1-F
HYAL1-R
5’-GGGAGCTATAGAAAATTGTCATGTCA-3’’5'-GATGTCAGTGTCTTCGATGTGGTA-3'
5'-GGGAGCTATAGAAAATTGTCATGTCA-3''
HYAL2-RHYAL2-F
HYAL2-R
5’-GCAGAATCGAAGCGTGGATAC-3’5'-CTAATGAGGTTTGTGAACCAGAATAT-3'
5'-GCAGAATCGAAGCGTGGATAC-3'
CD44-RCD44-F
CD44-R
5’-CCAAGATGATCAGCCATTCTGG-3’5'-AAGACATCTACCCCAGCAAC-3'
5'-CCAAGATGATCAGCCATTCTGG-3'
TLR4-RTLR4-F
TLR4-R
5’-TCAGGTCCAGGTTCTTGGTTGAG-3’5'-AGGATGATGCCAGGATGATGTC-3'
5'-TCAGGTCCAGGTTCTTGGTTGAG-3'
GAPDH-RGAPDH-F
GAPDH-R
5’-TGGTCATGAGTCCTTCCACGATA-3’5'-CCCATGTTCGTCATGGGTGT-3'
5'-TGGTCATGAGTCCTTCCACGATA-3'
F, forward; R, reverse; P, probeF, forward; R, reverse; P, probe
그리고 그 결과들을 도 4 내지 21에 나타내었다.And the results are shown in Figures 4 to 21.
도 4는 인간 폐 상피세포 미니 장기에서 헤파린의 항코로나 바이러스 효과를 나타낸 것으로, 헤라핀의 처리가 항코로나 바이러스 효과가 있음이 확인되었다. Figure 4 shows the anti-coronavirus effect of heparin on human lung epithelial cell mini-organs, and it was confirmed that treatment with heparin has an anti-coronavirus effect.
도 5 및 도 6은 헤파린의 농도에 따른 항코로나 바이러스 효과를 나타낸 그래프로, 헤라핀의 농도가 3.5~5nM일 때 가장 우수한 효과가 나타남을 확인하였다.Figures 5 and 6 are graphs showing the anti-coronavirus effect according to the concentration of heparin, and it was confirmed that the best effect was observed when the concentration of heparin was 3.5 to 5 nM.
도 7은 헤파린과, 헤라핀과 4-MU의 농도에 따른 항코로나 바이러스 효과를 나타낸 그래프로, 헤파린, 헤라핀과 4-MU를 동시사용하는 경우 유사한 수준의 효과가 나타남을 확인하였다. Figure 7 is a graph showing the anti-coronavirus effect according to the concentration of heparin, herapin, and 4-MU. It was confirmed that a similar level of effect was observed when heparin, herapin, and 4-MU were used simultaneously.
도 8은 Human Primary Epithelial Cell Models에서 Heparin의 농도에 따른 항바이러스 효과를 나타낸 그래프로, Heparin의 처리가 바이러스 감염성을 낮추며, 10㎍/ml에서 가장 우수한 효과가 있음을 확인하였따.Figure 8 is a graph showing the antiviral effect according to the concentration of heparin in Human Primary Epithelial Cell Models. It was confirmed that treatment with heparin lowers viral infectivity and has the best effect at 10㎍/ml.
도 9는 Human Primary Epithelial Cell Models에서 Heparin과 4-MU의 처리에 따른 HAS2/3 유전자 복제의 발현변화를 나타낸 그래프로, Heparin과 4-MU를 함께 처리한 C군에서 가장 우수한 효과가 나타남을 확인하였다.Figure 9 is a graph showing the expression change of HAS2/3 gene replication according to treatment with heparin and 4-MU in Human Primary Epithelial Cell Models, confirming that the best effect was observed in group C treated with heparin and 4-MU together. did.
도 10 내지 도 14는 각 Cell Models에서 4-MU의 처리에 따른 HAS2, HAS3, HY 1, Hy2, CD44의 유전자의 발현변화를 나타낸 그래프이다. 도면에서 HLF는 human lung fibroblast를, HEC는 human endothelial cell을, BEC는 bronchial epithelial cells를, NEC는 Non-treated EC를, SPEC는 spike protein-treated EC를, 4-MU-NEC-CoC는 4-MU-treated NEC CoC를 나타낸다.Figures 10 to 14 are graphs showing changes in gene expression of HAS2, HAS3, HY 1, Hy2, and CD44 according to treatment with 4-MU in each Cell Model. In the drawing, HLF represents human lung fibroblasts, HEC represents human endothelial cells, BEC represents bronchial epithelial cells, NEC represents non-treated EC, SPEC represents spike protein-treated EC, and 4-MU-NEC-CoC represents 4- Indicates MU-treated NEC CoC.
