WO2023030294A1 - Aiphanol作为VEGFR激酶抑制剂的应用 - Google Patents
Aiphanol作为VEGFR激酶抑制剂的应用 Download PDFInfo
- Publication number
- WO2023030294A1 WO2023030294A1 PCT/CN2022/115732 CN2022115732W WO2023030294A1 WO 2023030294 A1 WO2023030294 A1 WO 2023030294A1 CN 2022115732 W CN2022115732 W CN 2022115732W WO 2023030294 A1 WO2023030294 A1 WO 2023030294A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- aiphanol
- endothelial cells
- angiogenesis
- lymphangiogenesis
- preparation
- Prior art date
Links
- KDMFHGGHQLUIRH-OOODPRFPSA-N aiphanol Chemical compound COC1=C(O)C(OC)=CC([C@@H]2[C@H](OC3=CC=C(\C=C\C=4C=C(O)C=C(O)C=4)C=C3O2)CO)=C1 KDMFHGGHQLUIRH-OOODPRFPSA-N 0.000 title claims abstract description 147
- KDMFHGGHQLUIRH-ILBGXUMGSA-N aiphanol Natural products COc1cc(cc(OC)c1O)[C@H]2Oc3cc(C=Cc4cc(O)cc(O)c4)ccc3O[C@@H]2CO KDMFHGGHQLUIRH-ILBGXUMGSA-N 0.000 title claims abstract description 136
- 229940043355 kinase inhibitor Drugs 0.000 title claims abstract description 10
- 239000003757 phosphotransferase inhibitor Substances 0.000 title claims abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 42
- 230000033115 angiogenesis Effects 0.000 claims abstract description 40
- 108091008605 VEGF receptors Proteins 0.000 claims abstract description 38
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 claims abstract description 38
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 claims abstract description 31
- 238000002360 preparation method Methods 0.000 claims abstract description 26
- 201000010099 disease Diseases 0.000 claims abstract description 23
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 23
- 230000035168 lymphangiogenesis Effects 0.000 claims abstract description 23
- 230000035755 proliferation Effects 0.000 claims abstract description 14
- 210000004027 cell Anatomy 0.000 claims description 58
- 101000851030 Homo sapiens Vascular endothelial growth factor receptor 3 Proteins 0.000 claims description 33
- 230000015572 biosynthetic process Effects 0.000 claims description 33
- 102100033179 Vascular endothelial growth factor receptor 3 Human genes 0.000 claims description 32
- 230000002401 inhibitory effect Effects 0.000 claims description 32
- 101000851007 Homo sapiens Vascular endothelial growth factor receptor 2 Proteins 0.000 claims description 30
- 210000003556 vascular endothelial cell Anatomy 0.000 claims description 23
- 206010028980 Neoplasm Diseases 0.000 claims description 22
- 210000005073 lymphatic endothelial cell Anatomy 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 21
- 239000003814 drug Substances 0.000 claims description 18
- 101000851018 Homo sapiens Vascular endothelial growth factor receptor 1 Proteins 0.000 claims description 13
- 206010027476 Metastases Diseases 0.000 claims description 13
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 claims description 13
- 230000009401 metastasis Effects 0.000 claims description 13
- 208000007433 Lymphatic Metastasis Diseases 0.000 claims description 11
- 210000002889 endothelial cell Anatomy 0.000 claims description 10
- 230000005012 migration Effects 0.000 claims description 10
- 238000013508 migration Methods 0.000 claims description 10
- 210000005239 tubule Anatomy 0.000 claims description 9
- 230000003527 anti-angiogenesis Effects 0.000 claims description 8
- 230000002500 effect on skin Effects 0.000 claims description 6
- 230000009545 invasion Effects 0.000 claims description 6
- 210000003606 umbilical vein Anatomy 0.000 claims description 6
- 230000008685 targeting Effects 0.000 claims description 4
- 230000005747 tumor angiogenesis Effects 0.000 claims description 3
- 208000007766 Kaposi sarcoma Diseases 0.000 claims description 2
- 206010012601 diabetes mellitus Diseases 0.000 claims description 2
- 208000002780 macular degeneration Diseases 0.000 claims description 2
- 210000001365 lymphatic vessel Anatomy 0.000 abstract description 17
- 239000000203 mixture Substances 0.000 abstract description 17
- 230000002792 vascular Effects 0.000 abstract description 11
- 238000009472 formulation Methods 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 description 58
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 35
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 30
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 30
- 210000004204 blood vessel Anatomy 0.000 description 24
- 241000699666 Mus <mouse, genus> Species 0.000 description 22
- 241000699670 Mus sp. Species 0.000 description 21
- 108010082117 matrigel Proteins 0.000 description 19
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- 102100038232 Vascular endothelial growth factor C Human genes 0.000 description 18
- 230000012010 growth Effects 0.000 description 18
- 239000000243 solution Substances 0.000 description 18
- 108091000080 Phosphotransferase Proteins 0.000 description 17
- 102000020233 phosphotransferase Human genes 0.000 description 17
- 238000001727 in vivo Methods 0.000 description 16
- 229940079593 drug Drugs 0.000 description 15
- 241000252212 Danio rerio Species 0.000 description 14
- 108010073923 Vascular Endothelial Growth Factor C Proteins 0.000 description 14
- 210000001165 lymph node Anatomy 0.000 description 14
- 210000001519 tissue Anatomy 0.000 description 14
- PFMPOBVAYMTUOX-GOSISDBHSA-N 2-amino-1-ethyl-7-[(3r)-3-hydroxy-4-methoxy-3-methylbut-1-ynyl]-n-methyl-4-oxo-1,8-naphthyridine-3-carboxamide Chemical compound C1=C(C#C[C@@](C)(O)COC)N=C2N(CC)C(N)=C(C(=O)NC)C(=O)C2=C1 PFMPOBVAYMTUOX-GOSISDBHSA-N 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 150000001875 compounds Chemical class 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 229910052739 hydrogen Inorganic materials 0.000 description 11
- 150000003384 small molecules Chemical class 0.000 description 11
- 210000002257 embryonic structure Anatomy 0.000 description 10
- 239000001257 hydrogen Substances 0.000 description 10
- 102000005962 receptors Human genes 0.000 description 10
- 108020003175 receptors Proteins 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 239000003112 inhibitor Substances 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 8
- 241000287828 Gallus gallus Species 0.000 description 8
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 8
- 230000001772 anti-angiogenic effect Effects 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000003292 glue Substances 0.000 description 8
- 230000003993 interaction Effects 0.000 description 8
- 238000003032 molecular docking Methods 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 7
- 238000003556 assay Methods 0.000 description 7
- 230000001419 dependent effect Effects 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 235000013601 eggs Nutrition 0.000 description 7
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 7
- 239000008399 tap water Substances 0.000 description 7
- 235000020679 tap water Nutrition 0.000 description 7
- 230000009261 transgenic effect Effects 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 6
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 210000001015 abdomen Anatomy 0.000 description 6
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000001114 immunoprecipitation Methods 0.000 description 6
- 230000000968 intestinal effect Effects 0.000 description 6
- 239000003446 ligand Substances 0.000 description 6
- 238000004020 luminiscence type Methods 0.000 description 6
- 230000001926 lymphatic effect Effects 0.000 description 6
- 210000004088 microvessel Anatomy 0.000 description 6
- 239000008096 xylene Substances 0.000 description 6
- 206010006187 Breast cancer Diseases 0.000 description 5
- 208000026310 Breast neoplasm Diseases 0.000 description 5
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 5
- 108060001084 Luciferase Proteins 0.000 description 5
- 239000005089 Luciferase Substances 0.000 description 5
- 210000000577 adipose tissue Anatomy 0.000 description 5
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 239000007640 basal medium Substances 0.000 description 5
- 229960000397 bevacizumab Drugs 0.000 description 5
- 210000003711 chorioallantoic membrane Anatomy 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 230000035784 germination Effects 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 230000035876 healing Effects 0.000 description 5
- 230000002209 hydrophobic effect Effects 0.000 description 5
- 238000010166 immunofluorescence Methods 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 206010009944 Colon cancer Diseases 0.000 description 4
- 101000742596 Homo sapiens Vascular endothelial growth factor C Proteins 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 239000001768 carboxy methyl cellulose Substances 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 208000029742 colonic neoplasm Diseases 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000033001 locomotion Effects 0.000 description 4
- 239000012188 paraffin wax Substances 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 4
- 239000012679 serum free medium Substances 0.000 description 4
- 230000019491 signal transduction Effects 0.000 description 4
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 4
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 4
- 230000004614 tumor growth Effects 0.000 description 4
- 239000001993 wax Substances 0.000 description 4
- -1 1,4 -benzodioxane lignan Chemical class 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101000725401 Homo sapiens Cytochrome c oxidase subunit 2 Proteins 0.000 description 3
- 101000595923 Homo sapiens Placenta growth factor Proteins 0.000 description 3
- 101000605127 Homo sapiens Prostaglandin G/H synthase 2 Proteins 0.000 description 3
- 101000742579 Homo sapiens Vascular endothelial growth factor B Proteins 0.000 description 3
- 101000742599 Homo sapiens Vascular endothelial growth factor D Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102100035194 Placenta growth factor Human genes 0.000 description 3
- 102100038277 Prostaglandin G/H synthase 1 Human genes 0.000 description 3
- 101710120037 Toxin CcdB Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 102100038217 Vascular endothelial growth factor B Human genes 0.000 description 3
- 102100038234 Vascular endothelial growth factor D Human genes 0.000 description 3
- 210000000709 aorta Anatomy 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 210000003414 extremity Anatomy 0.000 description 3
- 239000003517 fume Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 239000012146 running buffer Substances 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 2
- SPMVMDHWKHCIDT-UHFFFAOYSA-N 1-[2-chloro-4-[(6,7-dimethoxy-4-quinolinyl)oxy]phenyl]-3-(5-methyl-3-isoxazolyl)urea Chemical compound C=12C=C(OC)C(OC)=CC2=NC=CC=1OC(C=C1Cl)=CC=C1NC(=O)NC=1C=C(C)ON=1 SPMVMDHWKHCIDT-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010037464 Cyclooxygenase 1 Proteins 0.000 description 2
- 108010037462 Cyclooxygenase 2 Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102100031968 Ephrin type-B receptor 2 Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102000038030 PI3Ks Human genes 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- 108050003267 Prostaglandin G/H synthase 2 Proteins 0.000 description 2
- 241000713311 Simian immunodeficiency virus Species 0.000 description 2
- 108010053100 Vascular Endothelial Growth Factor Receptor-3 Proteins 0.000 description 2
- 102000016663 Vascular Endothelial Growth Factor Receptor-3 Human genes 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- BNBQRQQYDMDJAH-UHFFFAOYSA-N benzodioxan Chemical group C1=CC=C2OCCOC2=C1 BNBQRQQYDMDJAH-UHFFFAOYSA-N 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229920003123 carboxymethyl cellulose sodium Polymers 0.000 description 2
- 229940063834 carboxymethylcellulose sodium Drugs 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- KRVSOGSZCMJSLX-UHFFFAOYSA-L chromic acid Substances O[Cr](O)(=O)=O KRVSOGSZCMJSLX-UHFFFAOYSA-L 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 230000005757 colony formation Effects 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000011978 dissolution method Methods 0.000 description 2
- 230000007783 downstream signaling Effects 0.000 description 2
- 230000003511 endothelial effect Effects 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical compound C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 description 2
- 239000003365 glass fiber Substances 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 229920002674 hyaluronan Polymers 0.000 description 2
- 229960003160 hyaluronic acid Drugs 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 210000005075 mammary gland Anatomy 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 150000008442 polyphenolic compounds Chemical class 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 229960001755 resorcinol Drugs 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229960000940 tivozanib Drugs 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- YFDSDPIBEUFTMI-UHFFFAOYSA-N tribromoethanol Chemical compound OCC(Br)(Br)Br YFDSDPIBEUFTMI-UHFFFAOYSA-N 0.000 description 2
- 229950004616 tribromoethanol Drugs 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 210000001325 yolk sac Anatomy 0.000 description 2
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- LDRHUYYUJOKLAQ-UHFFFAOYSA-N 5-[2-[3-(2-hydroxy-3,5-dimethoxyphenyl)-2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]ethenyl]benzene-1,3-diol Chemical compound OC1=C(C=C(C=C1OC)OC)C1OC2=C(OC1CO)C=CC(=C2)C=CC=1C=C(C=C(C=1)O)O LDRHUYYUJOKLAQ-UHFFFAOYSA-N 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 241000167854 Bourreria succulenta Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101100381481 Caenorhabditis elegans baz-2 gene Proteins 0.000 description 1
- 101100028791 Caenorhabditis elegans pbs-5 gene Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010027458 Metastases to lung Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108050003243 Prostaglandin G/H synthase 1 Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 101100372762 Rattus norvegicus Flt1 gene Proteins 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 206010045453 Umbilical cord short Diseases 0.000 description 1
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 210000001643 allantois Anatomy 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000002376 aorta thoracic Anatomy 0.000 description 1
- 210000002403 aortic endothelial cell Anatomy 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 238000012925 biological evaluation Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000019693 cherries Nutrition 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000010293 colony formation assay Methods 0.000 description 1
- 239000000039 congener Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 229960002986 dinoprostone Drugs 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000013401 experimental design Methods 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
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 102000057840 human FLT4 Human genes 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 229930013686 lignan Natural products 0.000 description 1
- 235000009408 lignans Nutrition 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 210000001077 lymphatic endothelium Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000012634 optical imaging Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 210000001243 pseudopodia Anatomy 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000006884 regulation of angiogenesis Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001875 tumorinhibitory effect Effects 0.000 description 1
- 230000004218 vascular function Effects 0.000 description 1
- 230000007998 vessel formation Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
Definitions
- the present invention relates to a new application of polyphenolic compound Aiphanol, in particular to the related application of Aiphanol as a VEGFR kinase inhibitor.
