CN115260098A - Application of ABCB5 inhibitor in preparation of multi-drug-resistance rheumatoid arthritis treatment drug - Google Patents
Application of ABCB5 inhibitor in preparation of multi-drug-resistance rheumatoid arthritis treatment drug Download PDFInfo
- Publication number
- CN115260098A CN115260098A CN202210648571.0A CN202210648571A CN115260098A CN 115260098 A CN115260098 A CN 115260098A CN 202210648571 A CN202210648571 A CN 202210648571A CN 115260098 A CN115260098 A CN 115260098A
- Authority
- CN
- China
- Prior art keywords
- sinomenine
- abcb5
- drug
- rheumatoid arthritis
- compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101000677872 Homo sapiens ATP-binding cassette sub-family B member 5 Proteins 0.000 title claims abstract description 138
- 102100021501 ATP-binding cassette sub-family B member 5 Human genes 0.000 title claims abstract description 128
- 239000003814 drug Substances 0.000 title claims abstract description 72
- 206010039073 rheumatoid arthritis Diseases 0.000 title claims abstract description 72
- 229940079593 drug Drugs 0.000 title claims abstract description 62
- 238000011282 treatment Methods 0.000 title claims abstract description 51
- 239000003112 inhibitor Substances 0.000 title claims abstract description 24
- 230000036457 multidrug resistance Effects 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title abstract description 32
- INYYVPJSBIVGPH-QHRIQVFBSA-N Sinomenine Chemical compound C([C@@H]1N(CC2)C)C3=CC=C(OC)C(O)=C3[C@@]32[C@@H]1C=C(OC)C(=O)C3 INYYVPJSBIVGPH-QHRIQVFBSA-N 0.000 claims abstract description 183
- INYYVPJSBIVGPH-UHFFFAOYSA-N 14-episinomenine Natural products C1CN(C)C2CC3=CC=C(OC)C(O)=C3C31C2C=C(OC)C(=O)C3 INYYVPJSBIVGPH-UHFFFAOYSA-N 0.000 claims abstract description 107
- RARWEROUOQPTCJ-RBUKOAKNSA-N cepharamine Natural products C1CC2=CC=C(OC)C(O)=C2[C@@]2(CCN3C)[C@]13C=C(OC)C(=O)C2 RARWEROUOQPTCJ-RBUKOAKNSA-N 0.000 claims abstract description 107
- 229930002966 sinomenine Natural products 0.000 claims abstract description 107
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims abstract description 102
- 229960000485 methotrexate Drugs 0.000 claims abstract description 102
- 150000001875 compounds Chemical class 0.000 claims description 47
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 42
- 238000000034 method Methods 0.000 claims description 20
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 15
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 13
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 claims description 12
- -1 amino compound Chemical class 0.000 claims description 11
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000001257 hydrogen Substances 0.000 claims description 10
- 229910052739 hydrogen Inorganic materials 0.000 claims description 10
- 150000002825 nitriles Chemical class 0.000 claims description 9
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical compound NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 claims description 8
- DPBLXKKOBLCELK-UHFFFAOYSA-N pentan-1-amine Chemical compound CCCCCN DPBLXKKOBLCELK-UHFFFAOYSA-N 0.000 claims description 8
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 7
- 238000006467 substitution reaction Methods 0.000 claims description 7
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 6
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 6
- 229910052794 bromium Inorganic materials 0.000 claims description 6
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- REXUYBKPWIPONM-UHFFFAOYSA-N 2-bromoacetonitrile Chemical compound BrCC#N REXUYBKPWIPONM-UHFFFAOYSA-N 0.000 claims description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 5
- 150000001412 amines Chemical class 0.000 claims description 5
- 150000002431 hydrogen Chemical class 0.000 claims description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 5
- 238000007335 nucleophilic acyl substitution reaction Methods 0.000 claims description 5
- YLVKIBKAGUPACT-UHFFFAOYSA-N phenylazaniumylideneazanide Chemical class [N]NC1=CC=CC=C1 YLVKIBKAGUPACT-UHFFFAOYSA-N 0.000 claims description 5
- BMVXCPBXGZKUPN-UHFFFAOYSA-N 1-hexanamine Chemical compound CCCCCCN BMVXCPBXGZKUPN-UHFFFAOYSA-N 0.000 claims description 4
- QSNSCYSYFYORTR-UHFFFAOYSA-N 4-chloroaniline Chemical compound NC1=CC=C(Cl)C=C1 QSNSCYSYFYORTR-UHFFFAOYSA-N 0.000 claims description 4
- XJEVFFNOMKXBLU-UHFFFAOYSA-N 4-methylsulfonylaniline Chemical compound CS(=O)(=O)C1=CC=C(N)C=C1 XJEVFFNOMKXBLU-UHFFFAOYSA-N 0.000 claims description 4
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 claims description 4
- WRDWWAVNELMWAM-UHFFFAOYSA-N 4-tert-butylaniline Chemical compound CC(C)(C)C1=CC=C(N)C=C1 WRDWWAVNELMWAM-UHFFFAOYSA-N 0.000 claims description 4
- WJYIASZWHGOTOU-UHFFFAOYSA-N Heptylamine Chemical compound CCCCCCCN WJYIASZWHGOTOU-UHFFFAOYSA-N 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- NWWWGAKVHCSAEU-UHFFFAOYSA-N 5-bromopentanenitrile Chemical compound BrCCCCC#N NWWWGAKVHCSAEU-UHFFFAOYSA-N 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 239000003995 emulsifying agent Substances 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 3
- 239000000314 lubricant Substances 0.000 claims description 3
- 239000000375 suspending agent Substances 0.000 claims description 3
- 239000002562 thickening agent Substances 0.000 claims description 3
- 239000000080 wetting agent Substances 0.000 claims description 3
- CQPGDDAKTTWVDD-UHFFFAOYSA-N 4-bromobutanenitrile Chemical compound BrCCCC#N CQPGDDAKTTWVDD-UHFFFAOYSA-N 0.000 claims description 2
- 239000007884 disintegrant Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 43
- 206010059866 Drug resistance Diseases 0.000 abstract description 17
- 241000700159 Rattus Species 0.000 description 84
- 210000004027 cell Anatomy 0.000 description 71
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 56
- 238000006243 chemical reaction Methods 0.000 description 39
- 210000002683 foot Anatomy 0.000 description 27
- 230000014509 gene expression Effects 0.000 description 27
- 210000000988 bone and bone Anatomy 0.000 description 26
- 230000006378 damage Effects 0.000 description 25
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 24
- 210000004369 blood Anatomy 0.000 description 24
- 239000008280 blood Substances 0.000 description 24
- 108020004414 DNA Proteins 0.000 description 22
- 229940125782 compound 2 Drugs 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 18
- 206010003246 arthritis Diseases 0.000 description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 15
- 238000012453 sprague-dawley rat model Methods 0.000 description 15
- 238000010186 staining Methods 0.000 description 15
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 14
- 238000005160 1H NMR spectroscopy Methods 0.000 description 14
- 241001465754 Metazoa Species 0.000 description 14
- 238000004062 sedimentation Methods 0.000 description 14
- 230000001225 therapeutic effect Effects 0.000 description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 230000002401 inhibitory effect Effects 0.000 description 12
- 238000011552 rat model Methods 0.000 description 12
- 206010061218 Inflammation Diseases 0.000 description 11
- 210000000610 foot bone Anatomy 0.000 description 11
- 230000004054 inflammatory process Effects 0.000 description 11
- 230000001404 mediated effect Effects 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 11
- 230000009261 transgenic effect Effects 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 10
- 210000003743 erythrocyte Anatomy 0.000 description 10
- 230000002757 inflammatory effect Effects 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- 230000002441 reversible effect Effects 0.000 description 10
- 230000008961 swelling Effects 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 9
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 9
- 241000701161 unidentified adenovirus Species 0.000 description 9
- 238000000684 flow cytometry Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 102100033350 ATP-dependent translocase ABCB1 Human genes 0.000 description 7
- 102000013691 Interleukin-17 Human genes 0.000 description 7
- 108050003558 Interleukin-17 Proteins 0.000 description 7
- 108010047230 Member 1 Subfamily B ATP Binding Cassette Transporter Proteins 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 238000007619 statistical method Methods 0.000 description 7
- UNILWMWFPHPYOR-KXEYIPSPSA-M 1-[6-[2-[3-[3-[3-[2-[2-[3-[[2-[2-[[(2r)-1-[[2-[[(2r)-1-[3-[2-[2-[3-[[2-(2-amino-2-oxoethoxy)acetyl]amino]propoxy]ethoxy]ethoxy]propylamino]-3-hydroxy-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-[(2r)-2,3-di(hexadecanoyloxy)propyl]sulfanyl-1-oxopropan-2-yl Chemical compound O=C1C(SCCC(=O)NCCCOCCOCCOCCCNC(=O)COCC(=O)N[C@@H](CSC[C@@H](COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC)C(=O)NCC(=O)N[C@H](CO)C(=O)NCCCOCCOCCOCCCNC(=O)COCC(N)=O)CC(=O)N1CCNC(=O)CCCCCN\1C2=CC=C(S([O-])(=O)=O)C=C2CC/1=C/C=C/C=C/C1=[N+](CC)C2=CC=C(S([O-])(=O)=O)C=C2C1 UNILWMWFPHPYOR-KXEYIPSPSA-M 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 102000004127 Cytokines Human genes 0.000 description 5
- 108090000695 Cytokines Proteins 0.000 description 5
- 210000000068 Th17 cell Anatomy 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 210000000548 hind-foot Anatomy 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 230000002018 overexpression Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- GLGNXYJARSMNGJ-VKTIVEEGSA-N (1s,2s,3r,4r)-3-[[5-chloro-2-[(1-ethyl-6-methoxy-2-oxo-4,5-dihydro-3h-1-benzazepin-7-yl)amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide Chemical compound CCN1C(=O)CCCC2=C(OC)C(NC=3N=C(C(=CN=3)Cl)N[C@H]3[C@H]([C@@]4([H])C[C@@]3(C=C4)[H])C(N)=O)=CC=C21 GLGNXYJARSMNGJ-VKTIVEEGSA-N 0.000 description 4
- SZUVGFMDDVSKSI-WIFOCOSTSA-N (1s,2s,3s,5r)-1-(carboxymethyl)-3,5-bis[(4-phenoxyphenyl)methyl-propylcarbamoyl]cyclopentane-1,2-dicarboxylic acid Chemical compound O=C([C@@H]1[C@@H]([C@](CC(O)=O)([C@H](C(=O)N(CCC)CC=2C=CC(OC=3C=CC=CC=3)=CC=2)C1)C(O)=O)C(O)=O)N(CCC)CC(C=C1)=CC=C1OC1=CC=CC=C1 SZUVGFMDDVSKSI-WIFOCOSTSA-N 0.000 description 4
- GHYOCDFICYLMRF-UTIIJYGPSA-N (2S,3R)-N-[(2S)-3-(cyclopenten-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propanoyl]amino]propanamide Chemical compound C1(=CCCC1)C[C@@H](C(=O)[C@@]1(OC1)C)NC([C@H]([C@@H](C1=CC=C(C=C1)OC)O)NC([C@H](C)NC(CN1CCOCC1)=O)=O)=O GHYOCDFICYLMRF-UTIIJYGPSA-N 0.000 description 4
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 description 4
- SGTNSNPWRIOYBX-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino}-2-(propan-2-yl)pentanenitrile Chemical compound C1=C(OC)C(OC)=CC=C1CCN(C)CCCC(C#N)(C(C)C)C1=CC=C(OC)C(OC)=C1 SGTNSNPWRIOYBX-UHFFFAOYSA-N 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 229940125797 compound 12 Drugs 0.000 description 4
- 229940126543 compound 14 Drugs 0.000 description 4
- 229940125758 compound 15 Drugs 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 239000012074 organic phase Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 210000001258 synovial membrane Anatomy 0.000 description 4
- 229960001722 verapamil Drugs 0.000 description 4
- 108091033409 CRISPR Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102100027581 Forkhead box protein P3 Human genes 0.000 description 3
- 101000861452 Homo sapiens Forkhead box protein P3 Proteins 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000003110 anti-inflammatory effect Effects 0.000 description 3
- 230000002917 arthritic effect Effects 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 229940125773 compound 10 Drugs 0.000 description 3
- 229940126214 compound 3 Drugs 0.000 description 3
- 229940125898 compound 5 Drugs 0.000 description 3
- 238000002591 computed tomography Methods 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 239000002552 dosage form Substances 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 238000003364 immunohistochemistry Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 229910052500 inorganic mineral Inorganic materials 0.000 description 3
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000011707 mineral Substances 0.000 description 3
- 235000010755 mineral Nutrition 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000003325 tomography Methods 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- QCBCPALLWXTPLW-SFHVURJKSA-N (2S)-2-(3,4-dihydroxyphenyl)-8,8-dimethyl-2,3,9,10-tetrahydropyrano[2,3-h]chromen-4-one Chemical compound C1([C@@H]2CC(=O)C=3C=CC4=C(C=3O2)CCC(O4)(C)C)=CC=C(O)C(O)=C1 QCBCPALLWXTPLW-SFHVURJKSA-N 0.000 description 2
- 101150010947 ABCB5 gene Proteins 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- 102000003777 Interleukin-1 beta Human genes 0.000 description 2
- 108090000193 Interleukin-1 beta Proteins 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000003435 antirheumatic agent Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 159000000011 group IA salts Chemical class 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000010212 intracellular staining Methods 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 150000004668 long chain fatty acids Chemical class 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 210000003289 regulatory T cell Anatomy 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- TUFFYSFVSYUHPA-UHFFFAOYSA-M rhodamine 123 Chemical compound [Cl-].COC(=O)C1=CC=CC=C1C1=C(C=CC(N)=C2)C2=[O+]C2=C1C=CC(N)=C2 TUFFYSFVSYUHPA-UHFFFAOYSA-M 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 210000005222 synovial tissue Anatomy 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 229960001603 tamoxifen Drugs 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- UVNPEUJXKZFWSJ-LMTQTHQJSA-N (R)-N-[(4S)-8-[6-amino-5-[(3,3-difluoro-2-oxo-1H-pyrrolo[2,3-b]pyridin-4-yl)sulfanyl]pyrazin-2-yl]-2-oxa-8-azaspiro[4.5]decan-4-yl]-2-methylpropane-2-sulfinamide Chemical compound CC(C)(C)[S@@](=O)N[C@@H]1COCC11CCN(CC1)c1cnc(Sc2ccnc3NC(=O)C(F)(F)c23)c(N)n1 UVNPEUJXKZFWSJ-LMTQTHQJSA-N 0.000 description 1
- OAVCWZUKQIEFGG-UHFFFAOYSA-O 2-(5-methyl-2H-tetrazol-1-ium-1-yl)-1,3-thiazole Chemical compound CC1=NN=N[NH+]1C1=NC=CS1 OAVCWZUKQIEFGG-UHFFFAOYSA-O 0.000 description 1