도 15는 HLF cell Models에서 4-MU의 처리 농도에 따른 has2유전자의 발현변화를 나타낸 그래프이고, 도 16은 HLF cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 Has2 유전자의 발현변화를 나타낸 그래프이고, 도 17은 HLF cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 CD44 유전자의 발현변화를 나타낸 그래프이며, 도 18은 HLF cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 TLR4 유전자의 발현변화를 나타낸 그래프이다. 이때, TNFα는 10ng/ml로 처리하였고, TGFβ는 1ng/ml 처리하였다.Figure 15 is a graph showing the change in expression of the has2 gene according to the treatment concentration of 4-MU in HLF cell Models, and Figure 16 is a graph showing the change in expression of the Has2 gene after treating cells treated with 4-MU with TGF&TNF in HLF cell Models. It is a graph shown, and Figure 17 is a graph showing the change in expression of the CD44 gene after treating cells treated with 4-MU in HLF cell Models with TGF&TNF, and Figure 18 is a graph showing changes in expression of the CD44 gene after treatment with TGF&TNF in cells treated with 4-MU in HLF cell Models. This is a graph showing the change in expression of the TLR4 gene after treatment. At this time, TNFα was treated at 10ng/ml, and TGFβ was treated at 1ng/ml.
도 19는 HEC cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 Has2 유전자의 발현변화를 나타낸 그래프이고, 도 20은 본 발명의 HEC cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 CD44 유전자의 발현변화를 나타낸 그래프이며, 도 21은 본 발명의 HEC cell Models에서 4-MU의 처리한 세포에 TGF&TNF를 처리 후 TLR4 유전자의 발현변화를 나타낸 그래프이다. 이때, TNFα는 10ng/ml로 처리하였고, TGFβ는 1ng/ml 처리하였다.Figure 19 is a graph showing the expression change of the Has2 gene after treating cells treated with 4-MU with TGF&TNF in HEC cell models, and Figure 20 is a graph showing changes in expression of the Has2 gene after treating cells treated with 4-MU with TGF&TNF in HEC cell models of the present invention. It is a graph showing the change in expression of the CD44 gene after treatment, and Figure 21 is a graph showing the change in expression of the TLR4 gene after treatment of TGF&TNF in cells treated with 4-MU in the HEC cell models of the present invention. At this time, TNFα was treated at 10ng/ml, and TGFβ was treated at 1ng/ml.
이상의 결과를 통해 본 발명에 의한 조성물은, SARS-CoV-2 바이러스에 대한 우수한 항바이러스 활성을 가짐으로써, RNA 바이러스 감염, 특히 COVID-19 감염증의 새로운 예방 및 치료 물질로 사용할 수 있음을 알 수 있었다.The above results showed that the composition according to the present invention has excellent antiviral activity against the SARS-CoV-2 virus and can be used as a new prevention and treatment material for RNA virus infections, especially COVID-19 infections. .
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시 예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.
Claims (5)
A composition for preventing or treating RNA virus infection, comprising 4-Methylumbelliferon and heparin as active ingredients.
상기 조성물은,
하이알루로난(hyaluronan)의 합성을 억제하는 활성을 가져, SARS-CoV-2 감염의 진행을 억제하는 것을 특징으로 하는 RNA 바이러스 감염 예방 또는 치료용 조성물.
According to paragraph 1,
The composition is,
A composition for preventing or treating RNA virus infection, which has the activity of inhibiting the synthesis of hyaluronan and inhibits the progression of SARS-CoV-2 infection.
상기 조성물은,
SARS-CoV-2의 스파이크 단백질에 결합능을 가지는 것을 특징으로 하는 RNA 바이러스 감염 예방 또는 치료용 조성물.
According to claim 1 or 2,
The composition is,
A composition for preventing or treating RNA virus infection, characterized in that it has the ability to bind to the spike protein of SARS-CoV-2.
상기 4-메틸움벨리페론은 50~200microM의 농도로 포함되고,
상기 헤파린은 3.5~5nM의 농도로 포함되는 것을 특징으로 하는 RNA 바이러스 감염 예방 또는 치료용 조성물.
According to paragraph 1,
The 4-methylumbelliferone is contained at a concentration of 50 to 200 microM,
A composition for preventing or treating RNA virus infection, wherein the heparin is contained at a concentration of 3.5 to 5 nM.