- VEGF Vascular Endothelial Growth Factor
- VEGFA is widely expressed in a variety of tissues and organs, and it is also the main factor that promotes the growth of tumor blood vessels; VEGFB is mainly expressed in muscle and heart; VEGFC is expressed in lymphatic vessels and is the first lymphatic growth factor discovered; VEGFD and VEGFC Similar in structure, but mainly expressed in normal tissues, including heart, lung, skeletal muscle, colon, etc.
- VEGF can strongly promote the mitosis of vascular endothelial cells and the formation of new blood vessels. It is the most effective stimulating factor of tumor angiogenesis and has a close relationship with the growth, development and metastasis of tumors. These biological functions of VEGF are realized by binding to VEGF receptors on the membrane of vascular endothelial cells to trigger intracellular signal transduction.
- VEGFR vascular endothelial growth factor receptors
- Flt-1 VEGFR1
- VEGFR2 VEGFR2
- VEGFR3 VEGFR3
- VEGF receptors belong to membrane proteins of the receptor tyrosine kinase superfamily, and their intracellular domains belong to the tyrosine kinase domain.
- VEGFA, VEGFB, and PlGF can combine with VEGFR1 to stimulate downstream signals;
- VEGFR2 mainly binds with VEGFA and VEGFE, and is the main receptor that mediates VEGF activity.
- the activation of VEGFR1 and VEGFR2 and related downstream signaling pathways can effectively promote the proliferation of vascular endothelial cells and the formation of new blood vessels.
- VEGFR3 combines with VEGFC and VEGFD, and is mainly closely related to the formation of lymphatic vessels (Shibuya, M.; Claesson-Welsh, L., Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis. Exp Cell Res 2006, 312, (5) , 549-60.).
- Aiphanol has the activity of inhibiting cyclooxygenase 1 (Cyclooxygenase-1, COX-1) and cyclooxygenase 2 (Cyclooxygenase-2, COX-2) (Banwell MG, Bezos A, Chand S, et al. Convergent synthesis and preliminary biological evaluations of the stilbenolignan(+/-)-aiphanol and various congeners.Org Biomol Chem.2003;1(14):2427-2429.Lisa I.Pilkington,David Barker.Synthesis and biology of 1,4 -benzodioxane lignan natural products.
- COX2 can promote the production of prostaglandin E2 (PGE2), and PGE2 can up-regulate the expression of VEGF. Therefore, inhibiting COX2 activity can down-regulate the expression of VEGF to a certain extent, and then indirectly affect angiogenesis (Wu G, Luo J, Rana JS, Laham R, Sellke FW, Li J. Involvement of COX-2 in VEGF-induced angiogenesis via P38and JNK pathways in vascular endothelial cells. Cardiovasc Res. 2006; 69(2): 512-9), suggesting that Aiphanol may participate in the anti-angiogenic process by inhibiting COX2.
- One object of the present invention is to provide a new application of the compound Aiphanol.
- Aiphanol can bind to VEGFR and has kinase inhibitory activity, and can be used as a VEGFR kinase inhibitor.
- the present invention finds that Aiphanol can directly bind to VEGFR2 and VEGFR3, and effectively inhibit the kinase activity of VEGFR1, VEGFR2 and VEGFR3; cell proliferation and movement;
- Aiphanol can inhibit angiogenesis in four in vivo models including vascular fluorescence transgenic zebrafish model, chicken embryo chorioallantoic membrane model, mouse aortic ring angiogenesis model, and glue plug experiment;
- Aiphanol can inhibit including Lymphangiogenesis in three models of 3D lymphoid spheroid formation experiment, glue plug experiment, and in vitro lymph node sprouting experiment; mouse colon cancer MC38 tumor-bearing growth model further verified that Aiphanol can participate in anti-angiogenesis by binding to and inhibiting the activity of VEG
- the present invention provides the use of Aiphanol in the preparation of preparations that directly bind to VEGF receptors and/or inhibit the kinase activity of VEGF receptors.
- the VEGF receptors include one or more of VEGFR1, VEGFR2, VEGFR3.
- the present invention also provides the application of Aiphanol in the preparation of preparations for inhibiting tubule formation of vascular endothelial cells and/or lymphatic endothelial cells.
- the present invention also provides the application of Aiphanol in preparing preparations for inhibiting proliferation, migration and invasion of vascular endothelial cells and/or lymphatic endothelial cells.
- the vascular endothelial cells are human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVEC), and the lymphatic endothelial cells are human dermal lymphatic endothelial cells (Human dermal lymphatic endothelial cells). endothelial cells, HDLEC).
- the present invention also provides the application of Aiphanol in the preparation of anti-lymphangiogenesis preparation.
- the formulations are intended to target VEGF receptors for anti-lymphangiogenesis.
- Some specific experiments of the present invention have confirmed that Aiphanol can target VEGFR3 to inhibit lymphatic angiogenesis, thereby inhibiting tumor lymphatic metastasis. It shows that Aiphanol can be used as an inhibitor of VEGFR kinase, and can be used for the treatment of related diseases by anti-lymphangiogenesis.
- the preparation for anti-lymphangiogenesis of the present invention can be further used to prevent (prevent and/or treat) lymphangiogenesis-related diseases through anti-lymphangiogenesis
- the lymphangiogenesis-related diseases can be, for example, Including Kaposi sarcoma, and/or tumor lymphatic metastasis, etc.
- the present invention also provides the application of Aiphanol in the preparation of preparations for targeting VEGF receptors and anti-angiogenesis.
- Some specific experiments of the present invention confirm that Aiphanol can simultaneously target the kinase activity of VEGFR1 and VEGFR2 to inhibit angiogenesis and tumor growth. It shows that Aiphanol can be used as an inhibitor of VEGFR kinase, and can be used for the treatment of related diseases through anti-angiogenesis.
- the preparation of the present invention for targeting VEGF receptors to prevent angiogenesis can be further used to prevent and treat angiogenesis-related diseases by anti-angiogenesis, and the angiogenesis-related diseases can include, for example, tumor angiogenesis Hyperplasia and metastasis, and/or diabetic macular degeneration, etc.
- the Aiphanol of the present invention as an inhibitor of VEGFR kinase, can be used for the treatment of related diseases by simultaneously anti-angiogenesis and anti-lymphangiogenesis.
- the preparations described in the present invention include experimental preparations (reagents), which are pharmaceutical preparations when used in living individuals.
- the present invention also provides a VEGFR kinase inhibitor comprising Aiphanol.
- the present invention also provides a medicine for preventing and treating lymphangiogenesis-related diseases and/or angiogenesis-related diseases, which includes the active component Aiphanol.
- the medicament may also include a pharmaceutically acceptable carrier.
- the present invention also provides a method for preventing and treating lymphangiogenesis-related diseases and/or angiogenesis-related diseases, the method comprising administering an effective amount of Aiphanol to a subject in need.
- Aiphanol targets VEGFR to inhibit angiogenesis and/or inhibit lymphangiogenesis.
- the surface plasmon resonance (SPR) test found that the compound Aiphanol has strong affinity with VEGFR2 and VEGFR3, and the Kd is 9.76 ⁇ M and 3.2 ⁇ M, respectively.
- Molecular docking analysis showed that the binding energies of Aiphanol to VEGFR2 and VEGFR3 were -10.57kcal/mol and -10.86kcal/mol, respectively. A strong binding ability.
- the ADP-Glo TM experiment evaluated the effect of Aiphanol on the activity of VEGFR2 and VGEFR3 kinases, and the IC50 values were 0.92 ⁇ M and 0.29 ⁇ M, respectively, indicating that the compound had a relatively high catalytic activity on the two kinases. strong inhibitory effect.
- Western blot analysis confirmed that Aiphanol can down-regulate the phosphorylation levels of VEGFR1/PI3K/AKT and VEGFR2/AKT/ERK signaling pathway-related molecules activated by VEGF in HUVEC, and the time and / or concentration dependence.
- Immunoprecipitation experiments verified that Aiphanol could downregulate VEGF-C-induced VEGFR3 phosphorylation in HDLEC.
- human umbilical vein endothelial cells (HUVEC) and human dermal lymphatic endothelial cells (HDLEC) are used as research models to evaluate the effects of Aiphanol on angiogenesis and lymphangiogenesis, respectively.
- the present invention adopts classical tubule formation experiments to detect and find that Aiphanol inhibits the tubular structure formation of two endothelial cells in a concentration-dependent manner.
- the effect of Aiphanol on the proliferation of HUVEC and HDLEC was detected, and the results showed that Aiphanol had a certain inhibitory effect on the proliferation of the two types of cells in a time- and concentration-dependent manner.
- the effect of Aiphanol on the migration of HUVEC and HDLEC was detected by the scratch healing experiment, and the results showed that Aiphanol could effectively inhibit the migration of the two kinds of cells, with a dose-effect trend.
- Migration and invasion experiments verified that Aiphanol could inhibit the migration and invasion of HUVEC and HDLEC, and the results were consistent with the scratch healing experiments.
- the anti-angiogenic effect is evaluated by analyzing the area of the zebrafish intestinal venous plexus (Subintestinal vessels, SIVs) after drug treatment in the vascular fluorescence transgenic zebrafish model, and the concentration is 2.8 ⁇ M, 8.3
- the areas of inferior intestinal vessels in the ⁇ M Aiphanol group were 22664 and 20331 pixels, respectively, compared with the area of inferior intestinal vessels (26775 pixels) in the control group, p ⁇ 0.01, and the anti-angiogenic effects were 15% and 24%, indicating that Aiphanol Can effectively inhibit the angiogenesis of SIVs in zebrafish embryos.
- Matrigel matrigel containing or not containing VEGF and Aiphanol was implanted subcutaneously in C57 mice.
- the expression of CD31 a marker that can reflect new capillaries, was significantly up-regulated, and Aiphanol could effectively inhibit the infiltration and growth of blood vessels into Matrigel in vivo.
- VEGF-induced angiogenesis and the expression of CD31 were significantly weakened. It was also found in the chicken embryo chorioallantoic membrane experiment that Aiphanol can effectively inhibit the growth of embryonic blood vessels, especially capillaries, in a dose-dependent manner.
- Aiphanol can significantly inhibit the formation of sprouted blood vessels by vascular endothelial cells in the aorta in a 3D culture system, as detected by the mouse aortic ring angiogenesis experiment, and the trend is dose-dependent.
- the results of the above four models all indicate that Aiphanol has good anti-angiogenic activity in vivo.
- lymphocyte sprouting experiments it is found through lymphocyte sprouting experiments that Aiphanol effectively antagonizes the formation of lymphatic vessels, and the inhibitory ability is stronger than that of the VEGFR3 specific inhibitor SAR131675.
- the level of lymphangiogenesis marker Lyve-1 in the Aiphanol group was significantly reduced, indicating that Aiphanol can down-regulate the infiltration and growth of lymphatic vessels induced by VEGF-C in mice.
- the fresh lymph nodes of mice were removed for 3D system culture, and the results of fluorescent staining showed that Aiphanol could effectively inhibit the formation of new lymphatic vessels and the expression of Lyve-1.
- the present invention also detects the effect of Aiphanol on the metastasis of tumor cells in vivo through the in vivo lymphatic metastasis model of mouse breast cancer cell 4T1-luc with luciferase. , The metastasis of kidney organs, the inhibitory ability is stronger than that of VEGFR3 specific inhibitor SAR131675.
- the above results show that Aiphanol has a good anti-lymphangiogenesis effect in vivo, and can exert anti-tumor lymphatic metastasis effect through this effect.
- Aiphanol can be combined with VEGFR and can be used as a VEGFR kinase inhibitor.
- Aiphanol can target VEGFR to inhibit angiogenesis and lymphangiogenesis, and then can be used for the prevention of angiogenesis-related diseases and lymphangiogenesis-related diseases with treatment.
- FIG. 1A to Figure 1D show the experimental results of Aiphanol directly binding to VEGFR2 and VEGFR3.
- FIG. 1A to Figure 2E show the experimental results of Aiphanol inhibiting the activity of VEGFR1, VEGFR2 and VEGFR3.