- WDBQJSCPCGTAFG-QHCPKHFHSA-N 4,4-difluoro-N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclohexane-1-carboxamide Chemical compound FC1(CCC(CC1)C(=O)N[C@@H](CCN1CCC(CC1)N1C(=NN=C1C)C(C)C)C=1C=NC=CC=1)F WDBQJSCPCGTAFG-QHCPKHFHSA-N 0.000 description 1
- BWGRDBSNKQABCB-UHFFFAOYSA-N 4,4-difluoro-N-[3-[3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-thiophen-2-ylpropyl]cyclohexane-1-carboxamide Chemical compound CC(C)C1=NN=C(C)N1C1CC2CCC(C1)N2CCC(NC(=O)C1CCC(F)(F)CC1)C1=CC=CS1 BWGRDBSNKQABCB-UHFFFAOYSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 102000041092 ABC transporter family Human genes 0.000 description 1
- 108091060858 ABC transporter family Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 229940123907 Disease modifying antirheumatic drug Drugs 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 206010060820 Joint injury Diseases 0.000 description 1
- 206010023232 Joint swelling Diseases 0.000 description 1
- 208000032912 Local swelling Diseases 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 206010048723 Multiple-drug resistance Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- NUGPIZCTELGDOS-QHCPKHFHSA-N N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclopentanecarboxamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CC[C@@H](C=1C=NC=CC=1)NC(=O)C1CCCC1)C NUGPIZCTELGDOS-QHCPKHFHSA-N 0.000 description 1
- LFZAGIJXANFPFN-UHFFFAOYSA-N N-[3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-thiophen-2-ylpropyl]acetamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CCC(C=1SC=CC=1)NC(C)=O)C LFZAGIJXANFPFN-UHFFFAOYSA-N 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 206010061363 Skeletal injury Diseases 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 208000036981 active tuberculosis Diseases 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000003356 anti-rheumatic effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 1
- JUMGSHROWPPKFX-UHFFFAOYSA-N brefeldin-A Natural products CC1CCCC=CC2(C)CC(O)CC2(C)C(O)C=CC(=O)O1 JUMGSHROWPPKFX-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000013077 scoring method Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000011537 solubilization buffer Substances 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000002437 synoviocyte Anatomy 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000011870 unpaired t-test Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/22—Bridged ring systems
- C07D221/28—Morphinans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/10—Spiro-condensed systems
- C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
Abstract
The invention discloses application of an ABCB5 inhibitor in preparation of a multi-drug resistance rheumatoid arthritis treatment drug, wherein the ABCB5 inhibitor comprises at least one of sinomenine and sinomenine derivatives. In the invention, the inventor discovers a novel sinomenine derivative, and discovers that the sinomenine derivative and sinomenine can be used for reversing the drug resistance of multi-drug resistance rheumatoid arthritis, and the sinomenine derivative has a higher multi-drug resistance rheumatoid arthritis treatment effect after being combined with methotrexate, and also discovers that the sinomenine derivative has a better treatment effect than sinomenine, and has excellent patent drug potential and application value.
Description
Technical Field
The invention belongs to the field of development of targeted therapeutic drugs, and particularly relates to application of an ABCB5 inhibitor in preparation of a multi-drug-resistance rheumatoid arthritis therapeutic drug.
Background
Rheumatoid Arthritis (RA) is a common autoimmune disease with about 3000 million patients worldwide. RA is a chronic disease, which can cause joint and bone injury, the disability rate reaches 30% -50%, and the disease is lingering and difficult to cure. The gradual loss of labor force and the decline of social participation can be caused after the illness, and the patient also needs to face the long-term medical expense expenditure, which brings great challenges to the life quality of the patient. Therefore, accurate early diagnosis (and rapid initiation of treatment) and rational treatment strategy design (careful control of inflammation, reduction or prevention of disease damage) are of great help to improve the condition and the standard of living of RA patients.
Non-steroidal anti-inflammatory drugs (NSAIDs), steroids and symptom-ameliorating antirheumatic drugs (DMARDs) are the three major RA interventions. However, these drugs have certain drawbacks, such as the use of DMARDs with some undesirable side effects. Moreover, the development of drug resistance is one of the main reasons that hinder the successful treatment of RA. Statistically, about 25% of RA patients have multiple drug resistance, and thus the use of drugs such as DMARDs has to be stopped. Among them, DMARDs represent the drug MTX because it is cheap and cheap. The American College of Rheumatology (ACR) and the european union of antirheumatics (EULAR) recommend the use of low doses of MTX for the treatment of patients newly diagnosed with RA. However, RA patients do not respond/develop resistance to MTX treatment. The rate was estimated to range from 30% to 50%. Drug efflux is one of the causes of MTX resistance, and is related to ABCB5 of the ABC transporter family.
Therefore, if a new drug can be developed to overcome the multidrug resistance of rheumatoid arthritis, it will be of great importance for the successful treatment of RA.
Disclosure of Invention
The present invention has been made to solve at least one of the above-mentioned problems occurring in the prior art. Therefore, the invention provides the application of the ABCB5 inhibitor in preparing a multi-drug resistant rheumatoid arthritis treatment drug. In the invention, the inventor discovers a sinomenine derivative, discovers that the sinomenine derivative has the function of inhibiting ABCB5 mediated MTX drug resistance reaction as well as sinomenine for the first time, and discovers that the sinomenine derivative can obviously improve multi-drug resistant rheumatoid arthritis through experiments, and the therapeutic activity of the sinomenine derivative is higher than that of sinomenine.
The invention provides a sinomenine derivative, 1. The sinomenine derivative is a compound formed by the substitution or nucleophilic acyl substitution reaction of a hydroxyl at the 4-position of sinomenine, and the structural formula of the sinomenine derivative is as follows:
or
Wherein in the formula I, R group is selected from hydrogen, substituted or unsubstituted anilino, nitrogen-containing alkane and imidazole;
or
No R group is present and the a position is linked to the C of the b position to form a ring, giving the structure described below:
in the formula II, R groups are selected from hydrogen and nitrile-containing alkane.
According to a first aspect of the invention, in some embodiments of the invention, in formula I, the R group is selected from the structures shown below:
according to a first aspect of the present invention, in some embodiments of the present invention, in said formula II, the R group is selected from nitrile-containing C1-8An alkane.
In some embodiments of the present invention, in formula II, the R group is selected from nitrile-containing C2-5An alkane.
In some embodiments of the invention, in formula II, the R group is selected from the structures shown below:
in some embodiments of the invention, the sinomenine derivative is specifically:
in the embodiment of the invention, the sinomenine derivative is prepared by reacting sinomenine and an amino compound under a specific environment, and compared with sinomenine, the sinomenine derivative has a stronger MTX (methyl thiazolyl tetrazolium) drug resistance reversal effect on rheumatoid arthritis, can be used for more remarkably treating diseases related to multi-drug resistance rheumatoid arthritis, and provides an excellent theoretical basis for development of related drugs for treatment or prevention of multi-drug resistance rheumatoid arthritis.
In a second aspect of the present invention, there is provided a method for preparing a sinomenine derivative as described in the first aspect of the present invention, comprising the steps of:
(1) Mixing sinomenine and amino compounds under the conditions of triethylamine and triphosgene or N, N' -carbonyl diimidazole to obtain the compound; or
(2) Mixing sinomenine and nitrile compound containing bromine under alkaline condition.
In some embodiments of the present invention, the reaction equation of the step shown in (1) can refer to the attached figures 9A and 9B of the specification. (2) The reaction equation of the steps shown in (A) can refer to the attached figure 9C of the specification.
According to a second aspect of the present invention, in some embodiments of the present invention, the amine-based compound is selected from aniline, 4-nitroaniline, 4-tert-butylaniline, 4-methanesulfonylaniline, 4-chloroaniline, benzylamine, n-pentylamine, n-hexylamine, n-heptylamine.
According to a second aspect of the present invention, in some embodiments of the present invention, the bromine-containing nitrile compound is selected from bromoacetonitrile, 4-bromobutyronitrile, 5-bromovaleronitrile.
In some embodiments of the present invention, the preparation of compounds 2-10 is performed by referring to the reaction equation in fig. 9A of the accompanying drawings, mixing triphosgene, sinomenine and triethylamine, stirring, and adding an amine compound.
In some embodiments of the invention, the amine-based compound is selected from the group consisting of aniline, 4-nitroaniline, 4-tert-butylaniline, 4-methanesulfonylaniline, 4-chloroaniline, benzylamine, n-pentylamine, n-hexylamine, n-heptylamine.
In some embodiments of the present invention, the preparation of compounds 11 and 12 is performed by referring to the reaction equation in fig. 9B of the accompanying drawings, and sinomenine, N' -carbonyldiimidazole and triethylamine are mixed and stirred.
In some embodiments of the present invention, the preparation of compounds 13-15 is performed by referring to the reaction equation in fig. 9C of the drawings, and sinomenine and a nitrile compound containing bromine are mixed under alkaline and dimethylformamide conditions.
In some embodiments of the invention, the alkaline conditions are established using a pH adjusting agent. The pH regulator is an alkaline pH regulator and comprises hydroxide, alkaline salt and alkaline organic matters. The alkaline salt comprises sodium carbonate, sodium bicarbonate, sodium monohydrogen phosphate and sodium phosphate. The alkaline organic matter comprises sodium alkoxide.
In some embodiments of the invention, the above reaction is accomplished in a specific solvent system.
In some embodiments of the invention, the solvent is dichloromethane or dimethylformamide.
In some embodiments of the invention, the molar ratio of sinomenine to amine-based compound is 1:0.1 to 20.
In some embodiments of the invention, the molar ratio of sinomenine to amine-based compound is 1:1 to 10.
In some embodiments of the present invention, the molar ratio of sinomenine to the bromine-containing nitrile compound is 1:0.5 to 2.
Of course, the person skilled in the art can also synthesize the sinomenine derivative according to the structural formula provided in the present invention by other methods, including but not limited to the above preparation methods.
In some embodiments of the present invention, the preparation method of sinomenine derivative further comprises extraction and purification.
In some embodiments of the invention, the reaction product is extracted with ethyl acetate, the combined organic phases are dried, filtered, concentrated, and purified by column chromatography.
In some embodiments of the present invention, the solvent in the chromatography column is dichloromethane and methanol, and the mixing volume ratio is 91 to 92.
Of course, the extraction and purification of the compound can be performed by other means according to the actual use requirement by those skilled in the art, including but not limited to the above method.
In a third aspect of the invention, the use of an ABCB5 inhibitor for the manufacture of a medicament for the treatment of multi-drug resistant rheumatoid arthritis is provided.
According to a first aspect of the invention, in some embodiments of the invention, the ABCB5 inhibitor comprises at least one of sinomenine, a sinomenine derivative according to the first aspect of the invention.
In some embodiments of the present invention, the sinomenine derivative is a compound formed by substitution or nucleophilic acyl substitution of the hydroxyl group at the 4-position of sinomenine.
In some embodiments of the present invention, the structural formula of the sinomenine derivative is:
or
Wherein in the formula I, R groups are selected from hydrogen, substituted or unsubstituted anilino, nitrogen-containing alkane and imidazole;
or
No R group is present and the a position is linked to the C of the b position to form a ring, giving the structure described below:
in the formula II, R groups are selected from hydrogen and nitrile-containing alkane.
In the embodiment of the invention, the inventor verifies the treatment effect of the ABCB5 inhibitor on multi-drug-resistance rheumatoid arthritis through cell level and animal experiments respectively, discovers that sinomenine and sinomenine derivatives have the effect of reversing the drug resistance of the multi-drug-resistance rheumatoid arthritis for the first time, and particularly discovers that the sinomenine derivatives with improved sinomenine configuration show more excellent treatment effect than sinomenine in the experiments, thereby providing favorable pharmacological basis and potential patent drug development reference for the treatment of the multi-drug-resistance rheumatoid arthritis.
In some embodiments of the invention, the multi-drug resistant rheumatoid arthritis is methotrexate resistant rheumatoid arthritis.