상기 조성물은,
약학 조성물, 의약외품 조성물 중 1종의 것임을 특징으로 하는 RNA 바이러스 감염 예방 또는 치료용 조성물.According to paragraph 1,
The composition is,
A composition for preventing or treating RNA virus infection, characterized in that it is one of a pharmaceutical composition and a quasi-drug composition.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020220031286 | 2022-03-14 | ||
| KR20220031286 | 2022-03-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20230134446A true KR20230134446A (en) | 2023-09-21 |
Family
ID=88189437
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020230033179A KR20230134446A (en) | 2022-03-14 | 2023-03-14 | Composition for preventing or treating coronavirus infection |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20230134446A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210123235A (en) | 2020-04-02 | 2021-10-13 | 조선대학교산학협력단 | Use of ACE2 as treatment of COVID-19 |
| KR20210133892A (en) | 2020-04-29 | 2021-11-08 | 한국과학기술원 | Composition for preventing or treating CoVid-19 |
-
2023
- 2023-03-14 KR KR1020230033179A patent/KR20230134446A/en not_active Application Discontinuation
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210123235A (en) | 2020-04-02 | 2021-10-13 | 조선대학교산학협력단 | Use of ACE2 as treatment of COVID-19 |
| KR20210133892A (en) | 2020-04-29 | 2021-11-08 | 한국과학기술원 | Composition for preventing or treating CoVid-19 |
Non-Patent Citations (1)
| Title |
|---|
| Polo V et al, European Journal of Internal Medicine, 2020 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hu et al. | The severe acute respiratory syndrome coronavirus nucleocapsid inhibits type I interferon production by interfering with TRIM25-mediated RIG-I ubiquitination | |
| Chen et al. | High glucose inhibits neural stem cell differentiation through oxidative stress and endoplasmic reticulum stress | |
| Shen et al. | LncRNA HOTAIR inhibited osteogenic differentiation of BMSCs by regulating Wnt/β-catenin pathway. | |
| Kokubo et al. | BDNF-mediated cerebellar granule cell development is impaired in mice null for CaMKK2 or CaMKIV | |
| EP4218773A1 (en) | Human functional corneal endothelial cell and application thereof | |
| Lin et al. | Roles of notch signaling pathway and endothelial-mesenchymal transition in vascular endothelial dysfunction and atherosclerosis. | |
| US11919933B2 (en) | Use of MYOG gene as target in preparation of drug for treating cardiomyocyte apoptosis-associated cardiovascular disease | |
| Stanifer et al. | Critical role of type III interferon in controlling SARS-CoV-2 infection, replication and spread in primary human intestinal epithelial cells | |
| Lee et al. | Genipin inhibits RANKL-induced osteoclast differentiation through proteasome-mediated degradation of c-Fos protein and suppression of NF-κB activation | |
| Yang et al. | Drug repurposing of itraconazole and estradiol benzoate against COVID‐19 by blocking SARS‐CoV‐2 spike protein‐mediated membrane fusion | |
| Chen et al. | Porcine reproductive and respiratory syndrome virus induces interleukin-1β through MyD88/ERK/AP-1 and NLRP3 inflammasome in microglia | |
| JP2012530685A (en) | Use of autophagy (type II cell apoptosis) inhibitors | |
| Karwelat et al. | Influenza virus‐mediated suppression of bronchial Chitinase‐3‐like 1 secretion promotes secondary pneumococcal infection | |
| Qu et al. | Vitamin C Treatment Rescues Prelamin A‐Induced Premature Senescence of Subchondral Bone Mesenchymal Stem Cells | |
| Iverson et al. | Membrane-tethered mucin 1 is stimulated by interferon and virus infection in multiple cell types and inhibits influenza a virus infection in human airway epithelium | |
| Andreu et al. | Dextran sulfate from Leuconostoc mesenteroides B512F exerts potent antiviral activity against SARS-CoV-2 in vitro and in vivo | |
| WO2012150707A1 (en) | Low molecular weight compound which promotes induction of differentiation of stem cells into insulin-producing cells and method for inducing differentiation of stem cells into insulin-producing cells using low molecular weight compound | |
| Shao et al. | Prx II reduces oxidative stress and cell senescence in chondrocytes by activating the p16-CDK4/6-pRb-E2F signaling pathway. | |
| KR20230134446A (en) | Composition for preventing or treating coronavirus infection | |
| WO2021191674A2 (en) | Methods for screening of anti-virus reagent using organoids | |
| dos Ramos Almeida et al. | Neutrophil Virucidal Activity Against SARS-CoV-2 Is Mediated by Neutrophil Extracellular Traps | |
| Wang et al. | Impact of human airway epithelial cellular composition on SARS-CoV-2 infection biology | |
| Tedbury et al. | Mechanisms by which the cystic fibrosis transmembrane conductance regulator may influence SARS‐CoV‐2 infection and COVID‐19 disease severity | |
| Nau et al. | Varenicline prevents SARS-CoV-2 infection in vitro and in rhesus macaques | |
| Salerno et al. | Inhibition of SARS-CoV-2 infection in human cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoskeleton architecture and contractility |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A302 | Request for accelerated examination | ||
| E902 | Notification of reason for refusal | ||
| E601 | Decision to refuse application |