- Figure 3A to Figure 3B show the experimental results of Aiphanol inhibiting tubule formation of vascular endothelial cells and lymphatic endothelial cells.
- Figure 4A to Figure 4F show the experimental results of Aiphanol inhibiting the proliferation and movement of vascular endothelial cells and lymphatic endothelial cells.
- FIGS 5A to 5D show the experimental results of the anti-angiogenic activity of Aiphanol in different models.
- Figure 6A to Figure 6C show the experimental results of the anti-lymphangiogenesis activity of Aiphanol in different models.
- Figure 7A to Figure 7D show the experimental results of Aiphanol exerting anti-tumor growth and metastasis effects by inhibiting angiogenesis and lymphangiogenesis.
- Example 1 Aiphanol directly binds to VEGFR2 and VEGFR3
- VEGFR2 is mainly expressed in vascular endothelial cells and is the main receptor of VEGF, and VEGFA/VEGFR2 is the most important pathway for inducing angiogenesis.
- the expression of VEGFR3 is mainly limited to the adult lymphatic endothelium, and is considered to control lymphangiogenesis. Its main ligand VEGF-C can promote the proliferation and migration of lymphatic endothelial cells after activation of VEGFR-3.
- VEGF-C/VEGFR3 is a regulator of lymphatic Generated key signaling pathways. In this embodiment, the binding ability of Aiphanol to VEGFR2 and VEGFR3 was detected by surface plasmon resonance technology.
- Ligand-receptor interaction is a process of molecular recognition, mainly including electrostatic interaction, hydrogen bond interaction, hydrophobic interaction, van der Waals interaction, etc. Through calculation, the binding mode and affinity between the two can be predicted.
- the interaction modes of the small molecule Aiphanol and different proteins were analyzed in detail by molecular docking technology.
- Protein preparation Import the co-crystal structure of VEGFR2 and its inhibitor Tivozanib (PDB ID: 4ASE); use the CLUSTALW program to compare the sequences of VEGFR2 and VEGFR3, and the results show that the sequence homology between the two is about 71.5%, and then use the SWISS-MODEL program to carry out homology modeling of VEGFR3 to obtain a reasonable three-dimensional structure model of human VEGFR3 protein that can be used for subsequent molecular recognition research.
- Use the Protein Preparation Wizard Panel module to process the three-dimensional protein model: delete B chain, dehydrate, hydrogenate, repair missing residues, optimize structure, minimize energy, etc. (OPLS2005 force field, RMSD).
- the high-precision (XP) mode of the Glide module is used for molecular docking, that is, the receptor and ligand molecules are docked with each other through geometric matching and energy matching. After outputting the results, 2D and 3D mapping are performed on the binding mode of the protein and the small molecule compound.
- the molecular docking results of Aiphanol and VEGFR2 showed that the binding energy between the small molecule and VEGFR2 was -10.57kcal/mol, indicating that the compound had a strong binding force with the protein.
- the small molecule can form 4 hydrogen bonds and 1 salt bridge with VEGFR2: the OH on the resorcinol in the Aiphanol structure acts as the hydrogen bond donor and the hydrogen bond acceptor Glu885 and Asp1046 respectively, and the distances are respectively.
- the OH on the hydroxyl group of the alcohol acts as a hydrogen bond donor to form a hydrogen bond with Lys920, and the distance is
- the O on the methoxy group acts as a hydrogen bond acceptor to form a hydrogen bond with Asn923 at a distance of
- the benzene ring on the resorcinol in the Aiphanol structure can form a salt bridge with Lys868 (Fig. 1B).
- the molecular docking results of Aiphanol and VEGFR3 showed that the binding energy was -10.86kcal/mol, and most of the small molecules were located in the hydrophobic cavity of VEGFR3.
- Small molecules mainly bind in the hydrophobic cavity composed of Leu851, Ala877, Lys879, Glu896, Leu900, Val925, Val927, Glu928, Phe929, Cys930, Lys931, Tyr932, Asn934, Asn937, Leu1044, Asp1055, Phe1056.
- the OH on the benzene ring at both ends of Aiphanol forms three hydrogen bonds with Asn934, Asn937 and Asp1055 respectively, and the hydroxymethyl group on benzo1,4-dioxane forms two hydrogen bonds with Cys930 and Lys931.
- the two benzene rings and the benzo1,4-dioxane form a comparatively strong structure with the hydrophobic amino acids around the pocket (Leu851, Ala877, Lys879, Glu896, Leu900, Val925, Val927, Glu928, Phe929, Tyr932, Leu1044, Phe1056).
- the strong hydrophobic interaction further enhanced the affinity between Aiphanol and VEGFR3 (Fig. 1D).
- Example 2 Aiphanol can effectively inhibit the activity of VEGFR1, VEGFR2 and VEGFR3
- the main experimental methods include ADP-Glo TM assay and western blot.
- ADP-Glo TM assay principle is a luminescent kinase detection method, which detects the amount of ADP formed in the kinase reaction to reflect the kinase activity.
- the remaining ATP in the kinase reaction will be first consumed by the ADP-Glo reagent; the ADP generated in the kinase reaction will be reduced to ATP by the kinase detection reagent; Under the action, it reacts with fluorescein to emit light, and the luminescent signal is positively correlated with the kinase activity.
- the fluorescence signal value was converted to kinase activity and IC50 value was calculated.
- RLUS Luminescence value of the sample well (compound to be tested)
- Bind the antibody to protein G Sepharose beads Take out 20 ⁇ L protein G Sepharose 4B (suspension volume in the original package), suspend in 500 ⁇ L PBS, centrifuge at 1500rpm for 30s, discard the supernatant, then add antibody and 500 ⁇ L PBST, and keep at room temperature Shake for 1h, centrifuge at 1500rpm for 30s, slowly discard the supernatant, and the precipitate is the Sepharose beads bound to the antibody, which is reserved for immunoprecipitation.
- TritonX100/PBS containing protease inhibitors such as Cocktail and PMSF was added to extract cell proteins.
- Add buffer add 2 ⁇ SDS loading buffer, centrifuge at 14000rpm for 10s, heat at 95°C for 10min, centrifuge at 14000rpm for 10s, absorb the supernatant for electrophoresis;
- the results of Western blot experiments in Figure 2A and Figure 2C verified that Aiphanol can inhibit the phosphorylation levels of VEGFR1/PI3K/AKT and VEGFR2/AKT/ERK signaling pathway-related molecules activated by VEGF in HUVECs in a time- and/or concentration-dependent manner.
- the ADP-Glo TM assay results in Figure 2B and Figure 2D show that Aiphanol can effectively inhibit the catalytic activity of VEGFR2 and VEGFR3 kinases, with IC 50 values of 0.92 ⁇ M and 0.29 ⁇ M, respectively.
- the IP experiment results in Figure 2E showed that Aiphanol inhibited VEGF-C-activated VEGFR3 phosphorylation in a concentration-dependent manner in HDLEC.
- Example 3 Aiphanol inhibits tubule formation of vascular endothelial cells and lymphatic endothelial cells
- Tube formation test method 50 ⁇ L/well of Matrigel matrigel was coated on a 96-well plate, and stood in a 37° C. incubator for 30 minutes until it solidified. Digest and resuspend HUVEC cells in serum-free medium containing VEGF, drug or solvent control, VEGF antibody Bevacizumab (Bevacizumab), VEGFR3 specific inhibitor SAR131675 as positive control group, take 100 ⁇ L of cell suspension/well on Matrigel surface. After culturing in the incubator for 6 hours, the formation of tubules was observed under an inverted microscope and photographed, and the number of meshes was quantitatively analyzed using the Angiogenesis analyzer of Image J.
- VEGF antibody Bevacizumab Bevacizumab
- VEGFR3 specific inhibitor SAR131675 as positive control group
- Figure 3A shows the tubule formation assay results of HUVECs.
- VEGF can stimulate cells to form three-dimensional tubular structures similar to vascular lumens
- Bevacizumab can inhibit the formation of tubular structures
- Aiphanol inhibits the formation of tubular structures of vascular endothelial cells in a concentration-dependent manner in a 3D culture system.
- the tubular structure of the cells was interrupted, the mesh structure was reduced, the cells formed semi-tubular shape, and even shriveled and scattered on Matrigel in the form of islands, indicating that Aiphanol can inhibit the formation of tubular structure of vascular endothelial cells.
- Figure 3B shows the results of the tubule formation assay of HDLEC.
- the results showed that VEGF-C can promote the formation of three-dimensional tubular structure similar to the lumen of lymphatic vessels, and SAR131675 has a certain inhibitory effect on the formation of lymphatic vessel network structure.
- Aiphanol dose-dependently inhibited the formation of lymphatic vessels induced by VEGF-C.
- the lymphatic endothelial cell junctions decreased, and the cells were distributed in short cords on the matrigel, and the tubular structure was interrupted or even disappeared, indicating that Aiphanol can inhibit lymphatic vessel formation.
- Tubular structures of skin cells form.
- Embodiment 4 Aiphanol inhibits the proliferation and movement of vascular endothelial cells and lymphatic endothelial cells
- the cells were digested and resuspended in drug-containing serum-free medium, and 800 ⁇ L of 10% FBS-containing medium was added to the lower chamber, and 2 ⁇ 10 4 cells/200 ⁇ L (migration), 4 ⁇ 10 4 cells/500 ⁇ L ( Invasion) serum-free culture solution was added to the upper chamber, placed in an incubator for 24 hours, fixed in ice methanol for 30 minutes, stained with 0.1% crystal violet for 30 minutes, wiped off the non-migrated cells in the upper layer of the chamber with a cotton swab, sealed with neutral gum and photographed And count the number of cells.
- Figure 4A to Figure 4B show the results of cell confluence detection, and the results show that Aiphanol has a certain inhibitory effect on the proliferation of HUVEC and HDLEC in a time- and concentration-dependent manner.
- Figure 4C to Figure 4D show the results of scratch healing experiments, the results showed that Aipahnol inhibited the migration of HUVEC and HDLEC in a dose-dependent manner.
- Figure 4E to Figure 4F show the results of Transwell experiments. The results show that Aiphanol can inhibit the migration and invasion of HUVEC and HDLEC, with a dose-effect trend. When the concentration of Aiphanol is 30 ⁇ M, the number of cell migration and invasion is significantly reduced. The results confirmed that Aiphanol can inhibit the proliferation and motility of two endothelial cells.
- vascular fluorescence transgenic zebrafish model vascular fluorescence transgenic zebrafish model
- glue plug experiment mouse aortic ring angiogenesis experiment
- chicken embryo chorioallantoic membrane experiment to analyze the level of angiogenesis after drug Aiphanol treatment and evaluate its anti-angiogenesis effect.
- the vascular fluorescence transgenic zebrafish obtained embryos by natural pairing. The embryos were cleaned at 6h and 24h after fertilization, dead embryos were removed, and suitable embryos were selected according to the development of the embryos.
- the experimental design was determined according to the results of the pre-experiment: a total of 4 groups (1) DMSO solvent control group; (2) VEGF induction group: 500ng/mL; (3) Aiphanol group: 15 ⁇ M; (4) VEGF+Aiphanol group, 4 animals in each group mouse.
- a volume of 400 ⁇ L Matrigel was injected subcutaneously into the right abdomen of C57 female mice. After 2 weeks, the mice were dissected, and the plugs were taken out to take pictures. HE staining and immunohistochemical methods were used to detect the microvessel density in the plugs.
- Paraffin embedding adjust the temperature of the wax box to 65°C, soak in wax I for 30 minutes, and soak in wax II for 30 minutes, pick up the tissue with tweezers and place it in a stainless steel mold, pour the wax liquid until it overflows slightly, adjust the tissue position to the center of the mold, and cover carefully Put on a preheated embedding box to avoid air bubbles, and let the paraffin tissue solidify at room temperature overnight.
- Block endogenous peroxidase activity add 3% H 2 O 2 dropwise on the surface of the slice, and leave it at room temperature for 15 minutes.
- Primary antibody reaction Discard the blocking solution, add primary antibody (CD31, 1:100, Cell Signaling Technology) dropwise, 50 ⁇ L/tablet, and react overnight at 4°C in a wet box.
- primary antibody CD31, 1:100, Cell Signaling Technology
- Secondary antibody reaction add HRP-labeled secondary antibody dropwise, 50 ⁇ L/tablet, and react at room temperature for 1 hour.
- DAB color development Freshly prepare DAB color development solution, drop it on the glass slide, stop the reaction immediately when brown particles appear with the naked eye, and control the color development time within 10 minutes.
- Matrigel was placed in a 4°C freezer overnight to slowly melt, and the tip of the gun and the 48-well plate were pre-cooled in advance.
- Adipose tissue and connective tissue Adipose tissue and connective tissue.
- Serum-free medium containing or not containing VEGF and Aiphanol was added, 500 ⁇ L/well. Replace with a new one every 2 days
- Figure 5A shows the results of the vascular fluorescence transgenic zebrafish model. Compared with the area of intestinal blood vessels (26775 pixels) in the solvent group, the areas of intestinal blood vessels in the Aiphanol group with concentrations of 2.8 ⁇ M and 8.3 ⁇ M were 22664 pixels and 20331 pixels, respectively. The anti-angiogenic effect was 15% and 24%, and the difference was statistically significant (p ⁇ 0.01). The results showed that Aiphanol could inhibit the angiogenesis of the inferior intestinal venous plexus in zebrafish embryos at both concentrations.
- VEGF can induce the infiltration and growth of blood vessels in the mouse body into Matrigel, the color of the plug turns red, and the formation of blood vessels decreases after treatment with Aiphanol.
- HE staining showed that there were more vascular endothelial cells in the VEGF group, and the infiltrating vascular endothelial cells decreased after combined with Aiphanol.
- CD31 staining the VEGF group formed larger blood vessels (brown, indicated by the red arrow), while the blood vessels were significantly reduced after combined with Aiphanol. The results suggest that Aiphanol can inhibit angiogenesis in vivo.
- FIG. 5C The results of mouse aortic ring angiogenesis experiments show that VEGF can promote the germination and growth of vascular endothelial cells in the aorta in a three-dimensional culture system, forming a slender and vigorous microvascular plexus, and Bevacizumab in the positive control group can inhibit angiogenesis to a certain extent Aiphanol can inhibit the formation of microvessels induced by VEGF, and has a dose-dependent trend. When the concentration reaches 30 ⁇ M, vascular endothelial cells basically lose the ability to form microvessels, showing a state of "microvascular plexus collapse".
- FIG. 5D The results of chicken embryo chorioallantoic membrane experiments show that VEGF can promote the formation of embryonic blood vessels, especially microvessels, Bevacizumab can effectively reduce the number of microvessels formed, and Aiphanol inhibits the formation of microvessels in the corresponding parts in a concentration-dependent manner.
- the number and diameter of blood vessels in and around the blood vessels are all down-regulated, and even lead to atrophy of the blood vessels in situ.
- Example 6 Aiphanol has anti-lymphangiogenesis activity in different models
- Lyve-1 Lymphatic Vessel Endothelial Receptor-1
- Lymphatic Vessel Endothelial Receptor-1 is the main receptor of hyaluronic acid (HA) in lymphatic endothelial cells, which is mainly limited to lymphatic endothelial cells and is generally expressed in initial lymphatic
- HA hyaluronic acid
- Upper matrix including lymphoid spheroids: Gently blow down the hanging drop spheroids with PBS and collect them in a 50mL centrifuge tube with gentle movements to avoid spheroid breakage; centrifuge at 170g for 5min; discard the supernatant, and use fetal bovine serum ( FBS) to resuspend the spheres; prepare the matrix according to step 3), replace the DMEM basal medium with the sphere-containing FBS to prepare the above matrix, and keep the other conditions unchanged; take the matrix mixture and add it to the upper layer of the 48-well plate, and put it in a 37°C incubator for 2 -3h to be solidified.
- FBS fetal bovine serum
- mice Seven-week-old C57 female mice were randomly divided into four groups, and the abdomen of the mice was shaved to expose the abdomen.
- Matrigel matrigel containing 0.5 ⁇ g/mL VEGF-C and/or 30 ⁇ M SAR131675/Aiphanol was prepared, stored on ice, and the Matrigel mixture was injected into the abdomen of mice subcutaneously with a pre-cooled 1mL syringe, 250 ⁇ L/mouse. After 8 days, the mice were dissected, the plugs were taken out and photographed, some of the plugs were quickly frozen in liquid nitrogen, and frozen sections were performed by professional pathologists. The level of Lyve-1 in the gel plug was analyzed by sectioning combined with immunofluorescence method.
- Matrigel was added to the upper layer, 60 ⁇ L/dish, fully solidified at 37°C for 3 hours. Add complete medium containing VEGF-C, drug or solvent control, culture for 7 days, and change the medium every 2-3 days.
- FIG. 6A results of lymphoid spheroid sprouting experiments show that VEGF-C can significantly induce lymphoid spheroid sprouting in 3D culture system and form abundant pseudopodia. Tube formation, Aiphanol has stronger inhibitory ability than SAR131675.
- FIG. 6B The results of the glue plug experiment showed that compared with the control group, the green fluorescence intensity of the VEGF-C group was enhanced, and the expression of the lymphangiogenesis marker Lyve-1 was up-regulated. Both SAR131675 and Aiphanol could down-regulate the level of Lyve-1, and the down-regulation effect of Aiphanol Stronger than SAR131675.
- Example 7 Aiphanol exerts anti-tumor growth and metastasis effects by inhibiting angiogenesis and lymphangiogenesis
- angiogenesis the drug-resistant mouse colon cancer cell line MC38 of Aiphanol was used as the research object, and MC38 was injected into the tumor-bearing mice subcutaneously, and the volume change of the tumor after administration was observed, and the drug effect was observed.
- lymphangiogenesis mouse breast cancer cells 4T1-luc with luciferase were injected into mouse mammary gland fat pads to carry out lymphatic metastasis experiments in vivo, and the effect of Aiphanol on tumor cell metastasis in vivo was observed.
- MC38 cells in the logarithmic growth phase were taken, digested with 0.25% trypsin to make them into single cells, and the cell density was adjusted with complete culture medium.
- Administration was divided into two groups: solvent control, Aiphanol (30mg/kg), 7 mice in each group.
- Oral administration, drug dissolution method dissolve the compound in DMSO, dilute in PBS, add a little 1M NaOH to induce dissolution, and administer once/100 ⁇ L/d for a total of 12 days.
- mice were killed by neck dissection, the tumors were dissected, and the tumor mass was weighed.
- mice were anesthetized with tribromoethanol, luciferase substrate was injected intraperitoneally, and the mice were placed into the IVIS Spectrum small animal in vivo optical imaging system for imaging. Then the mice were dissected, and the limbs, axillary lymph nodes, inguinal lymph nodes, heart, liver, spleen, lung, and kidney organs were removed for imaging, and the fluorescence intensity was counted and drawn.
- Figure 7B MC38 cell tumor-bearing growth test results show that the tumor mass in the Aiphanol group is lower than that in the control group, p ⁇ 0.01, and the tumor inhibition rate is 57.7%, indicating that Aiphanol has a good tumor inhibitory effect.
- Figure 7C The results of frozen section combined with immunofluorescence showed that in the orthotopic tumor tissue of the control group, there were obvious cord-like blood vessels with high fluorescence intensity, while in the Aiphanol group, the number of continuous cord-like blood vessels was reduced, and the fluorescence intensity was significantly weakened.
- the expression of CD31 was down-regulated. The above results indicated that Aiphanol inhibited the growth of tumor by inhibiting angiogenesis in tumor tissue.
- Figure 7D The results of in vivo lymphatic metastasis experiment of luciferase-labeled mouse breast cancer cell 4T1-luc showed that the fluorescence intensity of limb lymph nodes in the control group was high, indicating that the tumor cells had a high metastasis rate, and there was also some metastasis in lung tissue; VEGFR3
- the specific inhibitor SAR131675 has a certain inhibitory effect on tumor metastasis in limb lymph nodes, axillary and inguinal lymph nodes, spleen and kidney, but has no inhibitory effect on lung metastasis; SAR131675 has an obvious inhibitory effect on metastasis, and the inhibitory effect is stronger than that of SAR131675.
- the inhibitory effect is enhanced, and it also has a certain inhibitory effect on tumor metastasis in the spleen and kidney.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Diabetes (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Oncology (AREA)
- Ophthalmology & Optometry (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Epidemiology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Aiphanol作为VEGFR激酶抑制剂的应用。具体地，提供了Aiphanol在制备直接结合VEGF受体和/或抑制VEGF受体激酶活性的制剂中的应用。Aiphanol可结合VEGFR，能作为VEGFR激酶抑制剂，可靶向VEGFR抑制血管生成和抑制淋巴管生成，进而可用于血管增生相关性疾病、淋巴管生成相关疾病的预防与治疗。
Description
本发明是关于多酚类化合物Aiphanol的新应用，具体地说，是关于Aiphanol作为VEGFR激酶抑制剂的相关应用。
血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)是公认的主要诱导血管生成的物质。VEGF于1983年被Senger等人发现，并于1989年被分离并克隆出来。在哺乳动物中，VEGF家族主要包括5个成员，即VEGFA、VEGFB、VEGFC、VEGFD和placental growth factor(PlGF)。VEGFA在广泛表达于多种组织器官，同时它也是促进肿瘤血管生长的主要因子；VEGFB主要表达在肌肉和心脏；VEGFC在淋巴管上表达，是第一个发现的淋巴管生长因子；VEGFD与VEGFC结构类似，但主要表达在正常组织中，包括心、肺、骨骼肌、结肠等。VEGF能强烈地促使血管内皮细胞有丝分裂和新生血管形成，是肿瘤血管生成的最有效刺激因子，与肿瘤的生长发育和转移具有密切的关系。而VEGF这些生物学功能则是通过结合于血管内皮细胞膜上的VEGF受体引发细胞内信号转导而实现的。
VEGF受体(VEGFR)有三种：VEGFR1(Flt-1)、VEGFR2(KDR)和VEGFR3(Flt-4)，不同的配体及受体所介导的功能有所差异(Olsson,A.K.；Dimberg,A.；Kreuger,J.；Claesson-Welsh,L.,VEGF receptor signalling-in control of vascular function.Nat Rev Mol Cell Biol 2006,7,(5),359-71.)。VEGF受体属于受体酪氨酸激酶超家族膜蛋白，其胞内区属于酪氨酸激酶区，被VEGF激活后可自身磷酸化，进而启动下游信号通路。VEGFA、VEGFB和PlGF可以与VEGFR1结合激发下游信号；VEGFR2则主要与VEGFA和VEGFE结合，是介导VEGF活性的主要受体。VEGFR1和VEGFR2及下游相关信号通路的激活可有效促进血管内皮细胞的增殖与新生血管的形成。VEGFR3与VEGFC和VEGFD结合，主要与淋巴管的形成密切相关(Shibuya,M.；Claesson-Welsh,L.,Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis.Exp Cell Res 2006,312,(5),549-60.)。
多酚类化合物Aiphanol，化学名5-[2-[3-(hydroxy-3,5-dimethoxyphenyl)-2-hydroxymethyl-2,3-dihydrobenzo[1,4]dioxin-6-yl]vinyl]benzene-1,3-diol，结构见式(I)。
有文献报道Aiphanol具有抑制环氧合酶1(Cyclooxygenase-1，COX-1)和环氧合酶2(Cyclooxygenase-2，COX-2)的活性(Banwell MG，Bezos A，Chand S，et al.Convergent synthesis and preliminary biological evaluations of the stilbenolignan(+/-)-aiphanol and various congeners.Org Biomol Chem.2003；1(14):2427-2429.Lisa I.Pilkington,David Barker.Synthesis and biology of 1,4-benzodioxane lignan natural products.Nat Prod Rep.2015Sep23；32(10):1369-88)。因COX2可以促进前列腺素E2(PGE2)的产生，而PGE2可以上调VEGF的表达。因此抑制COX2活性能在一定程度上下调VEGF的表达，进而间接影响血管生成(Wu G，Luo J，Rana JS，Laham R，Sellke FW，Li J.Involvement of COX-2in VEGF-induced angiogenesis via P38and JNK pathways in vascular endothelial cells.Cardiovasc Res.2006；69(2):512-9)，提示Aiphanol可能通过抑制COX2参与抗血管生成过程。
但迄今为止，尚未见Aiphanol对VEGF或其受体作用的相关报道。
发明内容
本发明的一个目的在于提供化合物Aiphanol的新应用。
本案发明人在研究中发现，Aiphanol可结合VEGFR，并具有激酶抑制活性，可作为VEGFR激酶抑制剂。具体而言，本发明发现Aiphanol可以直接结合VEGFR2和VEGFR3，并有效抑制VEGFR1、VEGFR2和VEGFR3激酶活性；Aiphanol具有较强的体外抑制血管内皮细胞及淋巴管内皮细胞的小管形成作用，并且能抑制两种细胞的增殖及运动；Aiphanol能够抑制包括血管荧光转基因斑马鱼模型、鸡胚绒毛尿囊膜模型、小鼠主动脉环血管新生模型、胶栓实验四种体内模型的血管新生；Aiphanol可以抑制包括3D淋巴球体形成实验、胶栓实验、离体淋巴结发芽实验三种模型的淋巴管生成；小鼠结肠癌MC38荷瘤生长模型进一步验证了Aiphanol可通过结合并抑制VEGFR1、VEGFR2激酶活性参与抗血管生成，进而发挥抗肿瘤生长效应；在小鼠体内通过带有荧光素酶的乳腺癌细胞4T1-luc体内淋巴道转移模型证实了Aiphanol通过结合和抑制VEGFR3激酶活性参与抗淋巴管生成，可以抑制肿瘤细胞在体内的淋巴道转移。
从而，本发明提供了Aiphanol在制备直接结合VEGF受体和/或抑制VEGF受体激酶活性的制剂中的应用。
根据本发明的具体实施方案，本发明的应用中，所述VEGF受体包括VEGFR1、VEGFR2、VEGFR3中的一种或多种。
本发明还提供了Aiphanol在制备抑制血管内皮细胞和/或淋巴管内皮细胞小管形成的制剂中的应用。
本发明还提供了Aiphanol在制备抑制血管内皮细胞和/或淋巴管内皮细胞增殖及迁移侵袭的制剂中的应用。
根据本发明的具体实施方案，本发明的应用中，所述血管内皮细胞为人脐静脉内皮细胞(Human umbilical vein endothelial cells，HUVEC)，所述淋巴管内皮细胞为人真皮淋巴管内皮细胞(Human dermal lymphatic endothelial cells，HDLEC)。
本发明还提供了Aiphanol在制备用于抗淋巴管生成的制剂中的应用。所述制剂是用于靶向VEGF受体而抗淋巴管生成。本发明的一些具体实验证实，Aiphanol可靶向VEGFR3抑制淋巴管生成进而抑制肿瘤的淋巴道转移。表明Aiphanol可作为VEGFR激酶的抑制剂，通过抗淋巴管生成用于相关疾病的治疗。换而言之，本发明的用于抗淋巴管生成的制剂，可进一步用于通过抗淋巴管生成而防治(预防和/或治疗)淋巴管生成相关疾病，所述淋巴管生成相关疾病例如可以包括Kaposi肉瘤、和/或肿瘤淋巴道转移等。
本发明还提供了Aiphanol在制备用于靶向VEGF受体而抗血管生成的制剂中的应用。本发明的一些具体实验证实，Aiphanol可同时靶向VEGFR1、VEGFR2激酶活性抑制血管生成和肿瘤生长。表明Aiphanol可作为VEGFR激酶的抑制剂，通过抗血管生成用于相关疾病的治疗。换而言之，本发明的用于靶向VEGF受体而抗血管生成的制剂，可进一步用于通过抗血管生成而防治血管增生相关性疾病，所述血管增生相关性疾病例如可以包括肿瘤血管增生与转移、和/或糖尿病性黄斑变性等。
根据本发明的具体实施方案，本发明的Aiphanol作为VEGFR激酶的抑制剂，可通过同时抗血管生成和抗淋巴管生成用于相关疾病的治疗。
本发明中所述的制剂，包括实验用制剂(试剂)，当用于有生命的个体时其为药物制剂。
从而，本发明还提供了一种VEGFR激酶抑制剂，其包括Aiphanol。
本发明还提供了一种防治淋巴管生成相关疾病和/或血管增生相关性疾病的药物，其包括活性组分Aiphanol。所述药物还可包括药学上可接受的载体。
本发明还提供了一种防治淋巴管生成相关疾病和/或血管增生相关性疾病的方法，该方法包括给予有需要的受试者有效量的Aiphanol。具体地，其中，Aiphanol作为VEGFR 激酶抑制剂靶向VEGFR抑制血管生成和/或抑制淋巴管生成。
在本发明的一些具体实施方案中，表面等离子体共振(Surface plasmon resonance，SPR)实验检测发现化合物Aiphanol与VEGFR2、VEGFR3均有较强的亲和力，Kd分别为9.76μM、3.2μM。分子对接技术分析Aiphanol与VEGFR2、VEGFR3的结合能分别为-10.57kcal/mol、-10.86kcal/mol，Aiphanol与VEGFR2、VEGFR3的结合模式相似，化合物均是与两个蛋白的ATP口袋进行结合，表现出很强的结合能力。
在本发明的一些具体实施方案中，ADP-Glo
TM实验评估了Aiphanol对VEGFR2、VGEFR3激酶活性的影响，IC
50值分别为0.92μM、0.29μM，表明化合物对两种激酶的催化活性均有较强的抑制作用。
在本发明的一些具体实施方案中，用蛋白免疫印迹法分析证实，Aiphanol可以下调HUVEC中VEGF激活的VEGFR1/PI3K/AKT和VEGFR2/AKT/ERK信号通路相关分子的磷酸化水平，且呈时间和/或浓度依赖性。免疫沉淀实验验证了Aiphanol可以下调HDLEC中VEGF-C诱导的VEGFR3磷酸化。
在本发明的一些具体实施方案中，以人脐静脉内皮细胞(HUVEC)、人真皮淋巴管内皮细胞(HDLEC)为研究模型，分别评估Aiphanol对血管生成和淋巴管生成的影响。本发明采用经典的小管形成实验检测发现，Aiphanol呈浓度依赖性地抑制两种内皮细胞的管状结构形成。
在本发明的一些具体实施方案中，检测Aiphanol对HUVEC和HDLEC增殖的作用，结果显示Aiphanol对两种细胞的增殖均有一定抑制作用，呈时间和浓度依赖性。划痕愈合实验检测Aiphanol对HUVEC、HDLEC迁移的影响，结果显示Aiphanol能够有效抑制两种细胞的迁移，具有量效趋势。迁移实验和侵袭实验验证了Aiphanol可以抑制HUVEC和HDLEC的迁移和侵袭，结果与划痕愈合实验相符。
在本发明的一些具体实施方案中，通过在血管荧光转基因斑马鱼模型中分析药物处理后斑马鱼肠静脉丛(Subintestinal vessels，SIVs)的面积，评价其抗血管生成作用，浓度为2.8μM、8.3μM的Aiphanol组斑马鱼肠下血管面积分别为22664、20331像素，与对照组斑马鱼的肠下血管面积(26775像素)相比p<0.01，抗血管形成作用为15％、24％，表明Aiphanol能有效抑制斑马鱼胚胎中SIVs的血管生成。将含或不含VEGF及Aiphanol的Matrigel基质胶种植于C57小鼠皮下，VEGF组中可反映新生毛细血管的标记物CD31的表达明显上调，而Aiphanol能有效抑制体内血管向Matrigel中的浸润及生长，VEGF诱导的血管生成及CD31的表达明显减弱。在鸡胚绒毛尿囊膜实验中也发现，Aiphanol可有效抑制胚胎血管尤其是毛细血管的生长，呈剂量依赖性。同样地，通过小 鼠主动脉环血管新生实验检测，Aiphanol可以明显抑制主动脉中的血管内皮细胞在3D培养体系下形成芽状血管，且呈量效趋势。以上四种模型结果均表明了Aiphanol具有良好的体内抗血管生成活性。
在本发明的一些具体实施方案中，通过淋巴球体发芽实验发现，Aiphanol有效拮抗淋巴管的形成，且抑制能力强于VEGFR3特异性抑制剂SAR131675。进一步在胶栓实验中发现，Aiphanol组中淋巴管生成标志物Lyve-1水平明显减少，表明Aiphanol可以下调小鼠体内淋巴管向VEGF-C诱导的浸润及生长。摘取小鼠新鲜的淋巴结进行3D体系培养，荧光染色结果显示Aiphanol可以有效抑制新生淋巴管的形成和Lyve-1的表达。
在本发明的一些具体实施方案中，通过软琼脂集落形成实验发现，Aiphanol在体外并不能直接抑制鼠结肠癌细胞MC38的增殖，但在C57小鼠MC38荷瘤模型中，Aiphanol能明显抑制肿瘤的生长，抑瘤率为57.7％。冰冻切片法结合免疫荧光法，验证了Aiphanol能明显下调新生毛细血管标记物CD31的水平，以上结果表明Aiphanol可通过抑制血管生成发挥抗肿瘤生长作用。
本发明也通过带有荧光素酶的小鼠乳腺癌细胞4T1-luc的体内淋巴道转移模型，检测了Aiphanol对肿瘤细胞在体内转移的影响，结果显示，Aiphanol可以抑制肿瘤细胞在四肢淋巴结和脾、肾脏器的转移，抑制能力强于VEGFR3特异性抑制剂SAR131675。以上结果表明了Aiphanol在体内具有良好的抗淋巴管生成作用，并可通过该作用发挥抗肿瘤的淋巴道转移效应。
综上所述，本发明发现Aiphanol可结合VEGFR，能作为VEGFR激酶抑制剂，Aiphanol可靶向VEGFR抑制血管生成和抑制淋巴管生成，进而可用于血管增生相关性疾病、淋巴管生成相关疾病的预防与治疗。
图1A至图1D显示Aiphanol直接结合VEGFR2、VEGFR3的实验结果。
图2A至图2E显示Aiphanol抑制VEGFR1、VEGFR2和VEGFR3活性的实验结果。
图3A至图3B显示Aiphanol抑制血管内皮细胞及淋巴管内皮细胞的小管形成的实验结果。
图4A至图4F显示Aiphanol抑制血管内皮细胞及淋巴管内皮细胞的增殖、运动的实验结果。
图5A至图5D显示Aiphanol在不同模型中抗血管生成活性的实验结果。
图6A至图6C显示Aiphanol在不同模型中抗淋巴管生成活性的实验结果。
图7A至图7D显示Aiphanol通过抑制血管生成和淋巴管生成发挥抗肿瘤生长和转移作用的实验结果。
为了更清楚地理解本发明，下面通过具体实施例并配合附图进一步详细说明本发明，但本发明并不因此而受到任何限制。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。下列实施例中所用Aiphanol为委托其他公司按照现有技术方法合成且已确认其结构如前述式(I)所示(未进行手性拆分)。
实施例1、Aiphanol直接结合VEGFR2、VEGFR3
研究表明，VEGFR2主要表达在血管内皮细胞，是VEGF的主要受体，VEGFA/VEGFR2是诱导血管生成的最主要通路。VEGFR3的表达主要限于成体淋巴管内皮，并且被认为控制淋巴管生成，其主要配体VEGF-C激活VEGFR-3后可促进淋巴管内皮细胞的增殖和移动，VEGF-C/VEGFR3是调节淋巴管生成的关键信号通路。本实施例通过表面等离子体共振技术检测了Aiphanol与VEGFR2、VEGFR3的结合能力。
分子对接(Molecular docking)是依据配体与受体作用的“锁-钥原理”，模拟配体小分子与受体蛋白大分子相互作用的一种技术方法。配体与受体相互作用是分子识别的过程，主要包括静电作用、氢键作用、疏水作用、范德华作用等。通过计算，可以预测两者间的结合模式和亲和力。本实施例通过分子对接技术详细分析了小分子Aiphanol分别和不同蛋白的互作模式。
实验方法：
1.表面等离子体共振实验(SPR)
1)新鲜配制所需试剂(包括运行缓冲液、分析物溶液等)，用1.05×PBS-P稀释小分子储液(10mM)10倍，配制含5％DMSO的1mM小分子溶液。
2)用5％DMSO运行缓冲液将小分子浓度稀释到200μM作为最高进样浓度，Aiphanol按2倍比例稀释成不同的浓度。
3)准备运行缓冲液，打开Biacore T200仪器。
4)选择CM5芯片，将芯片放入仪器中。
5)进行受体蛋白偶联。
6)在上样架上按照检测顺序放入配制好的药物溶液、再生溶液等开始结合实验。
7)用Biacore T200Evaluation Software对结果进行Affinity/kinetics拟合，Model选 择1:1binding，点击fit进行数据的拟合，计算结合解离常数Kd。
8)根据原始数据在Graphpad prism中绘制结合曲线。
2.分子对接技术
1)准备蛋白：导入VEGFR2与其抑制剂替沃扎尼(Tivozanib)的共晶结构(PDB ID:4ASE)；利用CLUSTALW程序对VEGFR2和VEGFR3进行序列比对，结果显示二者序列同源性约为71.5％，随后采用SWISS-MODEL程序对VEGFR3进行同源建模，获取可用于后续分子识别研究的人源VEGFR3蛋白合理的三维结构模型。使用Protein Preparation Wizard Panel模块对蛋白三维模型进行处理：删除B链、去水、加氢、修补缺失残基、优化结构、能量最小化等(OPLS2005力场，
RMSD)。
2)定义蛋白的格点文件：用Receptor Grid Generation模块定义格点文件，以Tivozanib为中心生成格点文件，盒子大小为
3)准备配体：将小分子化合物的2D格式通过LigPrep模块进行能量最小化(OPLS2005力场，
RMSD)等处理，输出相应的3D结构。
4)分子对接：采用Glide模块的高精度(XP)模式进行分子对接，即受体和配体分子之间通过几何匹配和能量匹配互相对接。输出结果后，对蛋白与小分子化合物的结合模式进行2D、3D作图。
表面等离子体共振实验(Aiphanol与VEGFR2)结果显示，Aiphanol与VEGFR2蛋白的Kd为9.76μM，小分子与蛋白具有较强的结合能力(图1A)。
Aiphanol与VEGFR2的分子对接结果显示，小分子与VEGFR2的结合能为-10.57kcal/mol，表明化合物与蛋白有很强的结合力。小分子可以与VEGFR2形成4个氢键作用、1个盐桥作用：Aiphanol结构中间苯二酚上的OH分别作为氢键供体及氢键受体Glu885、Asp1046形成氢键作用，距离分别为
醇羟基上的OH作为氢键供体与Lys920形成氢键作用，距离为
甲氧基上的O作为氢键受体与Asn923形成氢键作用，距离为
此外，Aiphanol结构中间苯二酚上的苯环可以与Lys868形成盐桥作用(图1B)。
表面等离子体共振实验(Aiphanol与VEGFR3)结果显示，Aiphanol与VEGFR3蛋白的Kd为3.2μM，小分子与蛋白具有很强的结合能力(图1C)。
Aiphanol与VEGFR3的分子对接结果显示，结合能为-10.86kcal/mol，小分子大部分位于VEGFR3的疏水性空腔中。小分子主要结合在由Leu851、Ala877、Lys879、Glu896、Leu900、Val925、Val927、Glu928、Phe929、Cys930、Lys931、Tyr932、Asn934、Asn937、Leu1044、Asp1055、Phe1056组成的疏水性空腔中。其中Aiphanol 两端苯环上的OH分别与Asn934、Asn937和Asp1055形成3个氢键作用，同时苯并1，4-二氧六环上的羟甲基与Cys930和Lys931形成2个氢键。两个苯环和苯并1，4-二氧六环与口袋周围的疏水性氨基酸(Leu851、Ala877、Lys879、Glu896、Leu900、Val925、Val927、Glu928、Phe929、Tyr932、Leu1044、Phe1056)形成了较强的疏水作用，进一步增强了Aiphanol与VEGFR3的亲和力(图1D)。
实施例2、Aiphanol可有效抑制VEGFR1、VEGFR2和VEGFR3活性
本实施例检测了Aiphanol对VEGFR1、VEGFR2和VEGFR3三种激酶活性的影响。主要的实验方法包括ADP-Glo
TMassay和蛋白免疫印迹法。
ADP-Glo
TMassay原理：ADP-Glo
TM是一种发光法激酶检测方式，检测激酶反应中所形成的ADP的量来反应激酶活性。在该检测方法中，激酶反应中剩余的ATP会首先被ADP-Glo试剂消耗掉；激酶反应中生成的ADP则会被激酶检测试剂还原成ATP；最后ATP在Ultra-Glo
TM萤光素酶的作用下，与荧光素反应发光，发光信号与激酶活性正相关。将荧光信号值转换成激酶活性，计算IC
50值。
实验方法：
1.ADP-Glo
TMassay
1)使用激酶酶学缓冲液稀释酶，底物，ATP和化合物。
2)在384浅孔板中每孔加入1μL抑制剂或5％DMSO，2μL激酶(4ng)，2μL底物和ATP的混合物。
3)30℃孵育1h。
4)每孔加入5μL的ADP-Glo试剂。
5)30℃孵育40min后每孔加入10μL的激酶检测试剂。
6)30℃孵育30min后在多功能酶标仪中读取每孔冷光值。
7)计算(测得的各组冷光值均应减去HPE对照孔冷光值)
％Enzyme Activity＝RLUs-HPE/(ZPE-HPE)×100％
其中：
RLUS：样品孔的冷光值(待测化合物)
HPE：100％抑制孔冷光值
ZPE：0％抑制孔冷光值
2.蛋白免疫印迹法(Western Blot)
1)药物处理细胞后，加入RIPA细胞裂解液(含Cocktail、PMSF等蛋白酶抑制剂)提取细胞蛋白。
2)取适量蛋白裂解液(30-40μg)煮沸10min，上样，进行SDS-PAGE电泳，浓缩胶电压90V，分离胶电压120V，电泳至溴酚蓝到底端。
3)300mA冰浴条件下转膜3h(根据分子量大小调整)。
4)取出硝酸纤维素膜，5％脱脂奶粉(TBST配制)室温封闭1h。
5)加一抗工作液，4℃反应过夜。
6)二抗反应1h，最后进行发光检测。
3.免疫沉淀(Immunoprecipitation，IP)实验
1)将抗体结合到protein G Sepharose beads上：将20μL protein G Sepharose 4B(原包装中的悬浮体积)取出，悬浮于500μL PBS中，1500rpm离心30s，弃上清，再加入抗体及500μL PBST，室温摇1h，1500rpm离心30s，缓慢弃去上清，沉淀即为结合抗体的Sepharose beads，留作免疫沉淀用。
2)药物处理细胞后，加入3％TritonX100/PBS(含Cocktail、PMSF等蛋白酶抑制剂)提取细胞蛋白。
3)免疫沉淀反应：将细胞裂解液(留少许作为阳性对照)加入结合了抗体的protein G Sepharose beads中，4℃旋转反应3h。
4)洗去未结合的蛋白：1500rpm离心30s，用0.3％TritonX100/PBS洗沉淀反应5次，每次500μL洗2min。
5)加入缓冲液：加入2×SDS上样缓冲液，14000rpm快速离心10s后，95℃加热10min，14000rpm快速离心10s，吸取上清进行电泳；
6)后续操作同蛋白印迹法。
图2A和图2C蛋白免疫印迹实验结果验证了Aiphanol可以抑制HUVEC中VEGF激活的VEGFR1/PI3K/AKT和VEGFR2/AKT/ERK信号通路相关分子的磷酸化水平，且呈时间和/或浓度依赖性。图2B和图2D的ADP-Glo
TMassay实验结果显示，Aiphanol可有效抑制VEGFR2、VEGFR3激酶的催化活性，IC
50值分别为0.92μM、0.29μM。图2E的IP实验结果显示，Aiphanol在HDLEC中呈浓度依赖性地抑制VEGF-C激活的VEGFR3磷酸化。
实施例3、Aiphanol抑制血管内皮细胞及淋巴管内皮细胞的小管形成
本实施例中，采用经典的小管形成实验检测Aiphanol对人脐静脉内皮细胞(HUVEC)、人真皮淋巴管内皮细胞(HDLEC)的管状结构形成的作用。
小管形成实验方法：将Matrigel基质胶50μL/孔涂布于96孔板中，在37℃培养箱静置30min，待其凝固。将HUVEC细胞消化、重悬于含VEGF、药物或溶剂对照的 无血清培养液，VEGF抗体贝伐单抗(Bevacizumab)、VEGFR3特异性抑制剂SAR131675作阳性对照组，取100μL细胞悬液/孔铺于Matrigel表面。在培养箱培养6h后，于倒置显微镜下观察小管结构形成情况并拍照，使用Image J的Angiogenesis analyzer对网眼数进行定量分析。
图3A显示了HUVEC的小管形成检测结果。结果表明，VEGF可以刺激细胞形成类似血管管腔样的三维管状结构，Bevacizumab可以抑制管状结构的形成，Aiphanol呈浓度依赖性地抑制血管内皮细胞在3D培养体系下的管状结构形成，其处理后内皮细胞管状结构中断，网眼结构减少，细胞形成半管状，甚至皱缩呈孤岛样散在分布于基质胶上，表明Aiphanol可抑制血管内皮细胞的管状结构形成。
图3B显示了HDLEC的小管形成检测结果。结果表明，VEGF-C可以促进细胞形成类似淋巴管管腔样的三维管状结构，SAR131675对淋巴管网状结构的形成有一定的抑制作用。Aiphanol呈剂量依赖性地抑制VEGF-C诱导的淋巴管形成，Aiphanol组中淋巴内皮细胞连接减少，细胞呈现短的条索状分布于基质胶上，管状结构中断甚至消失，表明Aiphanol可抑制淋巴管内皮细胞的管状结构形成。
实施例4、Aiphanol抑制血管内皮细胞及淋巴管内皮细胞的增殖、运动
本实施例中，通过细胞汇合度检测、划痕愈合实验、Transwell实验检测了Aiphanol对血管内皮细胞及淋巴管内皮细胞增殖、运动的影响。
实验方法：
1.细胞汇合度检测
将细胞铺96孔板，2000个细胞/孔，次日加入不同浓度的Aiphanol，每组平行设四个复孔，置于培养箱中培养，分别在24h、48h、72h和96h的时间点取出孔板，置于细胞生长分析仪中检测汇合度，根据各时间点的细胞汇合度值绘制生长曲线。
2.划痕愈合实验
将细胞铺6孔板，待细胞汇合度达到90％以上时，用200μL枪尖缓慢划“十”字，确保划痕宽度一致。PBS清洗2遍，去除残余血清和游离的细胞碎片，之后换成含药物或溶剂对照的无血清培养基，每组平行设两个复孔，于倒置显微镜下拍摄0h的划痕宽度，放培养箱继续培养24h后再次拍摄划痕宽度，细胞的迁移距离＝划痕宽度(0h)-划痕宽度(24h)。
3.Transwell实验
将细胞消化、重悬于含药物的无血清培养液，在下室加入800μL含10％FBS的培养液，将含2×10
4个细胞/200μL(迁移)、4×10
4个细胞/500μL(侵袭)的无血清培养液加入 上室，置于培养箱培养24h，冰甲醇中固定30min，0.1％结晶紫染色30min，用棉签将小室上层未迁移的细胞擦净，中性树胶封片后拍照并统计细胞个数。
图4A至图4B显示了细胞汇合度检测结果，结果表明Aiphanol对HUVEC、HDLEC的增殖均有一定的抑制作用，呈时间和浓度依赖性。图4C至图4D显示了划痕愈合实验结果，结果表明Aipahnol呈剂量依赖性地抑制HUVEC、HDLEC的迁移。图4E至图4F显示了Transwell实验结果，结果表明Aiphanol可以抑制HUVEC、HDLEC的迁移和侵袭，具有量效趋势，当Aiphanol浓度为30μM时，细胞迁移和侵袭个数明显减少。结果证实了Aiphanol能抑制两种内皮细胞的增殖和运动。
实施例5、Aiphanol在不同模型中均具有显著抗血管生成活性
本实施例采用血管荧光转基因斑马鱼模型、胶栓实验、小鼠主动脉环血管新生实验、鸡胚绒毛尿囊膜实验四种模型分析药物Aiphanol处理后的血管形成水平评价其抗血管生成作用。
实验方法：
1.血管荧光转基因斑马鱼模型
1)血管荧光转基因斑马鱼采用自然配对的方式获得胚胎，分别在受精后6h及24h对胚胎进行清理，移除死亡胚胎，并根据胚胎的发育情况挑选合适的胚胎。
2)取受精后2天的胚胎，采用显微注射的方式将药物注入斑马鱼卵黄囊内，每组注射30尾斑马鱼。
3)注射后于28℃继续孵育24h，从每组30尾斑马鱼中随机取出10尾。
4)于显微镜下观察血管荧光转基因斑马鱼的肠下血管丛形成情况并拍照。
5)使用尼康NIS-Elements D 3.10高级图像处理软件对图像进行分析，根据肠下血管丛的面积进行统计学分析。
2.胶栓实验
根据预实验结果确定实验设计：共4组(1)DMSO溶剂对照组；(2)VEGF诱导组：500ng/mL；(3)Aiphanol组：15μM；(4)VEGF+Aiphanol组，每组4只鼠。在C57雌鼠皮下右侧腹部注射体积400μL Matrigel/只，2周后剖鼠，取出胶栓拍照，进行HE染色和免疫组化法检测胶栓中的微血管密度。
3.HE染色
1)准备载玻片：
(1)用铬酸洗液浸泡载玻片过夜。
(2)取出，自来水冲洗去除残余的铬酸洗液。
(3)蒸馏水冲洗一遍，浸泡于无水酒精中，装盒备用，随用随擦。
2)取胶栓，置于塑料包埋盒中并做好标记，使用梯度酒精(浓度分别为50％、70％、80％、90％、95％、100％、100％)脱水，每级1h；二甲苯Ⅰ0.5h，二甲苯Ⅱ0.5h，取出组织在通风橱稍晾干。
3)石蜡包埋：调整蜡箱温度为65℃，浸蜡Ⅰ30min，浸蜡Ⅱ30min，用镊子夹取组织放于不锈钢模具中，倒入蜡液至稍溢出，调整组织位置处于模具中央，小心盖上预热的包埋盒，避免气泡，将石蜡组织放室温过夜凝固。
4)切片：将石蜡组织进行4μm连续切片，贴于载玻片上，65℃烘烤至石蜡融化。
5)常规脱蜡:二甲苯Ⅱ15min，二甲苯Ⅰ15min。
6)梯度酒精水化：100％、100％、95％、90％、85％、80％、75％酒精各5min。
7)用蒸馏水清洗一次，5min/次，PBS清洗一次，5min/次。
8)苏木素染液染3min，自来水冲洗去除残余的苏木素，1％盐酸-酒精分色数秒，自来水冲洗，浸泡于回收的EDTA修复液5min返蓝，自来水冲洗，0.5％伊红染液染3min，自来水冲洗。
9)梯度酒精(由低到高)脱水10s，二甲苯透明两次各10s，
10)通风橱中晾干，中性树脂封片。
4.免疫组化法
1)-7)同HE染色:1)-7)。
8)阻断内源性过氧化物酶活性：在切片表面滴加3％H
2O
2，室温放置15min。
9)PBS洗3次，5min/次。
10)抗原修复：将切片浸泡于EDTA抗原修复液(pH＝9.0)，微波炉加热，功率100W，5min/次，共3次，冷却至室温。
11)PBS洗3次，5min/次。
12)封闭：用免疫组化笔圈出组织部位，在组织表面滴加10％山羊血/PBS，室温放置1h。
13)一抗反应：弃去封闭液，滴加一抗(CD31，1:100，Cell Signaling Technology)，50μL/片，湿盒中4℃反应过夜。
14)PBS洗3次，5min/次。
15)二抗反应：滴加HRP标记二抗，50μL/片，室温反应1h。
16)PBS洗3次，5min/次。
17)DAB显色：新鲜配制DAB显色液，滴加于玻片上，肉眼观察出现棕色颗粒立即终止反应，显色时间控制在10min以内。
18)自来水冲洗，苏木素染液染3min，自来水冲洗去除残余的苏木素，1％盐酸-酒精分色数秒，自来水冲洗，浸泡于回收的EDTA修复液5min返蓝，蒸馏水冲洗。
19)梯度酒精(由低到高)脱水10s，二甲苯透明两次各10s，
20)通风橱中晾干，中性树脂封片。
5.为小鼠主动脉环血管新生实验
1)Matrigel基质胶提前放4℃冷库过夜缓慢融化，枪尖及48孔板提前预冷。
2)在48孔板中铺基质胶120μL/孔，放37℃30min待凝固。
3)期间处死C57小鼠，解剖摘取胸主动脉，用冰PBS中冲洗并去除动脉周边的脂
肪组织及结缔组织。
4)用无菌手术刀将主动脉切成1mm厚的主动脉环，约8-10个。
5)将主动脉环铺于基质胶上，37℃10min。
6)再加入60μL基质胶覆盖主动脉环，放37℃3h，使其充分凝固。
7)加入含或不含VEGF及Aiphanol的无血清培养基，500μL/孔。隔2d换一次新
鲜的培养基。
8)在6-9d根据主动脉血管内皮细胞发芽情况拍照并统计发芽面积。
6.鸡胚绒毛尿囊膜实验模型
1)调整孵箱温度为37℃，关闭培养箱内的CO
2管道，放置3个盛满水的水盘以增加湿度，确保湿度为60％。
2)购买SPF级种蛋(CE5)，于37℃预热的1％新洁尔灭溶液中浸泡5min，用干燥的医用纱布将其表面残余的液体擦干，放入消毒好的种蛋孵育架上，转移至孵箱中培养。
3)每隔8h轻柔上下颠倒翻转种蛋一次，避免种蛋粘壳。
4)孵化至第4天，取出种蛋于超净台内，用75％医用酒精消毒种蛋表面，无菌纱布擦干。小心将蛋壳打开，使鸡胚落入下方的培养皿中，使卵黄囊面朝上并居中，以避免血管的生长发育受影响。
5)放入培养箱培养24h，使鸡胚逐渐适应培养环境，观察并清除死亡的鸡胚。
6)将药液浸湿玻璃纤维滤纸，在鸡胚尿囊膜上选择合适的血管部位，将浸有药物的滤纸轻柔放置在血管表面。
7)将种蛋放回培养箱，继续培养48h。
8)小心揭开玻璃纤维滤纸，在显微镜下观察实验部位及其周边的血管发育情况并拍照，统计大血管和毛细血管的总数目。
图5A血管荧光转基因斑马鱼模型结果显示，同溶剂组的肠下血管面积(26775像素)相比，浓度为2.8μM、8.3μM的Aiphanol组斑马鱼肠下血管面积分别为22664像素、20331像素，抗血管形成作用为15％、24％，差异具有统计学意义(p<0.01)，结果表明Aiphanol在两种浓度下均可以抑制斑马鱼胚胎中肠下静脉丛的血管生成。
图5B胶栓实验结果表明，VEGF能诱导小鼠体内血管向Matrigel中的浸润和生长，胶栓颜色变红，加入Aiphanol处理后血管形成减少。HE染色可见VEGF组中有较多的血管内皮细胞，与Aiphanol联用后，浸润的血管内皮细胞减少。在CD31染色中，VEGF组形成较大的血管(棕色，红箭头指示)，而与Aiphanol联用后，血管明显减少。结果提示Aiphanol能抑制体内血管的生成。
图5C小鼠主动脉环血管新生实验结果表明，VEGF能促进主动脉中血管内皮细胞在立体培养体系中的发芽及生长，形成修长且旺盛的微血管丛，阳性对照组Bevacizumab对血管形成有一定抑制作用，Aiphanol能抑制VEGF诱导的微血管形成，具有量效趋势，当浓度达到30μM时，血管内皮细胞基本丧失形成微血管的能力，呈“微血管丛凋零”状态。
图5D鸡胚绒毛尿囊膜实验结果表明，VEGF能促进胚胎血管尤其是微小血管的形成，Bevacizumab能有效降低形成的微血管数量，Aiphanol呈浓度依赖性地抑制相应部位微血管的形成，可见加药部位及周边的血管数量、管径大小均有下调，甚至会导致原位血管发生萎缩。
以上四种模型强有力的支持了Aiphanol具有良好的抗血管生成活性。
实施例6、Aiphanol在不同模型中均具有抗淋巴管生成活性
本实施例采用3D淋巴球体发芽实验、胶栓实验及离体淋巴结培养三种模型探究Aiphanol对淋巴管生成的效应。Lyve-1(Lymphatic Vessel Endothelial Receptor-1)，淋巴管内皮受体-1，是淋巴管内皮细胞透明质酸(HA)的主要受体，其主要局限于淋巴管内皮细胞，一般表达于初始淋巴管独特的松散连接处，在管腔和基底外侧质膜表面呈点状分布，并在核周内质网衍生的细胞内室有不同程度的滞留。常作为区分血管和淋巴管的常用标志物。故进行Lyve-1染色可以鉴别出淋巴管。
实验方法：
1.3D淋巴球体发芽实验
1)准备羧甲基纤维素钠：称取600mg羧甲基纤维素钠粉末置于100mL玻璃瓶 中，并放入磁力搅拌棒，一起高压灭菌；加热DMEM基础培养基至60℃；取50mL预热的基础培养基倒入灭菌后的玻璃瓶中，与羧甲基纤维素钠混合；磁力搅拌30min，然后4℃搅拌2h；5000g下离心2h；移取清澈并高粘度的上清置于50mL离心管中；4℃保存备用。
2)准备悬滴：收集状态良好的HDLEC，用完全培养基重悬，调整细胞密度为4×10
5/mL；将细胞悬液与羧甲基纤维素钠按4:1体积混匀；用排枪(剪枪尖)吸取混合液滴加在10cm皿盖内，1×10
3个细胞/25μL/滴；在10cm皿中加入PBS，盖上皿盖，防止液滴变干；置于培养箱培养24h。
3)准备下层基质(不含细胞)：用DMEM基础培养基稀释鼠尾胶原蛋白1型至1mg/mL(体积4:1)；将羧甲基纤维素钠和DMEM基础培养基按1:4体积混匀；最后将鼠尾胶原蛋白1型稀释液和羧甲基纤维素钠稀释液等体积混匀，铺于48孔板中，200μL/孔，放37℃1h待凝固，整个过程需轻柔缓慢，避免产生气泡。
4)上层基质(含淋巴球体)：用PBS将悬滴的球体轻柔吹下并收集于50mL离心管中，动作轻柔，避免球体破碎；170g下离心5min；弃去上清，用胎牛血清(FBS)重悬球体；按3)步骤配制基质，用含球体的FBS替换DMEM基础培养基配制上述基质，其余条件不变；取基质混合液加入48孔板中的上层，放37℃培养箱2-3h待凝固。
5)加入含VEGF-C、化合物或溶剂对照的完全培养基，培养24h后于倒置显微镜下观察并拍照。
2.胶栓实验
7周龄C57雌鼠随机分组，每组4只，在小鼠腹部剃毛，暴露腹部。配制好含有0.5μg/mL VEGF-C和/或30μM SAR131675/Aiphanol的Matrigel基质胶，冰上保存，用预冷1mL注射器吸取Matrigel混合液注入小鼠腹部皮下，250μL/只。8d后，解剖小鼠，取出胶栓并拍照，取部分胶栓迅速冻入液氮中，由专业病理师进行冰冻切片。切片结合免疫荧光法分析胶栓中Lyve-1的水平。
2-1.冰冻切片法结合免疫荧光法
1)收集新鲜胶栓组织，切成黄豆大小，装入2mL冻存管，在液氮中速冻10s。
2)专业病理师进行冰冻切片:用樱花冷冻切片包埋剂包埋组织，在冰冻切片机下4μm快速切片。
3)4％多聚甲醇固定切片5min，PBS洗3次，3min/次。
4)0.05％TritonX100/PBS室温通透20min，PBS洗3次，3min/次。
5)5％山羊血/PBS室温封闭1h。
6)弃去封闭液，滴加一抗(Lyve-1，1:500，Abcam)，4℃过夜。
7)取出切片，室温复温30min，PBS洗3次，3min/次。
8)滴加荧光标记二抗，室温反应1h，PBS洗3次，3min/次。
9)滴加DAPI工作液，室温染色5min，PBS洗3次，3min/次。
10)90％甘油/PBS封片，于激光共聚焦显微镜下拍照分析。
3.离体淋巴结培养
1)解剖7周龄C57雌鼠，用手术剪和镊子沿小鼠颈部皮下小心摘取颈部淋巴结，用手术剪剪成1mm厚的淋巴结小丁，含抗生素的冰PBS冲洗两次。
2)在Glass bottom cell culture dish中铺生长因子减少的Matrigel基质胶，120μL/皿，37℃凝固1h。将淋巴结小丁种植于Matrigel表面，37℃孵箱培养10min。
3)在上层加入基质胶，60μL/皿，37℃充分凝固3h。加入含VEGF-C、药物或溶剂对照的完全培养基，培养7d，隔2-3d换一次液。
4)缓慢弃去培养基，用PBS轻柔洗两次。
5)后续同免疫荧光染色步骤。
图6A淋巴球体发芽实验结果显示，VEGF-C可以明显诱导淋巴球体在3D培养体系中的发芽，形成丰富的伪足，VEGFR3特异性抑制剂SAR131675和Aiphanol均可以减少伪足形成的数量，抑制淋巴管的生成，Aiphanol的抑制能力强于SAR131675。
图6B胶栓实验结果显示，VEGF-C组与对照组相比绿色荧光强度增强，上调淋巴管生成标志物Lyve-1的表达，SAR131675和Aiphanol均能下调Lyve-1的水平，Aiphanol的下调作用强于SAR131675。
图6C离体淋巴结培养的结果显示，VEGF-C组中Lyve-1染色为强阳性，表明VEGF-C能促进淋巴结中的淋巴管内皮细胞在基质胶中形成密集的淋巴管丛，SAR131675能减少淋巴管形成的数量，但无法抑制管径的大小，相比下Aiphanol能明显下调淋巴管形成的数量和管径大小，淋巴管丛基本消失。
实施例7、Aiphanol通过抑制血管生成和淋巴管生成发挥抗肿瘤生长和转移作用
本实施例中，进一步在体内实验中检测Aiphanol对血管生成及抗淋巴管生成的影响。在血管生成方面，以Aiphanol的耐药细胞株小鼠结肠癌细胞MC38为研究对象，将MC38注入小鼠皮下荷瘤，观察给药后肿瘤的体积变化，观察药效。在淋巴管生成方面，将带有荧光素酶的小鼠乳腺癌细胞4T1-luc注入小鼠乳腺脂肪垫开展体内淋巴道转移实验，观察Aiphanol对肿瘤细胞在体内转移的作用。
主要方法：
1.软琼脂集落形成实验
1)取对数生长期的MC38细胞，用0.25％胰蛋白酶消化，使之成为单细胞，完全培养液调整细胞密度。
2)用蒸馏水分别制备1.2％和0.7％两个浓度的低熔点琼脂糖液，高压灭菌后，维持在40℃中不凝固。
3)按1：1的比例将1.2％的琼脂糖和2×DMEM培养基(含有2×抗生素和20％FBS)混合，取混合液加入6孔板中，1.5mL/孔，室温冷却凝固备用。
4)按l：1比例将0.7％的琼脂糖和2×DMEM培养基混合后，再加入含化合物或溶剂对照的细胞悬液，充分混匀，加入铺有1.2％琼脂糖底层的6孔板中，1000个细胞/1.5mL/孔，放培养箱孵育，次日沿壁轻柔加入完全培养基，1mL/孔，期间可补充少许培养基，确保培养过程中软琼脂不会变干即可。
5)置37℃、5％CO
2培养箱中孵育14d，于倒置显微镜下计数并统计结果。
2.小鼠结肠癌细胞MC38荷瘤生长实验
1)收集生长状态良好的MC38细胞，PBS洗2次，胰酶消化，使之成为单细胞，完全培养基终止消化，1200rpm离心5min，弃上清，细胞沉淀用PBS再洗一次，离心弃上清，预冷PBS重悬。
2)将MC38细胞和Matrigel基质胶按1:1体积混匀，用1mL预冷注射器吸取细胞混匀液注入7周龄C57雌鼠腋下，7×10
5个细胞/100μL/只。
3)一周后观察肿瘤体积达到100mm
3以上时，随机分组。
4)给药分两组:溶剂对照、Aiphanol(30mg/kg)，每组7只鼠。口服给药，溶药方式：DMSO溶解化合物，PBS稀释，加少许1M NaOH促溶，给药1次/100μL/d，共给药12d。
5)期间隔2-3d称量体重及测量肿瘤体积，肿瘤体积计算方式：Volume＝0.5×length×(width)^2
6)断颈法处死小鼠，剖瘤，称取肿瘤质量。
3.冰冻切片法结合免疫荧光法实验
方法同实施例6。
4.荧光素酶标记的小鼠乳腺癌细胞4T1-luc体内淋巴道转移模型
1)收集生长状态良好的4T1-luc细胞，PBS洗2次，胰酶消化，使之成为单细胞，完全培养基终止消化，1200rpm离心5min，弃上清，细胞沉淀用PBS再洗一次，离心 弃上清，细胞用PBS重悬后置于冰上保存。
2)在7周龄BALB/c雌鼠腹部剃毛，暴露腹部，三溴乙醇麻醉小鼠，用无菌手术剪在乳腺脂肪垫外侧剪一个小口，用无菌镊子轻柔往外掏出透明果冻样的乳腺脂肪垫，用胰岛素针吸取细胞悬液，小心注入乳腺脂肪垫中，1×10
6个细胞/50μL/只鼠。将乳腺脂肪垫轻柔埋入原来的部位，将剪开的伤口进行缝合，手术完成。
3)次日，随机分组，给药分四组:溶剂对照、SAR131675(30mg/kg)、Aiphanol(5mg/kg)、Aiphanol(30mg/kg)，每组4只鼠。口服给药，溶药方式：DMSO溶解化合物，PBS稀释，加少许1M NaOH促溶，给药1次/100μL/d，共给药28d。期间隔3d称一次体重。
4)三溴乙醇麻醉小鼠，腹腔注射荧光素酶底物，将小鼠放入IVIS Spectrum小动物活体光学成像系统成像。再将小鼠解剖，摘取四肢、腋窝淋巴结、腹股沟淋巴结、心、肝、脾、肺、肾脏器进行成像，统计荧光强度并绘图。
图7A软琼脂集落形成实验结果显示，加药组与对照组中集落的个数和大小相似，没有显著差异，表明Aiphanol在体外无法抑制MC38细胞的增殖，MC38细胞属于Aiphanol的耐药细胞株。
图7B MC38细胞荷瘤生长实验结果显示，Aiphanol组中的肿瘤质量低于对照组，p<0.01，抑瘤率为57.7％，表明Aiphanol有良好的抑瘤作用。
图7C冰冻切片法结合免疫荧光法实验结果显示，对照组的原位肿瘤组织中具有明显的条索状血管，荧光强度高，而Aiphanol组中连续的条索状血管减少，荧光强度明显减弱，CD31的表达下调，以上结果表明Aiphanol通过抑制肿瘤组织中的血管生成，进而抑制肿瘤的生长。
图7D荧光素酶标记的小鼠乳腺癌细胞4T1-luc体内淋巴道转移实验结果显示，对照组中四肢淋巴结的荧光强度高，表明肿瘤细胞的转移率高，在肺组织也有一定的转移；VEGFR3特异性抑制剂SAR131675对肿瘤在四肢淋巴结、腋下及腹股沟淋巴结、脾、肾中的转移有一定的抑制作用，但对肺转移无抑制作用；Aiphanol对肿瘤在四肢淋巴结、腋下及腹股沟淋巴结中的转移有明显的抑制作用，抑制作用强于SAR131675，随着化合物剂量增大，抑制作用增强，对肿瘤在脾、肾转移也有一定抑制作用。结果证明Aiphanol可以抑制肿瘤细胞在体内的淋巴道转移。
Claims (14)
- Aiphanol在制备直接结合VEGF受体和/或抑制VEGF受体激酶活性的制剂中的应用。
- 根据权利要求1所述的应用，其中，所述VEGF受体包括VEGFR1、VEGFR2、VEGFR3中的一种或多种。
- Aiphanol在制备抑制血管内皮细胞和/或淋巴管内皮细胞小管形成的制剂中的应用。
- Aiphanol在制备抑制血管内皮细胞和/或淋巴管内皮细胞增殖及迁移侵袭的制剂中的应用。
- 根据权利要求3或4所述的应用，其中，所述血管内皮细胞为人脐静脉内皮细胞(Human umbilical vein endothelial cells，HUVEC)，所述淋巴管内皮细胞为人真皮淋巴管内皮细胞(Human dermal lymphatic endothelial cells，HDLEC)。
- Aiphanol在制备用于抗淋巴管生成的制剂中的应用。
- 根据权利要求6所述的应用，其中，所述制剂进一步用于通过抗淋巴管生成而防治淋巴管生成相关疾病，所述淋巴管生成相关疾病包括Kaposi肉瘤和/或肿瘤淋巴道转移。
- 根据权利要求6或7所述的应用，其中，所述制剂是用于靶向VEGF受体而抗淋巴管生成。
- Aiphanol在制备用于靶向VEGF受体而抗血管生成的制剂中的应用。
- 根据权利要求9所述的应用，其中，所述制剂进一步用于通过抗血管生成而防治血管增生相关性疾病，所述血管增生相关性疾病包括肿瘤血管增生转移和/或糖尿病性黄斑变性。
- 一种VEGFR激酶抑制剂，其包括Aiphanol。
- 一种防治淋巴管生成相关疾病和/或血管增生相关性疾病的药物，其包括活性组分Aiphanol。
- 一种防治淋巴管生成相关疾病和/或血管增生相关性疾病的方法，该方法包括给予有需要的受试者有效量的Aiphanol。
- 根据权利要求13所述的方法，其中，Aiphanol作为VEGFR激酶抑制剂靶向VEGFR抑制血管生成和/或抑制淋巴管生成。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111002244.X | 2021-08-30 | ||
| CN202111002244.XA CN115721639A (zh) | 2021-08-30 | 2021-08-30 | Aiphanol作为VEGFR激酶抑制剂的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023030294A1 true WO2023030294A1 (zh) | 2023-03-09 |
Family
ID=85290696
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/115732 WO2023030294A1 (zh) | 2021-08-30 | 2022-08-30 | Aiphanol作为VEGFR激酶抑制剂的应用 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN115721639A (zh) |
| WO (1) | WO2023030294A1 (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108498500A (zh) * | 2017-02-27 | 2018-09-07 | 北京市肿瘤防治研究所 | Aiphanol在抗肿瘤中的新应用 |
-
2021
- 2021-08-30 CN CN202111002244.XA patent/CN115721639A/zh active Pending
-
2022
- 2022-08-30 WO PCT/CN2022/115732 patent/WO2023030294A1/zh active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108498500A (zh) * | 2017-02-27 | 2018-09-07 | 北京市肿瘤防治研究所 | Aiphanol在抗肿瘤中的新应用 |
Non-Patent Citations (4)
| Title |
|---|
| BANWELL MARTIN G., BEZOS ANNA, CHAND SATISH, DANNHARDT GERD, KIEFER WERNER, NOWE ULRIKE, PARISH CHRISTOPHER R., PAUL SAVAGE G., UL: "Convergent synthesis and preliminary biological evaluations of the stilbenolignan (±)-aiphanol and various congeners", ORGANIC & BIOMOLECULAR CHEMISTRY, vol. 1, no. 14, 1 January 2003 (2003-01-01), pages 2427 - 2429, XP093042438, ISSN: 1477-0520, DOI: 10.1039/B305106D * |
| CHEN SHANMEI, FENG JUNNAN, ZHAO CHUANKE, WANG LIXIN, MENG LIN, LIU CAIYUN, CAI SHAOQING, JIA YANXING, QU LIKE, SHOU CHENGCHAO: "Aiphanol, a native compound, suppresses angiogenesis via dual-targeting VEGFR2 and COX2", SIGNAL TRANSDUCTION AND TARGETED THERAPY, vol. 6, no. 1, 1 January 2023 (2023-01-01), pages 1 - 3, XP093042436, DOI: 10.1038/s41392-021-00739-5 * |
| CHEN SHAN-MEI, ZHAO CHUAN-KE, YAO LI-CHENG, WANG LI-XIN, MA YU-NAN, MENG LIN, CAI SHAO-QING, LIU CAI-YUN, QU LI-KE, JIA YAN-XING, : "Aiphanol, a multi-targeting stilbenolignan, potently suppresses mouse lymphangiogenesis and lymphatic metastasis", ACTA PHARMACOLOGICA SINICA, vol. 44, no. 1, 1 January 2023 (2023-01-01), GB , pages 189 - 200, XP093042433, ISSN: 1671-4083, DOI: 10.1038/s41401-022-00940-4 * |
| CHEN ZHIQIANG, WU ZUZE: "Research Progress of Antiviral and Antitumor Activities of Natural Polyhydroxystilbene Compounds", CHINESE TRADITIONAL AND HERBAL DRUGS, vol. 34, no. 6, 25 June 2003 (2003-06-25), pages 11 - 13, XP009544190, ISSN: 0253-2670 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115721639A (zh) | 2023-03-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Berta et al. | Apelin promotes lymphangiogenesis and lymph node metastasis | |
| Bracey et al. | Mitochondrial NLRP3 protein induces reactive oxygen species to promote Smad protein signaling and fibrosis independent from the inflammasome | |
| Muley et al. | Secreted frizzled-related protein 4: an angiogenesis inhibitor | |
| Huang et al. | Soluble delta-like 1 homolog (DLK1) stimulates angiogenesis through Notch1/Akt/eNOS signaling in endothelial cells | |
| Moccia et al. | Nicotinic acid adenine dinucleotide phosphate activates two‐pore channel TPC1 to mediate lysosomal Ca2+ release in endothelial colony‐forming cells | |
| Qi et al. | Glipizide, an antidiabetic drug, suppresses tumor growth and metastasis by inhibiting angiogenesis | |
| CN102294016B (zh) | 治疗血管相关疾病的包含肽的药物组合物 | |
| Song et al. | Sinomenine reduces growth and metastasis of breast cancer cells and improves the survival of tumor-bearing mice through suppressing the SHh pathway | |
| Lv et al. | In vitro and in vivo effects of tumor suppressor gene PTEN on endometriosis: an experimental study | |
| Kong et al. | PIGF and Flt-1 on the surface of macrophages induces the production of TGF-β1 by polarized tumor-associated macrophages to promote lung cancer angiogenesis | |
| Sauer et al. | α2-Macroglobulin enhances vasculogenesis/angiogenesis of mouse embryonic stem cells by stimulation of nitric oxide generation and induction of fibroblast growth factor-2 expression | |
| Lu et al. | Resveratrol reduces store-operated Ca2+ entry and enhances the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex | |
| Patnam et al. | Lymphangiogenesis guidance mechanisms and therapeutic implications in pathological states of the cornea | |
| WO2023030294A1 (zh) | Aiphanol作为VEGFR激酶抑制剂的应用 | |
| Zhu et al. | The cGAS-STING pathway promotes endometriosis by up-regulating autophagy | |
| CN101653438A (zh) | 黄芪中毛蕊异黄酮在制备血管保护、促进血管新生药物中的应用 | |
| CN110974835A (zh) | 雷公藤红素在抑制肿瘤血管生成拟态中的应用 | |
| CN107446024B (zh) | 一种可拮抗ddx3蛋白rna结合活性的多肽dip-13及其应用 | |
| CN111214660B (zh) | Pax4基因表达抑制剂在制备抑制纤维化的药物中的应用 | |
| Zhao et al. | Foxa1 mediates eccrine sweat gland development through transcriptional regulation of Na-K-ATPase expression | |
| CN113171459A (zh) | Fundc1在制备防治血管、肿瘤疾病药物中的用途 | |
| CN101379082A (zh) | 治疗血管相关疾病的包含肽的药物组合物 | |
| Xie et al. | Identification of a cytoplasmic linker protein as a potential target for neovascularization | |
| CN109846880A (zh) | 硬皮病标志物及蛋白抑制剂的应用 | |
| CN109954130A (zh) | 双靶向配体化力达霉素dtll联合吉西他滨在胰腺癌治疗中的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22863429 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 22863429 Country of ref document: EP Kind code of ref document: A1 |