The inventor finds that the MTX drug resistance of rheumatoid arthritis is mainly based on ABCB5 transporter mediated MTX efflux, thereby causing the drug resistance reaction of MTX. However, no effective medicine can reverse the MTX resistance of the rheumatoid arthritis, but the invention firstly proposes that the use of the sinomenine and sinomenine derivatives can effectively inhibit ABCB5, thereby effectively overcoming the external discharge of MTX and further inhibiting the MTX resistance of the rheumatoid arthritis.
In a fourth aspect of the invention, the use of an ABCB5 inhibitor in combination with methotrexate for the manufacture of a medicament for the treatment of multi-drug resistant rheumatoid arthritis is provided.
According to a second aspect of the invention, in some embodiments of the invention, the ABCB5 inhibitor comprises at least one of sinomenine, a sinomenine derivative according to the first aspect of the invention.
In some embodiments of the present invention, the sinomenine derivative is a compound formed by substitution or nucleophilic acyl substitution of the hydroxyl group at the 4-position of sinomenine.
In some embodiments of the present invention, the structural formula of the sinomenine derivative is:
or
Wherein in the formula I, R groups are selected from hydrogen, substituted or unsubstituted anilino, nitrogen-containing alkane and imidazole;
or
No R group is present and the a position is linked to the C of the b position to form a ring, giving the structure described below:
in the formula II, R groups are selected from hydrogen and nitrile-containing alkane.
In the embodiment of the invention, the inventor verifies the treatment effect of the ABCB5 inhibitor on multi-drug-resistance rheumatoid arthritis through cell level and animal experiments respectively, discovers that sinomenine and sinomenine derivatives have the effect of reversing the drug resistance of the multi-drug-resistance rheumatoid arthritis for the first time, and particularly discovers that the sinomenine derivatives with improved sinomenine configuration show more excellent treatment effect than sinomenine in the experiments, thereby providing favorable pharmacological basis and potential patent drug development reference for the treatment of the multi-drug-resistance rheumatoid arthritis.
In some embodiments of the invention, the multi-drug resistant rheumatoid arthritis is methotrexate resistant rheumatoid arthritis.
The inventor finds that the MTX drug resistance of rheumatoid arthritis is mainly based on ABCB5 transporter mediated MTX efflux, thereby causing the drug resistance reaction of MTX. However, no effective medicine can reverse the MTX resistance of rheumatoid arthritis, but the invention firstly proposes that the use of sinomenine and sinomenine derivatives can effectively inhibit ABCB5, thereby effectively overcoming the discharge of MTX. Therefore, the sinomenine and the sinomenine derivative are used as ABCB5 inhibitors and are combined with methotrexate to be applied to the treatment of multi-drug resistant rheumatoid arthritis, so that the drug resistance reaction of MTX can be effectively reversed, and the anti-inflammatory effect of MTX can be recovered.
In a fifth aspect of the present invention, there is provided a medicament for treating multi-drug resistant rheumatoid arthritis, the medicament comprising sinomenine and at least one of the sinomenine derivatives of the first aspect of the present invention.
In some embodiments of the present invention, the sinomenine derivative is a compound formed by substitution or nucleophilic acyl substitution of the hydroxyl group at the 4-position of sinomenine. The structural formula of the sinomenine derivative is as follows:
or
Wherein in the formula I, R groups are selected from hydrogen, substituted or unsubstituted anilino, nitrogen-containing alkane and imidazole;
or
Does not contain an R group, and the a position is connected with the C at the b position to form a ring, so that the following structure is obtained:
in the formula II, R groups are selected from hydrogen and nitrile-containing alkane.
In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
In some embodiments of the invention, the excipient comprises at least one of a solvent, wetting agent, emulsifier, thickener, excipient, suspending agent, disintegrant, filler, lubricant, or diluent.
Of course, those skilled in the art can reasonably add other adjuvants according to the actual use requirement to achieve the technical effect brought by the specific adjuvants, which include but are not limited to the above-mentioned solvents, wetting agents, emulsifiers, thickeners, excipients, suspending agents, disintegrating agents, fillers, lubricants or diluents.
In some embodiments of the invention, the dosage form of the medicament comprises at least one of powder, ampoules, solutions, tablets or capsules.
Of course, those skilled in the art can select other dosage forms according to the actual use requirement, so as to achieve the drug release or therapeutic effect at a specific position or a specific purpose, and the dosage forms include, but are not limited to, the above-mentioned powder, injection, solution, tablet or capsule.
In some embodiments of the invention, the medicament comprises a therapeutically effective amount of sinomenine, a sinomenine derivative, or a combination of both.
In some embodiments of the invention, the therapeutically effective amount of sinomenine is greater than or equal to 100mg/kg/Day.
In some embodiments of the invention, the therapeutically effective amount of the sinomenine derivative is greater than or equal to 30mg/kg/Day.
In the embodiment of the invention, the inventor verifies through experiments that the sinomenine derivative can achieve better MTX drug resistance reversal effect of rheumatoid arthritis at the use amount of 30mg/kg/Day, and even has stronger treatment efficacy compared with sinomenine of 100mg/kg/Day in certain aspects.
The beneficial effects of the invention are:
1. the sinomenine derivative is developed for the first time and found to have excellent multi-drug resistance rheumatoid arthritis treatment effect, no obvious side effect on test animals and high safety.
2. The preparation method of the sinomenine derivative is simple to operate, mild in reaction conditions, high in relative yield and capable of achieving an effective yield of 80%, and excessive impurities are not introduced.
3. The invention discovers for the first time that sinomenine and sinomenine derivatives can inhibit the release of MTX mediated by ABCB5 transporter, thereby reversing the drug resistance of multi-drug resistance rheumatoid arthritis excellently, and after being used together with MTX, the sinomenine and sinomenine derivatives show extremely strong inhibitory activity and therapeutic effect on cell and animal model levels, thereby playing an extremely important role in the development and development of the treatment drugs and treatment schemes of multi-drug resistance rheumatoid arthritis, and really widening the development and research directions of the multi-drug resistance rheumatoid arthritis drugs.
Drawings
FIG. 1 shows the construction of a RAFLS-resistant cell line with high ABCB5 expression and the detection of the drug efflux function of the RAFLS-resistant cell by flow cytometry, wherein (A) is the analysis of protein bands expressed by ABCB1 or ABCB5 in transient transfected RAFLS cells by an immunoblotting method; (B) Is an efflux flow cytometry map of rhodamine 123 (Rho 123) or MTX-FITC in ABCB1 and ABCB5 overexpressed RAFLS; (C) Bar graphs of efflux effect of Rho123 or MTX-FITC in ABCB1 and ABCB5 overexpressed RAFLS, data shown in the bar graphs were normalized to controls transfected with empty vector.
FIG. 2 is a flow cytometry graph (A) of Rho123 efflux by RA-FLS of sinomenine-inhibited high-expression ABCB5 and a flow bar graph (B) of Rho123 efflux by RA-FLS of sinomenine-inhibited high-expression ABCB5, verapamil is a positive control; wherein the data shown in the bar graph in (B) is normalized with a control that is not transfected with an empty vector.
FIG. 3 shows MTT assay verification that sinomenine reverses the drug resistance response of RA-FLS (A) and Jurkat cells (B) highly expressing ABCB5 to methotrexate.
FIG. 4 is a schematic diagram of the construction of a joint cavity injection ABCB5 adenovirus mediated drug resistant AIA rat model.
FIG. 5 is a DAB staining method for immunohistochemistry to study the expression level of ABCB5 in synovium of ABCB5 adenovirus-mediated drug-resistant AIA rats, wherein (A) is DAB staining graph of articular cavity synovium, and (B) is histogram of statistical analysis result of immunohistochemical staining score.
FIG. 6 is a graph of the effect of sinomenine in combination with methotrexate on foot swelling in drug-resistant AIA rats: (A) SD rat foot photograph (B) changes in foot volume of SD rat.
FIG. 7 is MicroCT analysis of bone destruction in drug-resistant AIA rats: the method comprises the following steps of (A) recovering a foot microcomputer tomography image of an SD rat, indicating a remarkable bone destruction site by a yellow arrow, (B) obtaining a histogram of a foot bone destruction score statistical analysis result of the SD rat, and (C) obtaining an evaluation table of the foot bone destruction degree of the SD rat.
FIG. 8 shows the expression of inflammation-associated cytokines in SD rat blood samples measured by RT-PCR, wherein the results were normalized using healthy groups as controls.
FIG. 9 shows the reaction equations of sinomenine derivatives in examples of the present invention, in which (A) is the reaction equations of compounds 2 to 10, (B) is the reaction equations of compounds 11 to 12, and (C) is the reaction equations of compounds 13 to 15.
FIG. 10 is a graph comparing the effect of various concentrations of sinomenine on efflux MTX-FITC function in ABCB5.
FIG. 11 is a graph comparing the effect of various concentrations of sinomenine on efflux MTX-FITC function of ABCB5 and its mutants, wherein (A), (B) and (C) are sinomenine at 100, 50 and 20 μ M in sequence.
FIG. 12 is a graph comparing the effect of sinomenine derivatives (compounds 2-8) on the efflux MTX-FITC function of ABCB5 and its mutants.
FIG. 13 is a graph comparing the effect of sinomenine derivatives (compounds 9-15) on efflux MTX-FITC function of ABCB5 and its mutants.
FIG. 14 shows a plasmid map (A) of a gene vector containing { CAG-loxP-stop-loxP-RatABCB5 CDs-SV40 late pA } and a schematic diagram (B) of the construction of the target; .
FIG. 15 is a plasmid map of Cre-ERT2 gene vector in an example of the present invention.
FIG. 16 is a comparison of the improvement effect of sinomenine and its derivatives in combination with methotrexate on the foot swelling of drug-resistant rheumatoid arthritis transgenic rats, wherein (A) is a photograph of the foot swelling of SD rats; (B) Is a line graph of the statistical analysis result of the change condition of the foot volume of the SD rat.
FIG. 17 shows the expression level of ABCB5 mRNA in rat blood samples measured by qPCR.
FIG. 18 shows the results of MicroCT detection of sinomenine and its derivatives in combination with methotrexate on drug-resistant rheumatoid arthritis transgenic rats, wherein (A) is a picture of recovery of foot bone structure by computerized tomography, (B) is a histogram of statistical analysis results of bone destruction scores, and (C) is a score comparison table.
FIG. 19 is a histogram of erythrocyte sedimentation rate analysis results of transgenic animal experiments combining sinomenine and derivatives thereof with methotrexate for treating drug-resistant rheumatoid arthritis.
FIG. 20 is a flow cytogram of fluorescent staining of FoxP3 in rat CD4+ cells in experiments on transgenic animals with resistance to drug-resistant rheumatoid arthritis by sinomenine and derivatives thereof in combination with methotrexate.
FIG. 21 is a flow cytogram of fluorescent staining of IL-17A in rat CD4+ cells in experiments on transgenic animals with resistance to drug-resistant rheumatoid arthritis treated by sinomenine and derivatives thereof in combination with methotrexate.
FIG. 22 is a histogram of statistical analysis of flow-type fluorescence signal detection of FOXP3 and IL-17A in rat CD4+ cells in a transgenic animal experiment of combining sinomenine and derivatives thereof with methotrexate treatment for drug-resistant rheumatoid arthritis, wherein (A) is FOXP3+Total CD4 of T cells+Histogram of percentage of cells; (B) Is IL-17A+Total CD4 of T cells+Histogram of percentage of cells; (C) Is CD4+The ratio of FOXP3 to IL-17 in cells reflects the equilibrium.
FIG. 23 is a heat map of the increase ratio of various inflammatory factors in rat serum samples in experiments on transgenic animals with sinomenine and derivatives thereof (compound 2, C2) in combination with methotrexate for treatment of drug-resistant rheumatoid arthritis.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are given by way of illustration only.
The test materials and reagents used are, unless otherwise specified, all consumables and reagents which are customary and commercially available.
Example 1 construction of ABCB5 efflux function study cell platform Using RAFLS-resistant cells highly expressing ABCB5
The ABCB5 sequence was synthesized with reference to NCBI AY230001.1 (the synthesis procedure was entrusted to Nanjing Genscript Biotech Inc.). BamH I was added to the 5 'end of the synthetic ABCB5 sequence and EcoR I restriction sites were added to the 3' end. The pcDNA 3.1-mRFP-ABCB5 plasmid was cloned by BamH I and EcoR I restriction enzymes. Single clones were verified by double digestion with BamH I and EcoR I and sequencing. Using liposomes (
3000, invitrogen) transfection reagent ABCB5 recombinant plasmid was transferred into rheumatoid arthritis joint fibroblast-like synoviocytes (RAFLS) and seeded in 6-well plates with 10 cells per well of RAFLS6And (4) respectively. After confluency, 5. Mu.g/mL Rho123 (Sigma-Aldrich, R8004) or 0.5. Mu.M MTX-FITC (Invitrogen, M1198 MP) was added to each well and incubated at 37 ℃ for 1h. Cells were washed 5 times with pre-chilled PBS to stop the accumulation of Rho 123. Cells were then resuspended in 400. Mu.LPBS for flow cytometry analysis. Intracellular fluorescence was measured using a flow cytometer at 488nm excitation wavelength and 525nm emission wavelength. Data acquisition and analysis were both performed using Cell Quest (BD Biosciences, san jose, CA, USA), and at least three independent experiments were performed for all experiments. The results are shown as the average of the fluorescence intensities.
Meanwhile, RAFLS cells partially transfected with ABCB1 (for reference, the transfection method is the same as ABCB 5) or ABCB5 recombinant plasmid were treated with RIPA lysate (CST, 9806). Protein concentrations were then determined using the Bio-Rad protein assay (Bio-Rad Laboratories, inc., hercules, calif., USA).
The detection procedure was identical to ABCB5 with ABCB1 as a control. Wherein, the sequence of ABCB1 refers to NCBI M14758.1.
The results are shown in FIG. 1.
Western Blot results show that ABCB1 and ABCB5 are successfully and highly expressed in cells. The efflux transport function of ABCB5 high-expression drug-resistant cell line drugs is normal by using Rho123, and the result shows that Rho123 is effectively excreted from the RAFLS overexpressed by the ABCB5, which indicates that substrates can be normally transported after the ABCB5 transfection is successful. While fluorescein-linked MTX was used for testing drug efflux activity in flow cytometry analysis. Since MTX is a substrate for ABCB5, MTX-FITC will accumulate intracellularly without transient expression of ABCB5. On the basis of the constructed ABCB5 high-expression drug-resistant cell line, the absorption and accumulation of MTX-FITC in RAFLS are obviously reduced due to the overexpression of ABCB5 in the cells, so that the fact that the ABCB5 with high expression in the cells can cause the drug MTX to be discharged outside can be shown.
Example 2 screening of ABCB5 inhibitors Using the efflux of rhodamine 123 (Rho 123) in ABCB5 highly expressed RAFLS cells
Based on the methods in the above examples, ABCB5 recombinant plasmids were transferred into RAFLS cells using lipofectamine.
The method comprises the following specific steps: inoculate 10 per well in 6-well plates3Recombinant RAFLS cells, after overnight culture. The ABCB5 recombinant plasmid was transferred into RAFLS cells using a lipofectamine, followed by exposure of the cells to different test drugs (final concentrations of 10. Mu.M Verapamil (Verapamul) or 100. Mu.M Sinomenine (Sinomenine), respectively) and then contained 5% CO at 37 ℃2And incubated for 24 hours. Rho123 was added at a final concentration of 5.0. Mu.g/mL and incubated at 37 ℃ for 1 hour. Washing the cells 5 times with pre-cooled PBS stops the accumulation of Rho 123. Cells were then resuspended in 400. Mu.LPBS for flow cytometry analysis. Intracellular fluorescence was measured using a flow cytometer at 488nm excitation wavelength and 525nm emission wavelength. Recombinant RAFLS exposed to verapamil after transfection was used as a positive control, untreated recombinant RAFLS cells after transfection were used as blank negative controls, and the mean values of control groups transfected with empty vector were used for normalization.
The results are shown in FIG. 2. Since ABCB5 mediated MTX efflux phenomenon is a very large resistance in the treatment of RA, it causes patients to show resistance to MTX. Under normal conditions, rho123 forms a signal accumulation after entering cells. After ABCB5 is highly expressed, rho123 is greatly discharged without drug intervention, so that the fluorescence signal is reduced. In contrast, when 100 μ M sinomenine is used for treating cells, the efflux phenomenon can be obviously inhibited, and the combination of the positive control verapamil shows that sinomenine can effectively inhibit the efflux of ABCB5 on the MTX.
Example 3 Sinomenine reverses the MTX-resistant response of highly expressed ABCB5 RAFLS-resistant cells
The ABCB5 recombinant plasmid was transferred into RAFLS and Jurkat cells using lipofectamine according to the method in example 2. Inoculate 4X 10 per well in 96-well plates3The ABCB 5-transfected RAFLS cells or Jurkat cells were cultured overnight and then 100. Mu.L of sinomenine was added at various concentrations (final concentrations of 100, 50 and 10. Mu.M), followed byThe cells were then exposed to different concentrations (0.039-100. Mu. Mol/L, DMSO dilution) of MTX for 72 hours (for Jurkat cells, the corresponding concentrations of sinomenine and different concentrations of MTX were given at the same time as seeding). At the end of the incubation, 10. Mu.L of 5.0mg/mL MTT was added to each well and incubated at 37 ℃ for 4 hours, followed by 100. Mu.L of solubilization buffer (0.01 mol/LHCl with 10% sodium dodecyl sulfate) and incubated overnight. The absorbance at 570nm (A570 nm) was measured for each well the following day. The control group was RAFLS cells or Jurkat cells without MTX treatment and the blank group was cell-free seeded.
And calculating the cell activity according to the absorbance, wherein the calculation formula is as follows:
wherein A treatment represents the A570nm value of the wells of RAFLS/Jurkat cells after MTX treatment; a control represents the A570nm values for RAFLS/Jurkat cell wells without MTX treatment; the A blank indicates the A570nm value for the cell-free well.
The results are shown in FIG. 3. Overexpression of ABCB5 increased the required dose of MTX. Whereas, following the intervention with 100 μ M sinomenine, the cytotoxicity of RAFLS by MTX was restored. And the activity of sinomenine shows dose dependence, which shows that the recovery effect of low-concentration sinomenine on the dosage of MTX is weakened.
In conclusion, the inventors found that sinomenine can be used as a potential ABCB5 inhibitor and shows excellent MTX resistance reversal activity in an in vitro test in a dose-dependent manner.
Example 4 therapeutic Effect of Sinomenine on MTX-resistant arthritis rat model
(1) Construction of adenovirus-mediated ABCB5 high-expression arthritis rat model:
in this example, the test rats used were male Sprague Dawley (SD) rats of 5 weeks old, purchased from the center of medical laboratory animals of Guangdong province, and weighing 80-120g. The breeding environment is as follows: in a room equipped with temperature control and an automatic ventilation system, the room was cycled 12 hours light/dark and was free to drink and drink. This example was approved by the animal ethics committee of the special administrative district health office of australia, china, and was conducted according to the guidelines of the animal care and user committee of the university of australia technologies.
General arthritis model: rat arthritis was induced using complete Freund's adjuvant (CFA, CAS: 9007-81-2) with the following specific steps: the first inflammation occurred about day 9 after CFA injection by intradermal injection of 100 μ LCFA into rat tail root. The rat paw volume was measured and recorded every three days.
High expression ABCB5 arthritis model: rat arthritis was induced using complete Freund's adjuvant (CFA, CAS: 9007-81-2), ABCB5 adenovirus (pAV [ Exp ] -CMV > { rABCB5[ XM _006225905.2] (ns) }: T2A: EGFP), vector construction and viral packaging was done by Seike Biotech, inc., product ID: AVP-VB180424-1073gvh, wherein the NCBI number of the ABCB5 gene inserted into the vector is XM _006225905.2. The method comprises the following specific steps: rat caudate was injected intradermally with 100 μ LCFA, and then 100 μ L of the above ABCB5 adenovirus was injected into rat articular cavities using a microsyringe within the day.
A total of 48 male rats were selected and randomly divided into 8 experimental groups of 6 rats per group according to Table 1.
TABLE 1
At the end of the treatment period (day 30 after CFA injection), rats were sacrificed, blood and part of the organs were collected, the right hind paw was photographed and frozen, and the left hind paw was amputated and fixed in 4% Paraformaldehyde (PFA).
A micro computer tomography (MicroCT) is used for analyzing the foot bone destruction condition of a rat, and the method comprises the following specific steps: the left hind paw was scanned using an in vivo mini CT scanner ((SkyScan 1176, bruker, belgium) with an Al 1mm filter, wherein the scan parameters were 35 micron resolution, 62kv,385 μ a,98ms exposure time, angular velocity 0.70, image reconstruction with nreco software after scan end, CTvox software for opening reconstructed data file and generating observable 3-dimensional picture, CTAn software analyzing scan data.
MicroCT scoring is performed based on five disease-related indicators examined by MicroCT (bone mineral density, bone volume fraction, cortical mineral density, trabecular number and total porosity), and the calculation formula of MicroCT scoring is as follows:
or
The MicroCT score is the average value of the sum of the five disease related index values processed by the method.
Meanwhile, the expression condition of the cytokines relevant to the blood inflammation of the rat is analyzed by using real-time fluorescence quantitative PCR, wherein the cytokines relevant to the inflammation are IL-1 beta, IL-6, TNF-alpha and IL-2.
The specific operation is as follows: the collected rat blood was lysed with 300-400 μ L of a ten-fold volume of erythrocyte lysate (Byunyian, C3702) on ice for 10 minutes, centrifuged at 500g at 4 ℃ for 5min, and the red supernatant was discarded. If the lysis of the erythrocytes is found to be incomplete, the lysis can be repeated. After lysis was complete, the cells were washed with PBS. After removal of red Blood cells, total RNA was extracted using the FavorPrepTM Blood/filtered Cell Total RNA Mini Kit (FAVORGEN, FABRK 001-2) according to the protocol. Taking 1 mu g of total RNA obtained by extraction, and carrying out reverse transcription to obtain cDNA. Using PerfectStart with cDNA obtained by reverse transcription as a templateTMGreen Qpcr SuperMix (Transgen, AQ 601) and designed specific primers were used for RT-PCR amplification. Gene expression levels were normalized to Actin (control) and 2 was used-ΔΔCTThe method is used for analysis. For each primer, 3 independent experiments were analyzed, with 3 replicates in each group.All data were statistically analyzed using unpaired t-test.
The specific primer sequences of the target genes are shown in Table 2.
TABLE 2
Meanwhile, the inventor also carries out immunohistochemical analysis (DAB staining) on ABCB5 protein on synovial membranes of rat articular cavities, and the specific steps are as follows: and decalcifying the left hind leg joint of the rat fixed by PFA, embedding paraffin, and slicing to prepare a rat articular cavity synovial paraffin slice. Dewaxing was then carried out with xylene for 28 minutes by passing different concentrations of ethanol and water, followed by antigen Retrieval at 97 ℃ for 20 minutes using a Target Retrieval Solution 50 × Low pH (Dako Envison FLEX, lot 20080033). ABCB5 antibody incubation and DAB staining were then performed using the Envision FLEX + Visualization Systems (Dako K8002) (see instructions for details), and the treated sections were photographed under a 40X microscope. And (3) carrying out immunohistochemical scoring on the positive areas in the photos, wherein the specific scoring method comprises the following steps: a positive reaction is defined as showing a brown signal in the picture. The staining index (range of values 0-12) was then determined by multiplying the fraction of staining intensity with the fraction of positive areas. Wherein the score judgment standard of the dyeing intensity is as follows: staining negative, score 0; the dyeing intensity is weak, 1 point; the dyeing intensity is medium, 2 points; the staining intensity is strong, 3 points. The fraction of the positive area is judged according to the positive cell proportion, and the specific standard is as follows: the proportion of positive cells is less than 5 percent, and 0 point is obtained; 5 to 25 percent, 1 minute; 26% to 50%,2 min; 51 to 75 percent and 3 minutes; greater than 75% and 4 min.
Example of calculation: one specimen containing 75% tumor cells, with moderate staining intensity scored 3 × 2=6, while the other 25% are negative cells, with weaker staining intensity scored 1 × 1=1, with a final score of 6+1=7.
In the statistical analysis, scores 0-7 were considered low expression and scores 8-12 were considered high expression.
The results are shown in FIGS. 4-8.
FIG. 4 is a schematic diagram of rat model construction.
FIG. 5 is a DAB staining immunohistochemistry result of articular cavity synovial tissue, and it was revealed from the photograph of DAB staining immunohistochemistry (brown is a positive signal) that substantially no positive signal appeared in the Healthy Ctrl, AIA model group, MTX and SIN treatment groups in the case of injection of only adenovirus empty vector in the adenovirus-mediated high expression ABCB5 arthritis rat model, which is shown as a blue background. And positive reactions appear in the ABCB5 group, the ABCB5+ MTX group, the ABCB5+ SIN group and the ABCB5+ MTX + SIN group after being infected by the ABCB5 high-expression adenovirus, which indicates that the adenovirus successfully mediates the high expression of the ABCB5 transporter in the synovial tissue of the joint cavity. At the same time, the results of the evaluation of the staining index based on the positive signals of immunohistochemical photographs also demonstrated that the ABCB5 transporter was highly expressed in the synovial membranes of the ABCB5 group, ABCB5+ MTX group, ABCB5+ SIN group and ABCB5+ MTX + SIN group.
The right hind paw of the rat is photographed before the animal is drawn, so that the foot of the rat in the AIA model group is found to be swollen, and the success of modeling is indicated. After receiving 2mg/kg/WeekMTX or 100mg/kg/Day treatment, the foot swelling of the rats was reduced. However, the action effect of the two anti-arthritis drugs is remarkably weakened under the condition of high expression of ABCB5, and the feet of rats still have severe swelling in the ABCB5+ MTX group and the ABCB5+ SIN group. However, MTX combined with SIN has a good treatment effect on rats with high-expression ABCB5 arthritis, and the feet of the combined drug group do not swell obviously. Meanwhile, the quantification of the foot swelling condition of the rat is realized based on a volume measuring instrument. By continuously recording the foot volume, a foot volume change curve (figure 6) is formed, and the improvement effect on the foot volume is obviously weakened when MTX and SIN are used alone in the presence of ABCB5 transporter, and the foot swelling condition of an arthritic rat is obviously reduced when the MTX and SIN are used in combination, so that the development condition of drug-resistant RA can be obviously improved by the MTX and SIN combined drug.
After the left hind paw of the rat model is reconstructed by Micro CT scanning, the foot bone tissue is photographed by software, and the actual bone destruction condition of the rat foot of different experimental groups can be intuitively observed (figure 7). It can be found that the feet of the rats in the healthy group have compact bones and complete structures. After AIA modeling, bone destruction occurred in the feet of the general arthritic rats (yellow arrows indicate the location of bone destruction). In this regard, MTX or SIN alone reduced bone destruction in the foot, but the intervention of ABCB5 significantly aggravated the site and severity of bone destruction. In this case, the improvement effect of MTX or SIN alone is completely lost due to ABCB5 overexpression, and the effect of reducing the destruction of the foot bones cannot be obtained. In the combination group, the rat model maintained substantially intact foot bone structure with fewer bone destruction sites in the case of ABCB5 overexpression. Meanwhile, bone destruction is quantified by means of Micro CT analysis software, 5 quantified bone parameters are normalized and then averaged to obtain a mean value, namely a Micro CT score (figure 7B), after ABCB5 is highly expressed, treatment of a single drug cannot relieve bone destruction of rats, and the combined use of MTX and SIN has a good protective effect on foot bones, and the bone score is close to that of a healthy group. Comparing the bone destruction rating table (fig. 7C), it can be found that the MTX or SIN single treatment group with ABCB5 high expression was rated as severe bone destruction, and the combination group was slightly bone destruction, indicating that the degree of bone destruction caused by drug-resistant RA due to ABCB5 high expression can be effectively improved only when the combination was used.
FIG. 8 shows the expression of the related inflammatory factors IL-1 β, IL-2, IL-6, TNF- α in rat model, which reflects the severity of inflammation in rats. According to the real-time fluorescent quantitative PCR result, the four inflammatory factors are found to be positively correlated with the inflammatory reaction of the rat. After AIA modeling, the expression of 4 inflammatory factors is increased, the expression of related inflammatory factors is reduced by MTX treatment, but the treatment effect of MTX is obviously reduced under the intervention condition of ABCB5. However, in the combination group of the inverse combination, the expression of 4 inflammatory factors is obviously inhibited. It shows that the inflammatory reaction caused by drug-resistant RA due to ABCB5 high expression can be effectively improved only when the drug combination is used.
In conclusion, the ABCB5 high-expression drug-resistant arthritis rat model constructed in the embodiment can find that the high expression of the ABCB5 transport protein can cause MTX drug-resistant reaction of the arthritis rat model, and mainly shows that the MTX has reduced inhibition effect on foot swelling, bone destruction and inflammatory factor expression and loses therapeutic activity. The combined use of sinomenine obviously reverses the drug resistance reaction of MTX, provides a new application of sinomenine in treating rheumatoid arthritis, namely the sinomenine is used as an inhibitor of ABCB5 transport protein to be applied to the clinical treatment of drug-resistant rheumatoid arthritis.
Example 5 Synthesis of Sinomenine derivatives
1. Synthesis of Compound 2:
aniline and the 4-hydroxy group of sinomenine (a compound having a structure shown in formula 1, available from douruifeng biotechnology limited) are connected to obtain the following sinomenine derivative (a compound having a structure shown in formula 2):
the preparation method comprises the following steps:
the reaction equation is shown in FIG. 9A. 2.0g of triphosgene was dissolved in 134mL of anhydrous dichloromethane, and 1.5g of sinomenine (compound represented by formula 1) and 3.8mL of triethylamine solution (dissolved in anhydrous dichloromethane, concentration: 0.4M) were slowly added dropwise while cooling on ice, followed by stirring and reaction for 1 hour. 0.65mL of aniline was added and the reaction was allowed to warm to room temperature. TLC was used to monitor until the reaction was complete. The reaction mixture was extracted 3 times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (dichloromethane: methanol = 92) to isolate and purify compound2 (i.e., sinomenine derivative) with a yield of 80%.
Compound2 was characterized with the following specific results:
1H NMR(600MHz,CDCl3)δ7.52(d,J＝7.8Hz,2H),7.31(t,J＝8.0Hz,2H),7.07(t,J＝7.4Hz,1H),6.92 (d,J＝8.4Hz,1H),6.79(d,J＝8.5Hz,1H),5.50(s,1H),3.94(d,J＝15.5Hz,1H),3.73(s,3H),3.48(s,3H), 3.36–3.33(m,1H),3.22–3.19(m,1H),3.07(d,J＝18.5Hz,1H),2.87(dd,J＝20.4,5.3Hz,1H),2.68(dd,J＝ 12.2,3.1Hz,1H),2.56(d,J＝15.1Hz,1H),2.52(s,3H),2.31–2.27(m,1H),2.03–1.98(m,1H),1.81(d,J＝ 12.6Hz,1H)ppm；
13C NMR(150MHz,CDCl3)δ192.2,152.8,151.7,150.2,139.6,137.6,129.3,129.1,125.9,123.9,122.0, 118.8,112.0,110.1,57.9,56.2,56.2,55.3,55.1,53.5,48.8,47.5,41.3,41.2,39.6,24.6ppm；HRMS-ESI(m/z) Calcd for C26H29N2O5[M+H]+:449.2071；Found 449.2084.。
2. synthesis of Compound 3:
the structural formula of the compound 3 is shown as a formula 3.
The preparation method comprises the following steps:
the reaction equation is shown in fig. 9A, and the preparation method steps are the same as compound2, except that: sinomenine (204.7 mg) was reacted with triphosgene (262.3 mg), triethylamine (0.37 mL) and 4-nitroaniline (132.4 mg) to give compound 3 in 41% yield.
1H NMR(600MHz,CDCl3)δ9.58(s,1H),8.02(d,J＝8.9Hz,2H),7.58(d,J＝9.1Hz,2H),6.91(d,J＝8.5 Hz,1H),6.63(d,J＝8.4Hz,1H),5.62(d,J＝1.4Hz,1H),3.94–3.88(m,1H),3.58(s,3H),3.56(s,3H),3.23– 3.20(m,1H),3.09(d,J＝18.4Hz,1H),3.05(s,1H),2.74(dd,J＝18.2,5.5Hz,1H),2.62(d,J＝15.7Hz,1H), 2.54(dd,J＝12.2,3.1Hz,1H),2.45(s,3H),2.20–2.13(m,1H),1.93(td,J＝12.5,4.7Hz,1H),1.73(d,J＝12.4 Hz,1H)ppm；
13C NMR(150MHz,CDCl3)δ195.0,152.5,150.7,150.2,144.5,142.8,138.6,130.1,129.8,125.7,124.9, 117.9,116.7,110.6,56.2,55.7,55.0,50.8,46.5,46.4,42.8,41.4,37.3,24.1ppm；HRMS-ESI(m/z)Calcd for C26H27N3O7[M+H]+:494.1922；Found494.1919.。
3. synthesis of Compound 4:
the structural formula of the compound 4 is shown as a formula 4.
The preparation method comprises the following steps:
the reaction equation is shown in fig. 9A, and the preparation method steps are the same as compound2, except that: sinomenine (132.6 mg) was reacted with triphosgene (174.9 mg), triethylamine (0.25 mL) and 4-tert-butylaniline (100. Mu.L) to give compound 4 in 57% yield.
1H NMR(600MHz,CDCl3)δ8.17(s,1H),7.46–7.40(m,2H),7.32(d,J＝8.6Hz,2H),6.91(d,J＝8.4Hz, 1H),6.76(d,J＝8.5Hz,1H),5.50(d,J＝1.2Hz,1H),3.94(d,J＝15.7Hz,1H),3.71(s,3H),3.48(d,J＝5.3Hz, 3H),3.29–3.24(m,1H),3.11(s,1H),3.07(d,J＝18.5Hz,1H),2.79(dd,J＝18.3,5.4Hz,1H),2.60(dd,J＝ 12.3,2.9Hz,1H),2.55(d,J＝15.7Hz,1H),2.48(s,3H),2.22(td,J＝12.3,3.0Hz,1H),1.92(td,J＝12.6,4.5Hz, 1H),1.81(d,J＝12.4Hz,1H),1.30(s,9H)ppm；
13C NMR(150MHz,CDCl3)δ193.7,152.5,151.0,150.9,146.4,139.4,135.4,130.3,129.2,125.8,125.3, 118.2,115.3,111.0,56.5,56.0,54.9,50.2,46.8,45.8,42.5,40.9,36.8,34.3,31.4,24.3ppm；HRMS-ESI(m/z) Calcd for C30H36N2O5[M+H]+:505.2697；Found 505.2699.。
4. synthesis of Compound 5:
the structural formula of the compound 5 is shown as a formula 5.
The preparation method comprises the following steps:
the reaction equation is shown in fig. 9A, and the preparation method steps are identical to compound2, except that: sinomenine (123.7 mg) was reacted with triphosgene (155.3 mg), triethylamine (0.22 mL), and 4-methanesulfonylaniline (104.1 mg) to give compound 5 in 52% yield.
1H NMR(600MHz,CDCl3)δ9.64(s,1H),7.77(d,J＝8.6Hz,2H),7.67(d,J＝8.8Hz,2H),6.93(d,J＝8.5 Hz,1H),6.71(d,J＝8.5Hz,1H),5.58(s,1H),3.94–3.89(m,1H),3.64(s,3H),3.53(s,3H),3.32–3.27(m, 1H),3.19–3.15(m,1H),3.10(d,J＝18.4Hz,1H),3.00(s,3H),2.81(dd,J＝18.3,5.3Hz,1H),2.63(d,J＝15.6 Hz,2H),2.50(s,3H),2.23(t,J＝11.0Hz,1H),1.99(td,J＝12.5,4.2Hz,1H),1.74(d,J＝12.5Hz,1H)ppm；
13C NMR(150MHz,CDCl3)δ194.7,152.5,150.9,150.5,143.5,138.7,134.2,129.9,129.1,128.6,125.7, 118.4,116.0,110.9,56.5,55.9,55.1,50.5,46.6,45.9,44.8,42.5,41.2,36.9,24.2ppm；HRMS-ESI(m/z)Calcd for C27H30N2O7S[M+H]+:527.1847；Found 527.1836.。
5. synthesis of Compound 6:
the structural formula of the compound 6 is shown as a formula 6.
The preparation method comprises the following steps:
the reaction equation is shown in fig. 9A, and the preparation method steps are identical to compound2, except that: sinomenine (122.3 mg) was reacted with triphosgene (158.2 mg), triethylamine (0.22 mL) and 4-chloroaniline (77.4 mg) to give compound 6 in 10% yield.
1H NMR(600MHz,CDCl3)δ8.28(s,1H),7.46(d,J＝8.7Hz,2H),7.30–7.26(m,2H),6.93(d,J＝8.4Hz, 1H),6.77(d,J＝8.4Hz,1H),5.54(s,1H),3.91(d,J＝15.6Hz,1H),3.74(s,3H),3.51(s,3H),3.20(s,1H),3.08 (d,J＝18.3Hz,1H),3.02(s,1H),2.73(dd,J＝18.2,5.3Hz,1H),2.55(t,J＝11.8Hz,2H),2.45(s,3H),2.17(td, J＝12.3,2.8Hz,1H),1.89(td,J＝12.5,4.5Hz,1H),1.76(d,J＝12.5Hz,1H)ppm；
13C NMR(150MHz,CDCl3)δ194.1,152.4,150.9,150.6,139.1,136.7,130.4,129.7,129.0,128.4,125.5, 119.7,115.9,111.0,56.4,56.1,54.9,50.5,46.6,46.3,42.8,41.1,37.2,24.2ppm；HRMS-ESI(m/z)Calcd for C26H27ClN2O5[M+H]+:483.1681；Found 483.1675.。
6. synthesis of compound 7:
the structural formula of the compound 7 is shown in a formula 7.
The preparation method comprises the following steps:
the reaction equation is shown in fig. 9A, and the preparation method steps are identical to compound2, except that: sinomenine (183.3 mg) was reacted with triphosgene (246.0 mg), triethylamine (0.46 mL) and benzylamine (96. Mu.L) to give compound 7 in 51% yield.
1H NMR(600MHz,CDCl3)δ7.37–7.32(m,4H),7.29–7.26(m,1H),6.88(d,J＝8.4Hz,1H),6.75(d, J＝8.4Hz,1H),6.21(t,J＝6.0Hz,1H),5.49(s,1H),4.52(dd,J＝15.1,6.8Hz,1H),4.35(dd,J＝15.1,5.5Hz, 1H),3.89(d,J＝15.8Hz,1H),3.71(s,3H),3.47(s,3H),3.32–3.29(m,1H),3.11(s,1H),3.08–3.02(m,1H), 2.80(dd,J＝18.2,5.2Hz,1H),2.59(d,J＝11.3Hz,1H),2.50(d,J＝15.9Hz,1H),2.48(s,3H),2.20(td,J＝12.3, 2.7Hz,1H),1.95–1.89(m,1H),1.81(d,J＝12.6Hz,1H)ppm；
13C NMR(150MHz,CDCl3)δ193.0,154.3,152.4,150.9,139.9,138.5,130.2,129.0,128.6,127.5,127.4, 125.1,114.8,111.0,56.6,56.0,54.9,50.0,46.9,45.5,45.2,42.4,40.6,36.6,24.3ppm；HRMS-ESI(m/z)Calcd for C27H30N2O5[M+H]+:463.2228；Found 463.2224.。
7. synthesis of compound 8:
the structural formula of the compound 8 is shown as a formula 8.
The preparation method comprises the following steps:
the reaction equation is shown in fig. 9A, and the preparation method steps are the same as compound2, except that: sinomenine (106.2 mg) was reacted with triphosgene (143.1 mg), triethylamine (0.27 mL) and n-pentylamine (60. Mu.L) to give compound 8 in 31% yield.
1H NMR(600MHz,CDCl3)δ6.91(d,J＝8.4Hz,1H),6.79(d,J＝8.5Hz,1H),5.61(t,J＝5.8Hz,1H), 5.50(t,J＝4.7Hz,1H),3.89(d,J＝15.8Hz,1H),3.75(s,3H),3.64(d,J＝13.5Hz,1H),3.49(s,3H),3.30–3.17 (m,2H),3.08–2.99(m,2H),2.87(d,J＝10.5Hz,1H),2.65(s,3H),2.58(dd,J＝15.7,7.8Hz,1H),2.39(dt,J＝ 26.7,13.4Hz,1H),2.18–2.11(m,1H),1.89(d,J＝12.7Hz,1H),1.60–1.54(m,2H),1.46(t,J＝7.3Hz,1H), 1.38–1.32(m,4H),0.92(t,J＝7.0Hz,3H)ppm；
13C NMR(150MHz,CDCl3)δ192.3,154.0,152.6,151.4,140.0,129.5,127.2,125.3,113.2,111.5,57.3, 56.9,55.0,49.3,47.3,42.2,41.8,41.3,40.0,35.5,29.5,28.7,22.3,14.1,11.2ppm；HRMS-ESI(m/z)Calcd for C25H34N2O5[M+H]+:443.2541；Found443.2537.。
8. synthesis of compound 9:
the structural formula of the compound 9 is shown as a formula 9.
The preparation method comprises the following steps:
the reaction equation is shown in fig. 9A, and the preparation method steps are the same as compound2, except that: sinomenine (83.1 mg) was reacted with triphosgene (109.9 mg), triethylamine (0.15 mL) and n-hexylamine (52. Mu.L) to give compound 9 in 35% yield.
1H NMR(600MHz,CDCl3)δ6.87(d,J＝8.4Hz,1H),6.74(d,J＝8.4Hz,1H),5.71(t,J＝5.7Hz,1H), 5.50(d,J＝1.7Hz,1H),3.89(d,J＝15.7Hz,1H),3.74(s,3H),3.48(s,3H),3.30–3.20(m,2H),3.20–3.17(m, 1H),3.05(d,J＝18.2Hz,1H),3.01(s,1H),2.72(dd,J＝18.1,5.4Hz,1H),2.55–2.48(m,2H),2.44(s,3H), 2.16(td,J＝12.2,3.4Hz,1H),1.89–1.83(m,1H),1.79(d,J＝12.2Hz,1H),1.59–1.53(m,2H),1.38(dd,J＝ 14.3,7.5Hz,2H),1.35–1.27(m,4H),0.90(t,J＝6.9Hz,3H)ppm；
13C NMR(150MHz,CDCl3)δ193.5,154.1,152.4,150.9,139.9,130.4,129.6,124.9,115.5,111.0,56.4, 56.1,54.9,50.3,46.7,46.2,42.7,41.3,40.9,37.0,31.5,29.9,26.3,24.2,22.6,14.1ppm；HRMS-ESI(m/z)Calcd for C26H36N2O5[M+H]+:457.2697；Found457.2694.。
9. synthesis of compound 10:
the structural formula of the compound 10 is shown as a formula 10.
The preparation method comprises the following steps:
the reaction equation is shown in fig. 9A, and the preparation method steps are identical to compound2, except that: sinomenine (167.5 mg) was reacted with triphosgene (226.3 mg), triethylamine (0.42 mL) and n-heptylamine (120. Mu.L) to give compound 10 in 34% yield.
1H NMR(600MHz,CDCl3)δ6.90(d,J＝8.4Hz,1H),6.77(d,J＝8.4Hz,1H),5.59(s,1H),5.48(t,J＝4.3 Hz,1H),3.89(d,J＝15.8Hz,1H),3.75(s,3H),3.49(s,4H),3.32(s,1H),3.24(ddq,J＝33.3,13.3,6.8Hz,2H), 3.07(d,J＝18.7Hz,1H),2.93(dd,J＝18.5,5.0Hz,1H),2.77(d,J＝10.7Hz,1H),2.59(s,3H),2.56(d,J＝15.8 Hz,1H),2.33(dd,J＝12.3,10.1Hz,1H),2.07(dd,J＝12.6,9.1Hz,1H),1.86(d,J＝12.9Hz,1H),1.60–1.53 (m,2H),1.39–1.25(m,8H),0.89(t,J＝6.9Hz,3H)ppm；
13C NMR(150MHz,CDCl3)δ192.7,154.0,152.6,151.3,140.0,129.8,127.8,125.2,113.8,111.3,57.1, 56.1,55.0,49.6,47.2,44.7,42.1,41.3,40.3,36.0,31.8,29.9,28.9,26.6,24.4,22.6,14.1ppm；HRMS-ESI(m/z) Calcd for C27H38N2O5[M+H]+:471.2854；Found 471.2858.。
10. synthesis of compounds 11 and 12:
the structural formula of the compound 11 is shown as a formula 11. The structural formula of the compound 12 is shown as a formula 12.
As shown in FIG. 9B, 30.7mg of sinomenine (compound represented by formula 1) was dissolved in 0.42mL of anhydrous dichloromethane, 67.4mg of N, N' -carbonyldiimidazole and 34. Mu.L of triethylamine were added thereto, and the mixture was stirred and reacted for 1 hour. TLC was used to monitor until the reaction was complete. The reaction mixture was extracted 3 times with deionized water and ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated and purified by column chromatography (dichloromethane: methanol =91: 9) to give compound 11 (shown in formula 11) and compound 12 (shown in formula 12), with a yield of compound 11 of 29% and compound 12 of 30%.
1H NMR(600MHz,CDCl3)δ7.68(s,1H),7.10(d,J＝0.7Hz,2H),6.63(d,J＝8.3Hz,1H),6.54(d,J＝8.2 Hz,1H),5.47(d,J＝2.1Hz,1H),4.35(d,J＝15.7Hz,1H),3.80(s,3H),3.49(s,3H),3.19(t,J＝4.1Hz,1H), 3.03–2.98(m,2H),2.71(dd,J＝18.4,5.3Hz,1H),2.55(ddd,J＝12.0,4.3,1.7Hz,1H),2.45(t,J＝4.7Hz,1H), 2.44(s,3H),2.08(td,J＝12.1,3.7Hz,1H),1.94–1.91(m,1H),1.89(dd,J＝12.3,4.6Hz,1H)ppm；
13C NMR(150MHz,CDCl3)δ194.2,152.4,145.0,144.7,135.1,130.2,122.5,118.3,115.1,109.0,56.7, 56.1,54.8,49.1,47.1,45.8,42.7,40.5,35.9,24.3ppm；HRMS-ESI(m/z)Calcd for C23H25N3O5[M+H]+: 424.1867；Found424.1884.。
1H NMR(600MHz,CDCl3)δ6.84(d,J＝8.4Hz,1H),6.78(d,J＝8.4Hz,1H),5.52(d,J＝2.0Hz,1H), 3.82(s,3H),3.76(s,1H),3.50(s,3H),3.31–3.27(m,1H),3.17(s,1H),3.11(d,J＝18.4Hz,1H),2.64–2.55(m, 2H),2.46(s,3H),2.18(td,J＝12.3,3.3Hz,1H),2.02(td,J＝12.4,4.7Hz,1H),1.59(d,J＝12.3Hz,1H)ppm；
13C NMR(150MHz,CDCl3)δ185.5,162.7,151.2,146.0,139.8,127.1,123.4,121.0,114.7,112.5,59.4, 57.1,56.3,55.2,46.4,42.8,40.7,38.0,35.1,22.5ppm；HRMS-ESI(m/z)Calcd for C20H21NO5[M+H]+:356.1492； Found356.1516.。
11. synthesis of compound 13:
the structural formula of the compound 13 is shown as the formula 13.
As shown in FIG. 9C, 116.5mg of sinomenine (compound of formula 1) was dissolved in 1.3mL of dimethylformamide, and 275. Mu.L of potassium hydroxide (2M) and 21. Mu.L of bromoacetonitrile were added thereto, followed by stirring and reacting for 2 hours. After the reaction mixture was diluted with ethyl acetate, deionized water was added, and extraction was performed 3 times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated and purified by column chromatography (dichloromethane: methanol = 92) to obtain compound 13 (shown in formula 13) in a yield of 72%.
1H NMR(600MHz,CDCl3)δ6.86–6.79(m,1H),6.73(d,J＝8.4Hz,1H),5.50(d,J＝1.9Hz,1H),4.99–4.90(m,1H),4.72(d,J＝15.8Hz,1H),3.86(d,J＝15.5Hz,1H),3.83(s,3H),3.48(s,3H),3.16(t,J＝4.2Hz, 1H),3.01(dd,J＝9.7,8.2Hz,2H),2.70(dd,J＝18.4,5.3Hz,1H),2.62(d,J＝15.5Hz,1H),2.54–2.50(m,1H), 2.41(s,3H),2.01–1.96(m,1H),1.96–1.90(m,1H),1.84(d,J＝12.3Hz,1H)ppm；
13C NMR(150MHz,CDCl3)δ194.2,152.5,150.5,146.1,130.3,129.7,124.2,116.5,115.4,111.2,57.5, 56.2,55.6,54.9,50.5,46.8,46.3,42.7,41.3,36.9,24.4ppm；HRMS-ESI(m/z)Calcd for C21H24N2O4[M+H]+: 369.1809；Found 369.1794.。
12. synthesis of compound 14:
the structural formula of the compound 14 is shown as a formula 14.
The reaction equation is shown in fig. 9C, and the preparation method step is similar to compound 13, except that: sinomenine is 115.9mg, potassium hydroxide (2M) is 275 mu L, bromoacetonitrile is replaced by 30 mu L4-bromobutyronitrile, and the compound 14 is obtained by reaction, wherein the yield is 50%.
1H NMR(600MHz,CDCl3)δ6.75(d,J＝8.4Hz,1H),6.70(d,J＝8.4Hz,1H),5.51(d,J＝2.1Hz,1H), 4.23–4.13(m,2H),3.98(d,J＝15.9Hz,1H),3.79(s,3H),3.49(s,3H),3.15(t,J＝4.1Hz,1H),3.00(d,J＝18.2 Hz,1H),2.98–2.96(m,1H),2.75–2.61(m,3H),2.54–2.48(m,2H),2.42(s,3H),2.16(qd,J＝7.2,1.0Hz, 2H),1.99(td,J＝12.1,3.2Hz,1H),1.90(td,J＝12.4,4.4Hz,1H),1.83–1.79(m,1H)ppm；
13C NMR(150MHz,CDCl3)δ193.8,152.5,151.2,147.0,130.1,130.0,123.0,120.0,115.6,111.0,69.0, 56.4,55.4,54.8,50.3,46.9,46.2,42.7,41.0,37.5,26.7,24.6,14.0ppm；HRMS-ESI(m/z)Calcd for C23H28N2O4 [M+H]+:397.2122；Found 397.2111.。
13. synthesis of compound 15:
the structural formula of the compound 15 is shown as the formula 15.
The reaction equation is shown in fig. 9C, and the preparation method step is identical to compound 13, except that: sinomenine 97.7mg, potassium hydroxide (2M) 275 uL, bromoacetonitrile 35 uL 5-bromovaleronitrile, compound 15 was obtained by reaction with a yield of 70%.
1H NMR(600MHz,CDCl3)δ6.74(d,J＝8.5Hz,1H),6.70(d,J＝8.4Hz,1H),5.49(d,J＝1.8Hz,1H), 4.16–4.11(m,1H),4.11–4.07(m,1H),4.07–4.03(m,1H),3.78(s,3H),3.49(s,3H),3.18(s,1H),3.02–2.97 (m,2H),2.75(dd,J＝18.4,5.2Hz,1H),2.55–2.49(m,4H),2.43(s,3H),2.02–1.96(m,3H),1.96–1.89(m, 3H),1.83(d,J＝12.4Hz,1H)ppm；
13C NMR(150MHz,CDCl3)δ193.8,152.6,151.4,147.5,130.0,122.9,119.9,115.4,111.2,70.4,56.5,55.6, 54.8,50.1,47.0,46.0,42.6,40.9,37.3,29.1,24.6,22.3,16.9ppm；HRMS-ESI(m/z)Calcd for C24H30N2O4 [M+H]+:411.2278；Found 441.2281.。
example 6 Sinomenine derivatives inhibit the efflux of ABCB5 and its mutants to MTX in 293T cells
In order to further illustrate that the sinomenine derivative also has the MTX-resistant arthritis treatment effect of sinomenine, the inventor takes the sinomenine derivative prepared in the above examples for experimental verification.
The method comprises the following specific steps:
(1) Site-directed mutagenesis of ABCB 5:
ABCB5 was site-directed mutated using a biosystem thermal cycler using Pfuultra II fusion HS DNA polymerase (align) and dNTP using the ABCB5 mammalian expression plasmid in the above example as a template and the following primers (Table 3). After PCR, gel purification and DpnI digestion were performed, and a single clone of the digested product was pink and verified by sequencing.
TABLE 3
Using liposomes (
3000, invitrogen) transfection reagent ABCB5 and its mutant recombinant plasmids were transferred into 293T cells and seeded in 6-well plates, 10 per well of 293T cells6And (4) respectively. After confluency, various doses of sinomenine or the sinomenine derivatives in the above examples were added to the cells for pretreatment for 24h. After completion, 0.5. Mu.M MTX-FITC (Invitrogen, M1198 MP) was added to each well and incubated at 37 ℃ for 1h. The cells were washed 5 times with pre-chilled PBS to stop the accumulation of MTX-FITC, and then resuspended in 400 μ LPBS for flow cytometry analysis. Normalization was performed with a control transfected with empty vector.
The results are shown in FIGS. 10, 11, 12 and 13.
By analyzing the effects of sinomenine and sinomenine derivatives on the efflux function of ABCB5 and mutants thereof by using flow cytometry, it was found that sinomenine has a dose-dependence on the inhibitory effect on the efflux MTX function of ABCB5, and does not exert the inhibitory effect at a low dose (20. Mu.M or less) (FIG. 10). From the results of the experiments, it was also found that the inhibitory effect of sinomenine on ABCB5 mutant was also reduced with the decrease of the dose, and no inhibitory activity on its transport function was exhibited at 20 μ M (fig. 11A, 11B and 11C). The inhibition effect of the sinomenine derivative (compound 2) is also shown to be dose-dependent, but the sinomenine derivative has better effect of inhibiting the efflux function of ABCB5 and mutants thereof under the condition of low dose of 20 mu M compared with sinomenine. Further experiments prove that other sinomenine derivatives (compounds 3-15) have similar using effects to the compound2 and show effective ABCB5 inhibition under the low dosage condition of 20 mu M or 50 mu M (figures 12-13), thereby indicating that the sinomenine derivatives have the same ABCB5 efflux function inhibition activity as sinomenine.
In summary, based on the above test results, in the embodiments of the present invention, the inventors found a novel sinomenine derivative which can effectively inhibit the efflux of ABCB5 to MTX in 293T cells highly expressing ABCB5 or a mutant thereof, and has an inhibitory activity superior to sinomenine, thereby being expected to exhibit a more excellent therapeutic effect than sinomenine in the treatment of drug-resistant rheumatoid arthritis.
Example 7 therapeutic Effect of sinomenine derivatives on MTX-resistant arthritis rat model
To further verify the actual therapeutic effect of sinomenine derivatives on drug-resistant rheumatoid arthritis in the above examples, the inventors used sinomenine or sinomenine derivatives in combination with methotrexate to examine the therapeutic effect on drug-resistant rheumatoid arthritis transgenic rats highly expressing ABCB5 on CD4+ cells in the examples.
Through CRISPR/Cas9 mediated gene engineering, ABCB5 transgenic SD Rat with { CAG-loxP-stop-loxP-Rat ABCB5 CDs-SV40 late pA } knocked in RO SA26 site is constructed, rat ROSA26 gene is located on Rat chromosome 4, and intron 1-2 (the specific sequence case is CCTTCCCTCCCTCGTGATCTGC-TTTCTGGAAGATAGGCGGG-representing insertion position) is selected as a target site. gRNA targeting sequences are as follows:
gRNA1 (antisense strand of the matching gene) GACTCCAGTTGCAGATCACGAGG (SEQ ID NO: 19);
gRNA2 (sense strand of the matching gene) AAGATAGGCGGGAGTCTTCTGGG (SEQ ID NO: 20).
The method comprises the following specific steps: the gRNA of Rat ROSA26 gene, a gene vector containing { CAG-loxP-stop-loxP-Rat ABCB5 CDs-SV40 late pA } (plasmid map shown in FIG. 14A), and Cas9 mRNA were co-injected into Rat zygotes to generate knock-in targeted F0 progenitors (see FIG. 14B). The F0 ancestor and a wild rat are hybridized to generate an F1 generation, the germ line transmission identification of the F0 ancestor can be realized by carrying out PCR identification and sequence analysis on the F1 generation, so that an F0 generation rat which can be stably inherited and a qualified F1 generation rat (the genotype of the F1 generation rat is heterozygote) are screened out, and then homozygote ABCB5 transgenic mice are obtained by a plurality of series breeding. Meanwhile, constructing a Cre recombinase tool mouse requires constructing and purifying a Cre-ERT2 mammalian gene expression vector (as shown in FIG. 15), introducing the expression vector into a fertilized egg by microinjection, and screening positive F0 pups. The ABCB5 homozygote transgenic mice and Cre tool mice are hybridized and bred to screen cubs with positive double genes. A large number of transgenic SD rats with double gene positivity are bred, and tamoxifen is given to the young animals by intraperitoneal injection at a dosage of 75mg/kg/Day when the young animals are about two weeks old, and the induction is continuously carried out for 5 days.
A control group of normal SD rats was set. The experimental rats were continued to be acclimatized for at least 3 weeks after induction was completed. Arthritis was induced in rats at a weight of about 120-150g using complete Freund's adjuvant (CFA, CAS: 9007-81-2).
A total of 40 rats, 15 ordinary SD rats and 25 ABCB5 transgenic SD rats were selected and randomly divided into 8 experimental groups of 5 rats per group according to Table 4.
TABLE 4
The experiment was carried out for 30 days, and the paw and body weight measurements were taken every 3 days. Rats were sacrificed on the last day of the experiment and relevant tissue samples were obtained.
Methotrexate (MTX) was found to be effective in inhibiting joint swelling in normal AIA arthritic rats by foot body measurement (fig. 16), but when ABCB5 was highly expressed, its therapeutic effect was cancelled, and the swelling of the feet of the rats was not significantly improved, indicating the generation of MTX resistance. In the use of high-dose sinomenine (100 mg/kg/Day) in combination with methotrexate, the combination of the sinomenine and methotrexate is found to reverse the drug resistance of MTX, so that the swelling of feet of rats is obviously reduced. However, this effect was not observed with the combination of low doses of sinomenine (30 mg/kg/Day), which was shown to be a significant reduction in the therapeutic effect of the combination. The sinomenine derivative (compound 2) prepared in the above examples also has the effect of inhibiting ABCB5 transporter, but unlike sinomenine, the sinomenine derivative can be combined with methotrexate under the condition of lower dose (30 mg/kg/Day) to still significantly inhibit the increase of foot swelling of rats, thereby showing better application prospect.
mu.L of anticoagulated Blood was collected from each group of rats, samples were treated with erythrocyte lysate (Biyuntian C3702), total RNA was extracted using FavorPrepTM Blood/filtered Cell Total RNA Mini Kit, cDNA was obtained by reverse transcription PCR, and expression of ABCB5 in Blood was detected in ViiA 7 Real-Time PCR system using PerfectStartTM Green Qpcr SuperMix (Transgen, AQ 601) and primers shown in Table 5 below.
TABLE 5
| Primer and method for producing the same | Nucleotide sequence (5 '-3') |
| ABCB5 R F1 | TCGTGGGGCAGACCTGATTG(SEQ ID NO:21) |
| ABCB5 R R1 | GGGGGACTTGGCGCTATTCA(SEQ ID NO:22) |
The results are shown in FIG. 17.
The results of qPCR detection show that after tamoxifen induction, ABCB5+ AIA + MTX + SIN100, ABCB5+ AIA + MTX + SIN30 and ABCB5+ AIA + MTX + Compound2 successfully express high expression of ABCB5, which indicates that the CD4+ cell-specific expression of the SD rat animal model of ABCB5 is successfully constructed.
Rat foot bone destruction was analyzed using micro computer tomography (MicroCT) and scanning was performed using an in vivo micro CT scanner (SkyScan 1176, bruker, belgium) on Al 0.5mm filters. Wherein, the scanning parameters are as follows: 35 micron resolution, 62kV,385 μ A,98ms exposure time, angular velocity 0.70. After scanning is finished, images are reconstructed by using NReco software, CTvox software is used for opening reconstructed data files and generating observable 3-dimensional pictures, and scanning data are analyzed by CTAn software.
The MicroCT score was based on five disease-related indicators examined by MicroCT (bone mineral Density (BMD), tissue bone Density (TMD), bone volume score (BV/TV), trabecular number (Tb.N) and total porosity), and was calculated as:
or
The MicroCT score is the average value of the sum of the five disease related indexes after the treatment.
The results of the MicroCT measurements are shown in FIG. 18.
FIG. 18A is a photograph of computerized tomography analysis, which shows that 30mg/kg/Day sinomenine derivatives combined with methotrexate can significantly improve the bone destruction in rats with arthritis. While sinomenine showed excellent therapeutic activity at the 100mg/kg/Day dose in combination, the activity disappeared at the low dose and the bones of the feet of the rats were damaged more (arrows in the figure indicate the site of bone destruction). FIG. 18B is a histogram of MicroCT scoring, which shows the quality of foot bone parameter index in different groups of rats, wherein low doses of sinomenine derivatives combined with methotrexate can reverse foot bone to mild bone destruction, and the effect is better than that of sinomenine with the same dose.
Meanwhile, the method for detecting the blood sedimentation rate of the experimental rat comprises the following specific steps: collecting vein blood specimen of experimental rat by using blood sedimentation tube, and reversing 180 degrees for 6-8 times after collection to make anticoagulant in the tube and blood fully mixed. Vertically fixing the blood sedimentation tube with the specimen on a special blood sedimentation rack, recording the initial time and the corresponding serial number, keeping the blood sedimentation tube at the room temperature of about 20 ℃, and reading the millimeter number of erythrocyte sedimentation after 3 hours, wherein the millimeter number (mm)/time (h) of erythrocyte sedimentation is equal to the blood sedimentation rate (mm/h).
In the field, a plurality of pathological conditions, such as acute inflammation, active tuberculosis, rheumatism active period, severe tissue damage, anemia, malignant tumor and the like can lead to the pathological increase of erythrocyte sedimentation rate, and the speed of the erythrocyte sedimentation can assist in observing the change of disease conditions. The results of the blood sedimentation in fig. 19 show that intervention in rheumatoid arthritis caused by overexpression of ABCB5 gene resulted in failure of treatment with MTX, resulting in a significant increase in the blood sedimentation rate. The use of high-dose sinomenine and low-dose sinomenine derivative (compound 2) in combination with methotrexate can reverse ABCB5 mediated drug resistance reaction, so that the blood sedimentation rate is at a lower level, thereby improving the related symptoms of model rats.
At the same time, 400 μ L of fresh anticoagulated blood was collected from each group of rats and the samples were treated with erythrocyte lysate (Biyuntian C3702). After viable Cell counting, appropriate amounts of cells were surface stained using APC/CY7-CD45, FITC-CD3, perCP/Cy5-CD4, APC-CD8 (BioLegend), and Cell Staining Buffer (BioLegend Cat. No. 420201) according to the Cell surface flow cytometer Staining protocol. Using True-NuclearTMAfter membrane rupture is fixed by the transformation Factor Buffer Set (Cat. No. 424401), intracellular staining is carried out by utilizing a PE-Foxp3 (BioLegend) fluorescent antibody, and a sample is collected on a flow cytometer. Taking appropriate amount of cells (1-2X 10)6cell/mL) was induced by stimulation with Brefeldin A (BioLegend Cat. No. 423302) for 6h, followed by addition of fluorescent antibodyAnd dyeing at 4 ℃ in dark for 45 minutes. Reuse of Cyto-FastTMCells were treated for 20 minutes for membrane disruption using a Fix/Perm Buffer Set (BioLegend Cat. No. 426803). Intracellular staining was performed for 30 minutes with PE/Cy7-IL-17A (BioLegend) fluorescent antibody diluted in cell staining buffer, and the cell fluorescent signal was analyzed using a flow cytometer.
The results are shown in fig. 20, 21 and 22.
Tregs are T cells that mediate immune tolerance, while Th17 cells mediate a series of inflammatory responses, the actions of which counteract the differentiation process. Under normal conditions, the two are in equilibrium, thus being beneficial to maintaining the immune homeostasis of the body. However, in inflammatory reactions, autoimmune diseases and transplant rejection reactions, the differentiation pattern of primitive T lymphocytes is changed due to the influence of certain inflammatory factors, and the differentiation develops towards the generation of more Th17 cells, so that the balance between Treg and Th17 is destroyed, a series of inflammatory reactions are caused, and the body is damaged. FIGS. 20 and 21 are flow cytograms of fluorescent staining for FoxP3, IL-17A in experimental rat CD4+ cells, and FIG. 22 is a histogram of statistical analysis of fluorescent signals (where FIG. 22C is the ratio of FoxP3, IL-17A fluorescent signals, reflecting the propensity of the Treg/Th17 equilibrium in the sample). From the figure, it can be seen that in the arthritis rat model, foxp3 shows a descending trend, IL-17A shows an increase, the proportion of Treg cells in blood of an inflammation rat is reduced, the proportion of Th17 cells is increased, and the balance is inclined to the Th17 cells. Effective therapeutic measures can reverse the phenomenon, for example, the administration of 30mg/kg/Day low-dose sinomenine derivative combined with MTX, the independent administration of MTX and the low-dose sinomenine combined administration show certain therapeutic effects, but in the test groups, the administration of 30mg/kg/Day low-dose sinomenine derivative combined with MTX can better maintain the balance of Treg/Th17 and inhibit the increase of Th17 cells.
Taking fresh non-anticoagulated blood, and standing for at least 30 minutes. Serum samples were obtained by centrifugation at 1000 Xg for 10 min. The signals were then collected by flow assay using a Rat injection custom Panel 10-plex (Biolegend) according to the protocol. And finally, data processing is carried out on a data analysis website specified by biolegend. And standardizing each index data by using the index mean value of the corresponding healthy control group, and deducting the basic multiple ratio 1 to obtain the cell factor content increase multiple ratio. The results are shown as the mean of the growth multiple ratio for each index.
The results are shown in FIG. 23.
In FIG. 23, the average increase in serum cytokine levels by more than 3-fold is shown as red and increase by more than 25-fold is shown as blue. The serum levels of various inflammatory factors were significantly elevated after AIA molding compared to healthy controls. The situation improved after MTX intervention. CD4+The anti-inflammatory effect of ABCB5 group MTX highly expressed by the cells is inhibited. The inhibition of the anti-inflammatory effect of MTX was reversed when 100mg/kg/Day sinomenine was used as the ABCB5 inhibitor in the treatment, whereas the low dose of 30mg/kg/Day sinomenine did not show this effect. In contrast, the sinomenine derivative (compound 2) is capable of inhibiting ABCB5 transporter at a lower dose, and the increase of inflammatory cytokines in blood is inhibited.
In conclusion, compared with sinomenine, the sinomenine derivative (compound 2) prepared in the above embodiment can combine with methotrexate to exert excellent treatment effect in treating drug-resistant rheumatoid arthritis rats with high CD4+ cell expression ABCB5, effectively inhibit ABCB5 efflux transport function, and reverse MTX resistance reaction, so that the sinomenine derivative can be used as an ABCB5 inhibitor to show excellent application prospect and great drug-forming potential in preparation of drug-resistant rheumatoid arthritis preparations.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Australian university of science and technology
Application of <120> ABCB5 inhibitor in preparation of multi-drug-resistance rheumatoid arthritis treatment drug
<130>
<160> 22
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence
<400> 3
agcgatgatg cactgtcaga a 21
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence
<400> 4
gcattggaag ttggggtagg a 21
<210> 5
<211> 24
<212> DNA
<213> Artificial sequence
<400> 5
aaatgggctc cctctcatca gttc 24
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence
<400> 6
tctgcttggt ggtttgctac gac 23
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
<210> 9
<211> 42
<212> DNA
<213> Artificial sequence
<400> 9
acaaggaaat ctgtgtgtaa ccaaaaatca aggcagcaac ac 42
<210> 10
<211> 42
<212> DNA
<213> Artificial sequence
<400> 10
gtgttgctgc cttgattttt ggttacacac agatttcctt gt 42
<210> 11
<211> 39
<212> DNA
<213> Artificial sequence
<400> 11
gtcaatgtca tctgtcatgt gagtgttaag ttcaccgat 39
<210> 12
<211> 39
<212> DNA
<213> Artificial sequence
<400> 12
atcggtgaac ttaacactca catgacagat gacattgac 39
<210> 13
<211> 39
<212> DNA
<213> Artificial sequence
<400> 13
taggcactta attccttacg ggtcaatgag atgaccatc 39
<210> 14
<211> 39
<212> DNA
<213> Artificial sequence
<400> 14
gatggtcatc tcattgaccc gtaaggaatt aagtgccta 39
<210> 15
<211> 33
<212> DNA
<213> Artificial sequence
<400> 15
gctccccaca aatgctgctg tctttcctcg ctc 33
<210> 16
<211> 33
<212> DNA
<213> Artificial sequence
<400> 16
gagcgaggaa agacagcagc atttgtgggg agc 33
<210> 17
<211> 31
<212> DNA
<213> Artificial sequence
<400> 17
gaaagacagt agcatttgcg gggagcagcg g 31
<210> 18
<211> 31
<212> DNA
<213> Artificial sequence
<400> 18
ccgctgctcc ccgcaaatgc tactgtcttt c 31
<210> 19
<211> 23
<212> DNA
<213> Artificial sequence
<400> 19
gactccagtt gcagatcacg agg 23
<210> 20
<211> 23
<212> DNA
<213> Artificial sequence
<400> 20
aagataggcg ggagtcttct ggg 23
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence
<400> 21
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence
<400> 22
Claims (12)
1. A sinomenine derivative is characterized in that the sinomenine derivative is a compound formed by the substitution or nucleophilic acyl substitution reaction of a hydroxyl at the 4-position of sinomenine, and the structural formula of the sinomenine derivative is as follows:
wherein in the formula I, R groups are selected from hydrogen, substituted or unsubstituted anilino, nitrogen-containing alkane and imidazole;
or
No R group is present and the a position is linked to the C of the b position to form a ring, giving the structure described below:
in the formula II, R groups are selected from hydrogen and nitrile-containing alkane.
2. The sinomenine derivative of claim 2, wherein in formula I, the R group is selected from the structures shown below:
3. the sinomenine derivative of claim 2, wherein in the formula II, the R group is selected from nitrile-containing C1-8An alkane; said nitrile C1-8Alkanes are preferably of the structure shown below:
4. a method for preparing sinomenine derivative as claimed in any one of claims 1 to 3, comprising the steps of:
mixing sinomenine and amino compound under the conditions of triethylamine and triphosgene or N, N' -carbonyl diimidazole to obtain the compound; or
Mixing sinomenine and nitrile compound containing bromine under alkaline condition.
5. The method according to claim 4, wherein the amine-based compound is selected from aniline, 4-nitroaniline, 4-tert-butylaniline, 4-methanesulfonylaniline, 4-chloroaniline, benzylamine, n-pentylamine, n-hexylamine, and n-heptylamine.
6. The method according to claim 4, wherein the bromine-containing nitrile compound is selected from bromoacetonitrile, 4-bromobutyronitrile, and 5-bromovaleronitrile.
Application of the ABCB5 inhibitor in preparing a multi-drug-resistance rheumatoid arthritis treatment drug.
Use of an ABCB5 inhibitor in combination with methotrexate for the manufacture of a medicament for the treatment of multi-drug resistant rheumatoid arthritis.
9. The ABCB5 inhibitor according to claim 7 or 8, wherein the ABCB5 inhibitor comprises at least one of sinomenine, a sinomenine derivative according to any one of claims 1 to 3.
10. The ABCB5 inhibitor according to claim 7 or 8, wherein said multi-drug resistant rheumatoid arthritis is methotrexate resistant rheumatoid arthritis.
11. A drug for the treatment of multi-drug resistant rheumatoid arthritis, characterized in that the drug contains at least one of sinomenine and the sinomenine derivative according to any one of claims 1 to 3.
12. The medicament of claim 11, further comprising pharmaceutically acceptable excipients, wherein the excipients comprise at least one of solvents, wetting agents, emulsifiers, thickeners, excipients, suspending agents, disintegrants, fillers, lubricants, or diluents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210648571.0A CN115260098A (en) | 2022-06-09 | 2022-06-09 | Application of ABCB5 inhibitor in preparation of multi-drug-resistance rheumatoid arthritis treatment drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210648571.0A CN115260098A (en) | 2022-06-09 | 2022-06-09 | Application of ABCB5 inhibitor in preparation of multi-drug-resistance rheumatoid arthritis treatment drug |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115260098A true CN115260098A (en) | 2022-11-01 |
Family
ID=83759193
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210648571.0A Pending CN115260098A (en) | 2022-06-09 | 2022-06-09 | Application of ABCB5 inhibitor in preparation of multi-drug-resistance rheumatoid arthritis treatment drug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115260098A (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1504469A (en) * | 2002-11-28 | 2004-06-16 | �й���ѧԺ�Ϻ�ҩ���о��� | Tetrandrine and tetrandrine compound, synthesis and uses thereof |
| CN1582931A (en) * | 2004-05-24 | 2005-02-23 | 上海医药工业研究院 | Oral sustained releasing micro-tablets of tuduranine and their preparation |
| WO2007143139A1 (en) * | 2006-05-31 | 2007-12-13 | The Brigham And Women's Hospital, Inc. | Abcb5 positive mesenchymal stem cells as immunomodulators |
| WO2011009015A1 (en) * | 2009-07-16 | 2011-01-20 | Mallinckrodt Inc. | (+) - morphinans as antagonists of toll-like receptor 9 and therapeutic uses thereof |
| CN102260212A (en) * | 2011-05-05 | 2011-11-30 | 南京大学 | Sinomenine derivatives, and preparation method and application thereof |
| WO2012008984A1 (en) * | 2010-07-16 | 2012-01-19 | Mallinckrodt Llc | (+)-morphinans as antagonists of toll-like receptor 9 and therapeutic uses thereof |
| CN102532024A (en) * | 2011-12-28 | 2012-07-04 | 赵爱国 | Sinomenine derivative |
| CN102964303A (en) * | 2012-11-28 | 2013-03-13 | 湖南大学 | Sinomenine derivate and preparation method and application thereof |
| CN109223804A (en) * | 2017-07-10 | 2019-01-18 | 澳门科技大学 | For treating the arthritic quinone methyl triterpene compound of refractory rheumatoid and pharmaceutical composition |
-
2022
- 2022-06-09 CN CN202210648571.0A patent/CN115260098A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1504469A (en) * | 2002-11-28 | 2004-06-16 | �й���ѧԺ�Ϻ�ҩ���о��� | Tetrandrine and tetrandrine compound, synthesis and uses thereof |
| CN1582931A (en) * | 2004-05-24 | 2005-02-23 | 上海医药工业研究院 | Oral sustained releasing micro-tablets of tuduranine and their preparation |
| WO2007143139A1 (en) * | 2006-05-31 | 2007-12-13 | The Brigham And Women's Hospital, Inc. | Abcb5 positive mesenchymal stem cells as immunomodulators |
| US20130315880A1 (en) * | 2006-05-31 | 2013-11-28 | Children's Medical Center Corporation | Abcb5 positive mesenchymal stem cells as immunomodulators |
| WO2011009015A1 (en) * | 2009-07-16 | 2011-01-20 | Mallinckrodt Inc. | (+) - morphinans as antagonists of toll-like receptor 9 and therapeutic uses thereof |
| US20170029432A1 (en) * | 2009-07-16 | 2017-02-02 | Mallinckrodt Llc | (+)-morphinans as antagonists of toll-like receptor 9 and therapeutic uses thereof |
| WO2012008984A1 (en) * | 2010-07-16 | 2012-01-19 | Mallinckrodt Llc | (+)-morphinans as antagonists of toll-like receptor 9 and therapeutic uses thereof |
| CN102260212A (en) * | 2011-05-05 | 2011-11-30 | 南京大学 | Sinomenine derivatives, and preparation method and application thereof |
| CN102532024A (en) * | 2011-12-28 | 2012-07-04 | 赵爱国 | Sinomenine derivative |
| CN102964303A (en) * | 2012-11-28 | 2013-03-13 | 湖南大学 | Sinomenine derivate and preparation method and application thereof |
| CN109223804A (en) * | 2017-07-10 | 2019-01-18 | 澳门科技大学 | For treating the arthritic quinone methyl triterpene compound of refractory rheumatoid and pharmaceutical composition |
Non-Patent Citations (1)
| Title |
|---|
| 曾铖 等: ""青藤碱联合甲氨蝶呤治疗类风湿关节炎有效性和安全性的Meta 分析"", 《中国中药杂志》, vol. 46, no. 1, 31 January 2021 (2021-01-31), pages 214 - 224 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4018884B2 (en) | Treatment of spinal muscular atrophy | |
| Wang et al. | ABCB6 mutations cause ocular coloboma | |
| US7767398B2 (en) | Means and methods for diagnosing and treating affective disorders | |
| Barish et al. | BICRA, a SWI/SNF complex member, is associated with BAF-disorder related phenotypes in humans and model organisms | |
| CN106102761A (en) | Prevention or therapeutic agent for kidney diaseases | |
| CN105555317A (en) | Structure, manufacturing and uses of human-derived cell-permeable peptides conjugated with specific biologically active cargo peptides | |
| CN108138234A (en) | The method for treating myeloproliferative disorders | |
| Windpassinger et al. | CDK10 mutations in humans and mice cause severe growth retardation, spine malformations, and developmental delays | |
| KR20220100912A (en) | Treatment for hematopoietic cell malignancies using genetically engineered T cells targeting CD70 | |
| CN111187835A (en) | Pancreatic cancer target ERBB2 and its application in diagnosis and treatment | |
| CN115260098A (en) | Application of ABCB5 inhibitor in preparation of multi-drug-resistance rheumatoid arthritis treatment drug | |
| US20140296327A1 (en) | Diagnosis and treatment of mesothelioma | |
| WO2020143424A1 (en) | Gastrointestinal stromal tumor target depdc5 and application thereof in diagnosis and treatment | |
| CN113456631B (en) | Small molecule drug targeting ACSL1 and application thereof in treatment of endometrial cancer | |
| WO2021219009A1 (en) | Diagnosis and treatment of cancers showing high expression of piwi and/or nmd complex protein | |
| CN106701902A (en) | FOXR2 gene and application of expression product to diagnosis and treatment of liver cancer | |
| CN107466236A (en) | Thymosin α1 is used for the purposes of cystic fibrosis therapies | |
| CN115074368B (en) | Construction and application of drug-resistant rheumatoid arthritis animal model | |
| WO2017091952A1 (en) | Use of akt2 in diagnosis and treatment of tumor | |
| Lingappan et al. | Molecular insights using spatial transcriptomics of the distal lung in Congenital Diaphragmatic Hernia | |
| US20070148129A1 (en) | Therapeutic agents and uses therefor | |
| CN116396965B (en) | Application of AHDC1 in construction of obese animal model, method and drug screening method | |
| CN109528700A (en) | Application of the small molecule compound QW07 in preparation treatment prostatic disorders drug | |
| JP6465379B2 (en) | Model animal, prevention, improvement or treatment of lifestyle-related diseases, antifeedant, screening method, detection agent for lifestyle-related diseases, and detection method for lifestyle-related diseases | |
| JP2008131918A (en) | USE OF PROTEIN PHOSPHATASE 2Cepsilon (PP2Cepsilon) HAVING AMPK DEPHOSPHORIZATION ENZYME ACTION |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |