CN113604214A - High-stability oncolytic peptide fluorescent probe and preparation method and application thereof - Google Patents
High-stability oncolytic peptide fluorescent probe and preparation method and application thereof Download PDFInfo
- Publication number
- CN113604214A CN113604214A CN202110910097.XA CN202110910097A CN113604214A CN 113604214 A CN113604214 A CN 113604214A CN 202110910097 A CN202110910097 A CN 202110910097A CN 113604214 A CN113604214 A CN 113604214A
- Authority
- CN
- China
- Prior art keywords
- oncolytic
- peptide
- fluorescent probe
- polypeptide
- tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 205
- 230000000174 oncolytic effect Effects 0.000 title claims abstract description 122
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 77
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 70
- 229920001184 polypeptide Polymers 0.000 claims abstract description 59
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000003814 drug Substances 0.000 claims abstract description 13
- 229940043267 rhodamine b Drugs 0.000 claims abstract description 9
- 201000010099 disease Diseases 0.000 claims abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 8
- 206010028980 Neoplasm Diseases 0.000 claims description 43
- 210000004881 tumor cell Anatomy 0.000 claims description 38
- 238000000034 method Methods 0.000 claims description 27
- 239000007790 solid phase Substances 0.000 claims description 15
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 238000001308 synthesis method Methods 0.000 claims description 12
- 230000002194 synthesizing effect Effects 0.000 claims description 10
- 239000008194 pharmaceutical composition Substances 0.000 claims description 9
- -1 9-fluorenylmethyloxycarbonyl Chemical group 0.000 claims description 8
- 238000006482 condensation reaction Methods 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000005520 cutting process Methods 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 5
- 230000001225 therapeutic effect Effects 0.000 claims description 5
- 239000000796 flavoring agent Substances 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
- 125000000539 amino acid group Chemical group 0.000 claims description 3
- 239000011230 binding agent Substances 0.000 claims description 3
- 230000000295 complement effect Effects 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 239000007884 disintegrant Substances 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 3
- 239000003112 inhibitor Substances 0.000 claims description 3
- 239000000314 lubricant Substances 0.000 claims description 3
- 239000000700 radioactive tracer Substances 0.000 claims description 3
- 239000004094 surface-active agent Substances 0.000 claims description 3
- 239000000375 suspending agent Substances 0.000 claims description 3
- 239000003086 colorant Substances 0.000 claims description 2
- 239000007859 condensation product Substances 0.000 claims description 2
- 235000013355 food flavoring agent Nutrition 0.000 claims description 2
- 230000000259 anti-tumor effect Effects 0.000 abstract description 29
- 230000001464 adherent effect Effects 0.000 abstract description 23
- 108010029554 LTX-315 Proteins 0.000 abstract description 22
- GGAKLYWEFZCVIT-TVEKFXMRSA-N (2S)-2,6-diamino-N-[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1,6-diamino-1-oxohexan-2-yl]amino]-1-oxo-3,3-diphenylpropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxohexan-2-yl]hexanamide Chemical compound C=1C=CC=CC=1C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CC=CC=C1 GGAKLYWEFZCVIT-TVEKFXMRSA-N 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 15
- 238000002474 experimental method Methods 0.000 abstract description 15
- 230000009471 action Effects 0.000 abstract description 4
- 230000004048 modification Effects 0.000 abstract description 4
- 238000012986 modification Methods 0.000 abstract description 4
- 230000032683 aging Effects 0.000 abstract description 3
- 230000007547 defect Effects 0.000 abstract description 3
- 231100000673 dose–response relationship Toxicity 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 35
- 239000000243 solution Substances 0.000 description 26
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 25
- 150000001413 amino acids Chemical class 0.000 description 16
- 201000011510 cancer Diseases 0.000 description 15
- 239000011347 resin Substances 0.000 description 15
- 229920005989 resin Polymers 0.000 description 15
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 239000007787 solid Substances 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 9
- 230000002147 killing effect Effects 0.000 description 9
- 230000007246 mechanism Effects 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 8
- 210000000170 cell membrane Anatomy 0.000 description 8
- 238000009833 condensation Methods 0.000 description 8
- 230000005494 condensation Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 7
- 230000005284 excitation Effects 0.000 description 7
- 238000003384 imaging method Methods 0.000 description 7
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 6
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 6
- 150000001408 amides Chemical class 0.000 description 6
- 125000002091 cationic group Chemical group 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000009826 distribution Methods 0.000 description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 6
- 238000004007 reversed phase HPLC Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 5
- 206010025323 Lymphomas Diseases 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 239000002775 capsule Substances 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 125000006239 protecting group Chemical group 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 239000000232 Lipid Bilayer Substances 0.000 description 4
- 230000001093 anti-cancer Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000010511 deprotection reaction Methods 0.000 description 4
- 239000002552 dosage form Substances 0.000 description 4
- 125000001165 hydrophobic group Chemical group 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 206010061289 metastatic neoplasm Diseases 0.000 description 4
- 239000012046 mixed solvent Substances 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229920000881 Modified starch Polymers 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 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 3
- 230000004913 activation Effects 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 229940041181 antineoplastic drug Drugs 0.000 description 3
- 239000001768 carboxy methyl cellulose Substances 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000011503 in vivo imaging Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 201000007270 liver cancer Diseases 0.000 description 3
- 208000014018 liver neoplasm Diseases 0.000 description 3
- 230000001394 metastastic effect Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000000149 penetrating effect Effects 0.000 description 3
- 229920001223 polyethylene glycol Chemical group 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 101000798100 Bos taurus Lactotransferrin Proteins 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- 239000007821 HATU Substances 0.000 description 2
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 2
- HSHXDCVZWHOWCS-UHFFFAOYSA-N N'-hexadecylthiophene-2-carbohydrazide Chemical compound CCCCCCCCCCCCCCCCNNC(=O)c1cccs1 HSHXDCVZWHOWCS-UHFFFAOYSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 239000002202 Polyethylene glycol Chemical group 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 206010068771 Soft tissue neoplasm Diseases 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229940072440 bovine lactoferrin Drugs 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 235000010947 crosslinked sodium carboxy methyl cellulose Nutrition 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000009881 electrostatic interaction Effects 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000002489 hematologic effect Effects 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 2
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 2
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 231100001231 less toxic Toxicity 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000000845 maltitol Substances 0.000 description 2
- 235000010449 maltitol Nutrition 0.000 description 2
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 2
- 229940035436 maltitol Drugs 0.000 description 2
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 2
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 2
- 239000008108 microcrystalline cellulose Substances 0.000 description 2
- 229940016286 microcrystalline cellulose Drugs 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 2
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 239000000230 xanthan gum Substances 0.000 description 2
- 235000010493 xanthan gum Nutrition 0.000 description 2
- 229920001285 xanthan gum Polymers 0.000 description 2
- 229940082509 xanthan gum Drugs 0.000 description 2
- FTLYMKDSHNWQKD-UHFFFAOYSA-N (2,4,5-trichlorophenyl)boronic acid Chemical compound OB(O)C1=CC(Cl)=C(Cl)C=C1Cl FTLYMKDSHNWQKD-UHFFFAOYSA-N 0.000 description 1
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 1
- RUVJFMSQTCEAAB-UHFFFAOYSA-M 2-[3-[5,6-dichloro-1,3-bis[[4-(chloromethyl)phenyl]methyl]benzimidazol-2-ylidene]prop-1-enyl]-3-methyl-1,3-benzoxazol-3-ium;chloride Chemical compound [Cl-].O1C2=CC=CC=C2[N+](C)=C1C=CC=C(N(C1=CC(Cl)=C(Cl)C=C11)CC=2C=CC(CCl)=CC=2)N1CC1=CC=C(CCl)C=C1 RUVJFMSQTCEAAB-UHFFFAOYSA-M 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- 208000036762 Acute promyelocytic leukaemia Diseases 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 244000144730 Amygdalus persica Species 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 206010004146 Basal cell carcinoma Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 238000011748 CB6F1 mouse Methods 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000006332 Choriocarcinoma Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 229920002785 Croscarmellose sodium Polymers 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- SHWNNYZBHZIQQV-UHFFFAOYSA-J EDTA monocalcium diisodium salt Chemical compound [Na+].[Na+].[Ca+2].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O SHWNNYZBHZIQQV-UHFFFAOYSA-J 0.000 description 1
- 208000032027 Essential Thrombocythemia Diseases 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 229920002907 Guar gum Polymers 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 239000004354 Hydroxyethyl cellulose Substances 0.000 description 1
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 206010023347 Keratoacanthoma Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 206010024291 Leukaemias acute myeloid Diseases 0.000 description 1
- 208000035561 Leukaemic infiltration brain Diseases 0.000 description 1
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 1
- 208000028018 Lymphocytic leukaemia Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 208000025205 Mantle-Cell Lymphoma Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 239000004368 Modified starch Substances 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 1
- 208000008601 Polycythemia Diseases 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 208000003837 Second Primary Neoplasms Diseases 0.000 description 1
- 201000010208 Seminoma Diseases 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 244000228451 Stevia rebaudiana Species 0.000 description 1
- 206010042971 T-cell lymphoma Diseases 0.000 description 1
- 208000027585 T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 201000006083 Xeroderma Pigmentosum Diseases 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 208000009621 actinic keratosis Diseases 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000000181 anti-adherent effect Effects 0.000 description 1
- 239000003911 antiadherent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 210000003445 biliary tract Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 210000000621 bronchi Anatomy 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229960005069 calcium Drugs 0.000 description 1
- 235000001465 calcium Nutrition 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000007910 chewable tablet Substances 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229960001681 croscarmellose sodium Drugs 0.000 description 1
- 229960000913 crospovidone Drugs 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000007919 dispersible tablet Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 210000003162 effector t lymphocyte Anatomy 0.000 description 1
- 239000007938 effervescent tablet Substances 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 239000002662 enteric coated tablet Substances 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- MVPICKVDHDWCJQ-UHFFFAOYSA-N ethyl 3-pyrrolidin-1-ylpropanoate Chemical compound CCOC(=O)CCN1CCCC1 MVPICKVDHDWCJQ-UHFFFAOYSA-N 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 238000000695 excitation spectrum Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229940014259 gelatin Drugs 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000000665 guar gum Substances 0.000 description 1
- 235000010417 guar gum Nutrition 0.000 description 1
- 229960002154 guar gum Drugs 0.000 description 1
- 239000007902 hard capsule Substances 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 description 1
- 229940071826 hydroxyethyl cellulose Drugs 0.000 description 1
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 1
- 229940031574 hydroxymethyl cellulose Drugs 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 229940071676 hydroxypropylcellulose Drugs 0.000 description 1
- 210000005008 immunosuppressive cell Anatomy 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000003093 intracellular space Anatomy 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 210000003228 intrahepatic bile duct Anatomy 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000000244 kidney pelvis Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000000867 larynx Anatomy 0.000 description 1
- XMGQYMWWDOXHJM-UHFFFAOYSA-N limonene Chemical compound CC(=C)C1CCC(C)=CC1 XMGQYMWWDOXHJM-UHFFFAOYSA-N 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 150000002632 lipids Chemical group 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229940031703 low substituted hydroxypropyl cellulose Drugs 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 208000003747 lymphoid leukemia Diseases 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 235000019426 modified starch Nutrition 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 206010028537 myelofibrosis Diseases 0.000 description 1
- 208000025113 myeloid leukemia Diseases 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 230000009826 neoplastic cell growth Effects 0.000 description 1
- 208000007538 neurilemmoma Diseases 0.000 description 1
- 230000000955 neuroendocrine Effects 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 231100001221 nontumorigenic Toxicity 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 239000006191 orally-disintegrating tablet Substances 0.000 description 1
- 239000007968 orange flavor Substances 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 210000003800 pharynx Anatomy 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- INAAIJLSXJJHOZ-UHFFFAOYSA-N pibenzimol Chemical compound C1CN(C)CCN1C1=CC=C(N=C(N2)C=3C=C4NC(=NC4=CC=3)C=3C=CC(O)=CC=3)C2=C1 INAAIJLSXJJHOZ-UHFFFAOYSA-N 0.000 description 1
- 229950008882 polysorbate Drugs 0.000 description 1
- 229920000523 polyvinylpolypyrrolidone Polymers 0.000 description 1
- 235000013809 polyvinylpolypyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000010241 potassium sorbate Nutrition 0.000 description 1
- 239000004302 potassium sorbate Substances 0.000 description 1
- 229940069338 potassium sorbate Drugs 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 201000006402 rhabdoid cancer Diseases 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- 229940085605 saccharin sodium Drugs 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 206010039667 schwannoma Diseases 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000015424 sodium Nutrition 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 229960003885 sodium benzoate Drugs 0.000 description 1
- 229950004777 sodium calcium edetate Drugs 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 229940045902 sodium stearyl fumarate Drugs 0.000 description 1
- 229940001482 sodium sulfite Drugs 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 229960000776 sodium tetradecyl sulfate Drugs 0.000 description 1
- GGHPAKFFUZUEKL-UHFFFAOYSA-M sodium;hexadecyl sulfate Chemical compound [Na+].CCCCCCCCCCCCCCCCOS([O-])(=O)=O GGHPAKFFUZUEKL-UHFFFAOYSA-M 0.000 description 1
- NWZBFJYXRGSRGD-UHFFFAOYSA-M sodium;octadecyl sulfate Chemical compound [Na+].CCCCCCCCCCCCCCCCCCOS([O-])(=O)=O NWZBFJYXRGSRGD-UHFFFAOYSA-M 0.000 description 1
- UPUIQOIQVMNQAP-UHFFFAOYSA-M sodium;tetradecyl sulfate Chemical compound [Na+].CCCCCCCCCCCCCCOS([O-])(=O)=O UPUIQOIQVMNQAP-UHFFFAOYSA-M 0.000 description 1
- 239000007901 soft capsule Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 201000010700 sporadic breast cancer Diseases 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 229940032147 starch Drugs 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000007939 sustained release tablet Substances 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 239000000979 synthetic dye Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 208000001608 teratocarcinoma Diseases 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- AAAQKTZKLRYKHR-UHFFFAOYSA-N triphenylmethane Chemical compound C1=CC=CC=C1C(C=1C=CC=CC=1)C1=CC=CC=C1 AAAQKTZKLRYKHR-UHFFFAOYSA-N 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 208000024051 villous adenoma of colon Diseases 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Analytical Chemistry (AREA)
- Optics & Photonics (AREA)
- Epidemiology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Materials Engineering (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a high-stability oncolytic peptide fluorescent probe and a preparation method and application thereof, belonging to the technical field of biological medicine and disease treatment. The invention synthesizes a series of novel oncolytic peptide fluorescent probes, which are formed by connecting LTX-315 and rhodamine B by different connecting groups, and experiments prove that the novel oncolytic peptide fluorescent probes prepared by the invention not only can emit red fluorescence, but also greatly increase the activity of oncolytic peptide on adherent cells. Meanwhile, the dose-response curve shows that the antitumor activity of the modified polypeptide is obviously improved, and the antitumor activity is about 10 times of that of LTX-315. The aging curve shows that the structural modification of the oncolytic peptide increases the stability of the oncolytic peptide, prolongs the action time and solves the defects of low activity, incapability of tracing and the like of LTX-315 on adherent cells. Therefore, it has good practical application value.
Description
Technical Field
The invention belongs to the technical field of biological medicine and disease treatment, and particularly relates to a high-stability oncolytic peptide fluorescent probe, and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
In 2020, there are about 1930 million new cancer cases and nearly 1000 million cancer patients die due to ineffective treatment. Chemotherapy remains a conventional and important systemic treatment in tumor treatment options, but is limited by adverse reactions and drug resistance, so that a new effective anticancer drug with small toxic and side effects is urgently needed to be found.
Oncolytic peptides are a class of antineoplastic drugs derived from or inspired by natural antimicrobial peptides (AMPs) that exhibit high antitumor activity while being less toxic to normal cells. Importantly, the oncolytic peptides mediate anti-cancer effects regardless of the genetic and epigenetic characteristics of the tumor cells, which is largely determined by their unique physicochemical properties, in particular, the net positive charge on the surface of the oncolytic peptide and the specific distribution of hydrophobic residues are critical for oncolytic peptide activity. The oncolytic peptide is combined with the cell membrane of the tumor cell due to electrostatic interaction, and hydrophobic residues are inserted into the cell membrane to destroy a lipid bilayer, promote the lysis of the membrane and finally cause the death of the tumor cell.
The cationic oncolytic peptide LTX-315 obtained by modifying natural antimicrobial peptide bovine lactoferrin has high anti-tumor activity (4-hour IC) on suspended tumor cells50Only 8.3 +/-1.2 mu M) is a cationic oncolytic peptide with wide prospect. LTX-315 disrupts the cell membrane and mitochondrial membrane of tumor cells, and after cell membrane disruption, the cellular contents flow out, releasing danger-associated molecular pattern molecules (danger-associated molecular pattern molecules) that trigger a strong immune response, which reflects the ability of LTX-315 to kill tumor cells and induce a protective immune response. The treatment of LTX-315 can reduce the number of immunosuppressive cells, increase the abundance of effector T cells, and finally achieve the effect of remodeling the tumor microenvironment.
The inventors found that although LTX-315 had a strong activity on suspended tumor cells, the peptide synthesized by the former human had a poor activity on adherent tumor cells with a 24-hour IC50About 150 μ M, and the specific mechanism of anticancer has been unknown, which has limited its application.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a high-stability oncolytic peptide fluorescent probe and a preparation method and application thereof. The invention synthesizes a series of novel oncolytic peptide fluorescent probes, which are formed by connecting different connecting groups of LTX-315 and rhodamine B and are verified by experiments, the novel oncolytic peptide fluorescent probes prepared by the invention not only can emit red fluorescence, but also greatly increase the inhibitory activity of oncolytic peptide on adherent tumor cells, and a dose-effect curve shows that the antitumor activity of the modified polypeptide is obviously improved, and the antitumor activity is about 10 times of that of LTX-315. Meanwhile, the aging curve shows that the structural modification of the oncolytic peptide greatly increases the stability of the oncolytic peptide, prolongs the action time and solves the defects of low activity, incapability of tracing and the like of LTX-315 on adherent cells. Therefore, the high-stability oncolytic peptide fluorescent probe has good practical application value.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, a high-stability oncolytic peptide fluorescent probe is provided, which comprises the following amino acid residue sequence:
WKW-212 rhodamine B-5-amino-3-oxopentanoic acid-KKWWKKW (Dip) K-NH2
WKW-217 rhodamine B-GABA-KKWWKKW (Dip) K-NH2
WKW-223 rhodamine B-11-amino-3, 6, 9-trioxaundecanoic acid-KKWWKKW (Dip) K-NH2
WKW-385 rhodamine B-AEEA-KKWWKKW (Dip) K-NH2
The oncolytic peptide fluorescent probe can emit red fluorescence, and realizes the space-time positioning and tracing of the oncolytic peptide. Meanwhile, the anti-tumor activity and stability of the oncolytic peptide are greatly improved, and particularly the killing activity to adherent tumor cells is remarkably improved.
In a second aspect of the present invention, there is provided a nucleotide encoding the oncolytic peptide fluorescent probe, comprising any one of the following groups:
(a) a nucleotide encoding a polypeptide having the amino acid sequence;
(b) a nucleotide complementary to the nucleotide of (a).
The third aspect of the invention provides a synthesis method of the oncolytic peptide fluorescent probe, wherein the synthesis method comprises the steps of synthesizing polypeptide by adopting a solid-phase polypeptide synthesis method, and carrying out condensation reaction on the polypeptide, a connecting group and a fluorescent group for connection.
Specifically, the polypeptide is synthesized by a solid-phase polypeptide synthesis method (Fmoc-SPPS) based on 9-fluorenylmethyloxycarbonyl.
In a fourth aspect of the present invention, an application of the above-mentioned oncolytic peptide fluorescent probe in preparation of a medicament for preventing and/or treating (adjunctively treating) tumor-related diseases is provided.
Also, it is noted that tumors are used in the present invention as known to those skilled in the art, which include benign tumors and/or malignant tumors. Benign tumors are defined as cellular hyperproliferation that fails to form aggressive, metastatic tumors in vivo. Conversely, a malignant tumor is defined as a cell with various cellular and biochemical abnormalities capable of forming a systemic disease (e.g., forming tumor metastases in distant organs).
In a fifth aspect of the invention, a composition is provided, which comprises the above-described high-stability oncolytic peptide fluorescent probe.
In a sixth aspect of the invention, a formulation is provided, which comprises a high-stability oncolytic peptide fluorescent probe and pharmaceutically acceptable excipients and/or carriers.
In a seventh aspect of the present invention, there is provided a method for preventing and/or treating a tumor, the method comprising: comprising administering to a subject a therapeutically effective dose of the above-described high stability oncolytic peptide fluorescent probe, the above-described composition or the above-described formulation.
In an eighth aspect of the present invention, there is provided the use of the above-described high-stability oncolytic peptide fluorescent probe as a non-therapeutic tumor cell inhibitor and/or tracer. According to the invention, the "non-therapeutic purpose" is, for example, inhibition of tumor cell proliferation and promotion of tumor cell death in vitro, and in particular, the killing activity of the high-stability oncolytic peptide fluorescent probe on adherent tumor cells is higher; meanwhile, the high-stability oncolytic peptide fluorescent probe can emit red fluorescence so as to realize space-time positioning and tracing of the oncolytic peptide, has longer red fluorescence wavelength and stronger penetrating power, is applied to imaging of cells, tissues and living animals, and can be used as a tracing agent so as to explore the anti-tumor activity and action mechanism of the oncolytic peptide at the level of molecules, cells, tissues and animals.
The beneficial technical effects of the technical scheme are as follows:
1) according to the technical scheme, the time-space positioning and tracing of the oncolytic peptide are realized by enabling the oncolytic peptide to emit red fluorescence. The red fluorescence has longer wavelength and stronger penetrating power, can be widely applied to cell imaging, living animal imaging and the like, and the fluorescent probe is an important tool for researching the action mechanism and positioning of the oncolytic peptide at the cell and animal level.
2) The technical scheme can greatly improve the anti-tumor activity of the oncolytic peptide. The synthetic oncolytic peptide fluorescent probe greatly improves the killing activity of the oncolytic peptide on adherent tumor cells. Wherein, IC of each peptide50(24 hours) 14.7. + -. 2.7. mu.M (WKW-212), 15.2. + -. 0.9. mu.M (WKW-217), 14.3. + -. 1.4. mu.M (WKW-223) and 13.8. + -. 0.3. mu.M (WKW-385), respectively, compared to the IC of the classical oncolytic peptide LTX-31550The killing activity of the oncolytic peptide fluorescent probe on adherent tumor cells is improved by more than 10 times according to the experiment, wherein the killing activity is as high as 152 +/-5.9 mu M.
3) The technical scheme can greatly improve the stability of the oncolytic peptide. The oncolytic peptides such as LTX-315 and the like developed by the predecessors have relatively simple structure, are easily degraded by protease in vivo, and have poor biological stability and short half-life. According to the project, groups such as rhodamine B, polyethylene glycol and the like are introduced to the oncolytic peptide, so that the degradation of the oncolytic peptide by protease can be prevented, the enzymolysis stability of the oncolytic peptide is improved, and the application value is high. The aging curve shows that the anti-tumor activity of the oncolytic peptide fluorescent probe can be maintained for more than 72 hours; while the antitumor activity of traditional oncolytic peptides such as LTX-315 and the like begins to decline in 12 hours, and almost disappears in 48 hours.
In conclusion, the cationic oncolytic peptide LTX-315 developed by the predecessor has lower killing activity on adherent tumor cells, poorer enzymolysis stability, shorter half-life and undefined antitumor mechanism. Aiming at the problems, the invention designs and synthesizes a novel oncolytic peptide fluorescent probe, and the probe can emit red fluorescence, has higher killing activity aiming at adherent tumor cells, and has very high enzymolysis and anti-tumor cell stability. The fluorescent probe not only can be used as a tool molecule for exploring the anti-tumor activity and action mechanism of the oncolytic peptide at the molecular, cell and animal levels, but also can be used as a potential anti-tumor lead polypeptide for developing anti-tumor polypeptide medicaments, so the fluorescent probe has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of a solid phase polypeptide synthesis method of the present invention.
FIG. 2 is a structural formula, a primary amino acid sequence, an analytical reversed-phase high performance liquid chromatogram and an ESI-MS mass spectrum of WKW-212 in example 1 of the present invention.
FIG. 3 is a structural formula, a primary amino acid sequence, an analytical reversed-phase high performance liquid chromatogram and an ESI-MS mass spectrum of WKW-217 in example 1 of the present invention.
FIG. 4 is a structural formula, a primary amino acid sequence, an analytical reversed-phase high performance liquid chromatogram and an ESI-MS mass spectrum of WKW-223 in example 1 of the present invention.
FIG. 5 is a structural formula, a primary amino acid sequence, an analytical reversed-phase high performance liquid chromatogram and an ESI-MS mass spectrum of WKW-385 in example 1 of the present invention.
FIG. 6 is a spectrum of excitation and emission spectra of oncolytic peptide fluorescent probes WKW-385 in example 1 of the present invention.
FIG. 7 is a graph showing the relationship between the molar concentration and the fluorescence intensity of oncolytic peptide fluorescent probes WKW-385 in example 1 of the present invention.
FIG. 8 shows oncolytic peptide probes WKW-385 in HepG in example 1 of the present invention2Profile in adherent hepatoma cells.
FIG. 9 is a distribution diagram of oncolytic peptide probes WKW-385 in tumor-bearing mice according to example 1 of the present invention.
FIG. 10 shows the oncolytic peptide fluorescent probe pair HepG in example 1 of the present invention2Inhibition of adherent hepatoma cell proliferation.
FIG. 11 is a graph showing changes in morphology of adherent tumor cells caused by oncolytic peptide fluorescent probes WKW-385 in example 1 of the present invention.
FIG. 12 is a graph showing the time course of the inhibitory effect of the oncolytic peptide fluorescent probe on adherent tumor cells in example 1 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Interpretation of terms:
peptide: from amino groups (-NH) of amino acids2) And carboxyl (-COOH) to form a peptide bond, wherein the compound formed by dehydration condensation of 10-100 amino acid molecules is called polypeptide. The polypeptide drug has the advantages of higher activity, stronger stability, smaller toxic and side effect, less dosage and the like, has obvious treatment benefits for tumors, autoimmune diseases, hypertension and certain cardiovascular and metabolic diseases, and has wide development and application prospects.
An oncolytic peptide: oncolytic peptides are a class of antineoplastic drugs derived from or inspired by natural antimicrobial peptides (AMPs) that exhibit high antitumor activity while being less toxic to normal cells. Importantly, oncolytic peptides exert anti-cancer effects regardless of the phenotypic or genetic characteristics of tumor cells, which is largely determined by their unique physicochemical properties, in particular, the net positive charge on the surface of the oncolytic peptide and the specific distribution of hydrophobic residues are critical for oncolytic peptide activity. The oncolytic peptide is combined with the cell membrane of the tumor cell due to electrostatic interaction, and hydrophobic residues are inserted into the cell membrane to break down a lipid bilayer, promote the lysis of the membrane and finally cause the death of the tumor cell.
Polypeptide fluorescent probe: after being excited by exciting light, the fluorescent material emits light in the ultraviolet-visible-near infrared region and is called as fluorescence. The polypeptide fluorescent probe is a molecule which can emit fluorescence and is formed by combining polypeptide and a fluorescent group in a covalent bond mode.
And (3) rhodamine B: is a typical artificial synthetic dye of triphenylmethane, the overall charge is positive charge, and the molecular weight is 479.029. It has bright peach red color, the aqueous solution is blue red, and has strong fluorescence after dilution, and is a common fluorescent group.
IC50: the concentration required for half the maximal inhibitory effect of the drug. The smaller the value, the stronger the inhibitory activity.
The following models explain the mechanism of oncolytic peptide membrane-breaking killing of tumor cells.
A barrel wall model. Monomeric oncolytic peptides accumulate on cells and subsequently undergo conformational changes and aggregation to form barrel-like multimers. In this model, aggregation forces the polypeptide into the hydrophobic center of the cell membrane, and then the hydrophobic chains of the oncolytic peptide contact the acyl chains of the membrane, aligning with the lipid core of the bilayer, weakening the cell membrane. Whereas the hydrophilic part of the peptide forms water pores, which widen as the number of aggregated peptides increases.
A carpet model. This model describes the interaction of a positively charged alpha helix with a negatively charged phospholipid on the outer membrane, which is covered with a peptide to form a "carpet" of peptides. The oncolytic peptide remains parallel to the cell surface without insertion into the lipid bilayer, but as the concentration of peptide increases, the phospholipid spins and reorients itself, a process that increases the fluidity of the membrane, thus impairing the barrier properties of the membrane, disrupting the lipid bilayer and forming micelles.
And (4) an annular hole model. This is a two-stage model, peptides are inactive at low concentrations, distributed parallel to the bilayer, while they are converted to the active form at high concentrations and perpendicular to the bilayer, irreversibly destroying the membrane. The instability of the newly formed circular pore allows the peptide to enter the membrane, enter the intracellular space and inhibit various life processes, such as DNA replication and protein synthesis, leading to tumor cell death.
As mentioned above, the cationic oncolytic peptide LTX-315 obtained by modifying bovine lactoferrin as a natural antimicrobial peptide has high anti-tumor activity on suspended tumor cells, but has poor activity on adherent tumor cells, and the specific anti-cancer mechanism of the cationic oncolytic peptide is not clear, so that the application of the cationic oncolytic peptide is limited.
In view of the above, in an exemplary embodiment of the present invention, a high-stability oncolytic peptide fluorescent probe is provided, which comprises the following amino acid residue sequence:
WKW-212 rhodamine B-5-amino-3-oxopentanoic acid-KKWWKKW (Dip) K-NH2
WKW-217 rhodamine B-GABA-KKWWKKW (Dip) K-NH2
WKW-223 rhodamine B-11-amino-3, 6, 9-trioxaundecanoic acid-KKWWKKW (Dip) K-NH2
WKW-385 rhodamine B-AEEA-KKWWKKW (Dip) K-NH2
The oncolytic peptide fluorescent probe can emit red fluorescence, and realizes the space-time positioning and tracing of the oncolytic peptide; meanwhile, the anti-tumor activity and stability of the oncolytic peptide are greatly improved, and especially the killing activity to adherent tumor cells is remarkably improved.
In still another embodiment of the present invention, there is provided a nucleotide encoding the high-stability oncolytic peptide fluorescent probe, which comprises any one of the following groups:
(a) a nucleotide encoding a polypeptide having the amino acid sequence;
(b) a nucleotide complementary to the nucleotide of (a).
In another embodiment of the present invention, there is provided a method for synthesizing the above-described oncolytic peptide fluorescent probe, the method comprising: synthesizing polypeptide by solid phase polypeptide synthesis method; and carrying out condensation reaction connection on the polypeptide, a connecting group and a fluorescent group.
Specifically, the polypeptide is synthesized by a solid-phase polypeptide synthesis method (Fmoc-SPPS) based on 9-fluorenylmethyloxycarbonyl.
Unless otherwise specified, the present invention used Rink Amide Am resin (degree of substitution 0.32mmol/g) to synthesize the target polypeptide having an Amide at the nitrogen terminus, and all amino acids used were Fmoc-L-type amino acids.
More specifically, the synthesis method of the oncolytic peptide fluorescent probe comprises the following steps:
s1, synthesizing the polypeptide by a solid-phase polypeptide synthesis method based on 9-fluorenylmethyloxycarbonyl;
s2, carrying out condensation reaction on the polypeptide prepared in the step S1, a connecting group and a fluorescent group;
s3, adding a peptide cutting reagent into the condensation product prepared in the step S2 for peptide cutting treatment, and separating and purifying to obtain the oncolytic peptide fluorescent probe.
The specific method of step S1 includes:
pre-activating and activating Rink Amide Am resin, and then removing Fmoc protecting groups by using piperidine-containing DMF (dimethyl formamide) solution for deprotection; and (3) condensing the amino acids until all the amino acids are completely condensed, removing the Fmoc protecting group of the last amino acid, and then cleaning.
In another embodiment of the present invention, the pre-activation treatment method comprises: alternately cleaning the resin with DMF and DCM, and soaking for 1-2 hours at room temperature;
the activation treatment method comprises the following steps: the pretreated resin is immersed in a DMF/DCM mixed solution (1:1, volume ratio, v: v), and is subjected to a shaking activation treatment at 25-30 deg.C (preferably 28 deg.C) for 0.5-2 hours (preferably 1 hour).
The deprotection method comprises the following steps: the Fmoc protecting group is removed in DMF solution containing 20% piperidine (v: v) under the condition of 25-30 deg.C (preferably 28 deg.C), and the deprotection operation is carried out twice for 5 minutes and 10 minutes respectively.
The condensation reaction comprises the following specific steps: each amino acid condensation reaction is carried out twice under the condition of 25-30 ℃ (preferably 28 ℃), and the time is 20 minutes and 30 minutes respectively. The mixture ratio of the reactant amino acid is Fmoc-L-type amino acid: HCTU: DIPEA is 3 equivalent times: 2.8 times equivalent: 6 times equivalent.
The cleaning step comprises: the resin is washed with DMF and DCM alternately, and finally washed clean with DCM, and the solvent remaining in the resin is removed completely (e.g. by pumping with a water pump or an oil pump).
The specific method of the step S2 includes:
and (3) carrying out condensation reaction on the polypeptide prepared in the step S1 and a fluorescent group, wherein the reactant ratio is rhodamine B: HATU: HOAT: DIPEA is 6 equivalent times: 2.8 times equivalent: 3 times of equivalent: 6 times equivalent. Solid-phase condensation is carried out for three times at 25-30 ℃ (preferably 28 ℃), and the reaction time is 30 minutes, 40 minutes and 50 minutes respectively. During the fluorophore linkage, rhodamine B must be in excess (6-fold equivalent of resin). After the DIPEA reagent was added, the condensation solution had to be added to the synthesis tube within two minutes.
In step S3, the specific method of the peptide cutting process includes:
a peptide-cleaving reagent containing TFA (TFA: TIPS: water: phenol: 88:2:5:5(v: v: v)) is added to the synthesis tube to cleave the polypeptide for 2.5 to 3 hours at a reaction temperature of 25 to 30 ℃ (preferably 28 ℃). After the reaction is finished, concentrating the reaction product, then adding anhydrous ether (precooled to 4-10 ℃ in advance) which is ice-bathed in advance into the concentrated reaction solution, and precipitating the target polypeptide; subsequent centrifugation yielded a white precipitate of the crude peptide. And repeatedly cleaning with ice-bath anhydrous ether, centrifuging twice, ventilating and drying the obtained solid precipitate, and volatilizing the organic solvent to obtain powdery crude peptide.
The specific separation and purification method comprises the following steps:
the crude peptide was dissolved in a mixed solvent of acetonitrile and water containing 0.1% TFA, and lyophilized to obtain a flocculent crude peptide solid. Dissolving the crude peptide solid in a mixed solvent of acetonitrile and water containing 0.1% TFA, separating and purifying the crude peptide solution by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) to obtain a pure target polypeptide solution, and freeze-drying the polypeptide solution to obtain the pure solid target polypeptide.
The target polypeptide can be analyzed and identified by analytical reversed-phase high performance liquid chromatography, ESI-MS and the like. The solid target polypeptide is sealed and stored in a refrigerator at the temperature of 20 ℃ below zero for later use.
In another embodiment of the present invention, the application of the above-mentioned oncolytic peptide fluorescent probe in the preparation of a medicament for preventing and/or treating tumor-related diseases is provided.
Also, it is to be noted that the terms "tumor" or "cancer" are used interchangeably and mean the presence of cells having the typical characteristics of oncogenic cells, such as uncontrolled proliferation, immortalization, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological characteristics. Cancer cells are typically in the form of tumors, but such cells may be present alone in the animal, or may be non-tumorigenic cancer cells, such as leukemia cells. These terms include solid tumors, soft tissue tumors, or metastatic lesions. As used herein, the term "tumor" includes premalignant tumors as well as malignant tumors. In certain embodiments, the tumor is a solid tumor, a soft tissue tumor, or a metastatic lesion. The term also refers to solid tumors named for the cell types that form solid tumors, hematological, myeloid, or lymphoid cancers. Examples of solid tumors include, but are not limited to, sarcomas and carcinomas. Examples of hematological cancers include, but are not limited to, leukemia, lymphoma, and myeloma. These terms include, but are not limited to, a primary cancer originating at a particular site in the body, metastatic cancer that spreads from its point of origin to other regions of the body, recurrence of the original primary cancer after remission, and a second primary cancer that is a new primary cancer of a person with a different type of past cancer history than the latter.
In yet another embodiment of the present invention, the cancer is selected from the group consisting of benign or malignant tumors: lung (including small cell lung cancer and non-small cell lung cancer), bronchus, prostate, breast (including sporadic breast cancer and cowden patient), pancreas, gall bladder, gastrointestinal tract, colon, rectum, colon cancer, colorectal cancer, thyroid, liver, biliary tract, intrahepatic bile duct, hepatocyte, adrenal gland, stomach, central or peripheral nervous system (including astrocytoma, neuroblastoma, glioma, glioblastoma and schwannoma), neuroendocrine, endometrial, kidney, renal pelvis, bladder, uterus, cervix, vagina, ovary, multiple myeloma, esophagus, nose, neck or head, brain, mouth and pharynx, larynx, small intestine, melanoma, villous adenoma of the colon, sarcomas (including osteosarcoma, fibrosarcoma or rhabdomyosarcoma and kaposi's sarcoma), neoplasias, epithelial tumors, breast cancer, basal cell carcinoma, Squamous cell carcinoma, actinic keratosis, polycythemia, essential thrombocythemia, thyroid follicular cancer, leukemia (including acute myeloid leukemia, chronic myeloid leukemia, lymphocytic leukemia, myeloid leukemia, meningeal leukemia, and promyelocytic leukemia), lymphoma (including non-hodgkin's lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, B cell lymphoma, T cell lymphoma, hairy cell lymphoma, and burcky's lymphoma), myelodysplastic syndrome, choriocarcinoma, rhabdoid cancer, seminoma, teratocarcinoma, xeroderma pigmentosum; retinoblastoma, keratoacanthoma, myeloid myelofibrosis, Waldenstrom's disease, and Barret adenocarcinoma.
In another embodiment of the present invention, a pharmaceutical composition comprising the above-described oncolytic peptide fluorescent probe is provided.
Pharmaceutical compositions of the compounds of the present invention may be administered in any manner selected from: oral, aerosol inhalation, rectal, nasal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, with oral, intramuscular, intraperitoneal or intravenous administration being preferred.
In yet another embodiment of the present invention, a pharmaceutical formulation is provided comprising an oncolytic peptide fluorescent probe and a pharmaceutically acceptable carrier.
The pharmaceutical preparation can be any pharmaceutically acceptable pharmaceutical dosage form, such as tablets (including dispersible tablets, sustained release tablets, enteric-coated tablets, effervescent tablets, orally disintegrating tablets, chewable tablets, irregular tablets and the like), hard capsules (including gastric-soluble capsules, enteric-coated capsules and sustained release capsules), soft capsules (including gastric-soluble capsules and enteric-coated capsules), dropping pills, micro-pills, granules, dry suspension, powder, oral liquid (including solution, suspension and emulsion), injection (including powder injection for injection and injection liquid) and the like, and the pharmaceutical dosage forms are recorded and described in Chinese pharmacopoeia.
Other suitable pharmaceutical carriers such as diluents, fillers, disintegrants, surfactants, suspending agents, binders, lubricants, coloring agents, flavoring agents, etc. may optionally be included according to the pharmaceutical dosage form. The term "optionally contains" means that one or more of them may be selected or not selected.
Suitable fillers or diluents used include lactose, mannitol, sorbitol, microcrystalline cellulose, starch, modified starch, dextrin, cyclodextrin and its derivatives, calcium phosphate, sucrose, polyethylene glycol (polyethylene glycols of various molecular weights), pregelatinized starch, xylitol, fructose, maltitol, dextran, glucose, calcium sulfate, calcium hydrogen phosphate, and the like; the disintegrant includes sodium carboxymethylcellulose, croscarmellose sodium, sodium carboxymethyl starch, low substituted hydroxypropyl cellulose, crospovidone, pregelatinized starch, corn starch, sodium croscarmellose, microcrystalline cellulose, calcium carboxymethylcellulose, and the like; the surfactant includes sodium lauryl sulfate, sodium tetradecyl sulfate, sodium hexadecyl sulfate, sodium octadecyl sulfate, polysorbate (common name: tween including its various types), sorbitan fatty acid (common name: span including its various types), and the like; the binder comprises polyvinylpyrrolidone, starch slurry, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, guar gum, xanthan gum, and the like; suspending agents include, but are not limited to, hydroxypropylmethyl cellulose, ethyl cellulose, gum arabic, xanthan gum, sodium carboxymethyl cellulose, and the like; such lubricants include magnesium stearate, stearic acid, talc, sodium stearyl fumarate, and the like. In addition, pH adjusting agents or buffers such as phosphate buffer, citric acid, sodium citrate, acetate buffer, dilute hydrochloric acid, sodium carbonate, sodium hydroxide, and the like; preservatives such as sodium benzoate, potassium sorbate, methylparaben, propylparaben, and the like; stabilizers and antioxidants such as sodium calcium edetate, sodium sulfite, vitamin C, vitamin E, and the like; taste modifiers may also be included, for example maltitol, aspartame, stevia, fructose, sucrose, saccharin sodium, flavors such as orange flavor, strawberry flavor, and the like.
Of course, the pharmaceutical carrier is not limited to the above, and may further include other conventional and appropriate additives or pharmaceutical excipients, such as wetting agents, which can be selected from water, ethanol, water-ethanol solution, etc., according to the different dosage forms.
In still another embodiment of the present invention, there is provided a method for preventing and/or treating tumor, the method comprising: comprising administering to a subject a therapeutically effective dose of the above-described high stability oncolytic peptide fluorescent probe, the above-described pharmaceutical composition or the above-described pharmaceutical formulation.
The subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment. A "therapeutically effective dose" refers to an amount that results in an improvement in any parameter or clinical symptom. The actual dosage may vary from patient to patient and does not necessarily refer to the total amount of all disease symptoms eliminated, and can be determined by methods well known in the art.
In yet another embodiment of the present invention, there is provided the use of the above-described high stability oncolytic peptide fluorescent probe as a non-therapeutic tumor cell inhibitor and/or tracer. According to the invention, the "non-therapeutic purpose" is, for example, inhibition of tumor cell proliferation and promotion of tumor cell death in vitro, and in particular, the killing activity of the high-stability oncolytic peptide fluorescent probe on adherent tumor cells is higher; meanwhile, the high-stability oncolytic peptide fluorescent probe can emit red fluorescence so as to realize space-time positioning and tracing of the oncolytic peptide, has longer red fluorescence wavelength and stronger penetrating power, is applied to imaging of cells, tissues and living animals, and can be used as a tracing agent so as to explore the anti-tumor activity and action mechanism of the oncolytic peptide at the level of molecules, cells, tissues and animals.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
In this example, all target polypeptides were prepared using the solid phase peptide synthesis technique based on 9-fluorenylmethyloxycarbonyl (Fmoc-SPPS). In the process of synthesizing the polypeptide, the target polypeptide with Amide at the nitrogen terminal is synthesized by using Rink Amide Am resin (the substitution degree is 0.32mmol/g) and all the amino acids are Fmoc-L-type amino acids unless otherwise specified.
The scale of the synthesized polypeptide is generally 0.15 mmol. As shown in FIG. 1, a basic scheme for polypeptide synthesis is presented, comprising:
solid phase polypeptide synthesis procedure: 470mg of Rink Amide Am resin (1 equivalent) was weighed out and the resin was then washed alternately with DMF and DCM and soaked for 1-2 hours at room temperature to pre-activate the resin. 8 ml of a DMF/DCM mixture (1:1 by volume, v: v) was added to soak the resin and activated by shaking in a constant temperature shaker at 28 ℃ for 1 hour. The Fmoc protecting group was then removed using a solution of 20% piperidine (v: v) in DMF. Deprotection was carried out twice at 28 ℃ for 5 min and 10 min, respectively. The amino acids were condensed at 28 ℃ twice for 20 min and 30 min each. The mixture ratio of the reactant amino acid is Fmoc-L-type amino acid: HCTU: DIPEA is 3 equivalent times: 2.8 times equivalent: 6 times equivalent. After the condensation of all amino acids was completed, the Fmoc protecting group of the last amino acid was removed, followed by alternate washing with DMF and DCM, and finally the resin was washed clean with DCM, and the residual solvent in the resin was thoroughly drained with a water pump, an oil pump.
Synthesis of oncolytic peptide fluorescent probe: after the solid phase condensation of the polypeptide fragments and the linker groups is completed using the solid phase polypeptide synthesis procedure described above, the condensation of the fluorophore is performed. The mixture ratio of reactants is rhodamine B: HATU: HOAT: DIPEA is 6 equivalent times: 2.8 times equivalent: 3 times of equivalent: 6 times equivalent. Solid phase condensation was carried out three times at 28 ℃ for 30 minutes, 40 minutes and 50 minutes, respectively. During the fluorophore linkage, rhodamine B must be in excess (6-fold equivalent of resin). After the DIPEA reagent was added, the condensation solution had to be added to the synthesis tube within two minutes.
And (3) cutting peptide: to the synthesis tube, a peptide-cleaving reagent containing TFA (TFA: TIPS: water: phenol: 88:2:5:5(v: v: v: v)) was added to cleave the polypeptide for 2.5 to 3 hours at 28 ℃. The cleaved peptide solution was retained and transferred to a three-necked flask, and the cleaved peptide solution was concentrated to 2 ml using high purity nitrogen bubbling. Then, anhydrous ether (precooled to 4-10 ℃ in advance) in an ice bath is added into the concentrated reaction solution, and the target polypeptide is precipitated. Subsequent centrifugation through a centrifuge resulted in a white precipitate of the crude peptide. And repeatedly cleaning with ice-bath anhydrous ether, centrifuging twice, standing the obtained solid precipitate in a fume hood for 1 hour, and volatilizing the organic solvent to obtain powdery crude peptide.
Separation and purification: the crude peptide was dissolved in a mixed solvent of acetonitrile and water containing 0.1% TFA, and lyophilized in a vacuum lyophilizer to obtain a flocculent crude peptide solid. Dissolving the crude peptide solid in a mixed solvent of acetonitrile and water containing 0.1% TFA, separating and purifying the crude peptide solution by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) to obtain a pure target polypeptide solution, and then putting the polypeptide solution into a vacuum freeze dryer for freeze drying to obtain the pure solid target polypeptide.
And analyzing and identifying the target polypeptide by using analytical reversed-phase high-performance liquid chromatography, ESI-MS and the like. The structural formula, the primary amino acid sequence, the analytical reverse phase high performance liquid chromatogram and the ESI-MS mass spectrogram of the 4 prepared oncolytic peptide fluorescent probes are shown in figures 2-5. The solid target polypeptide is sealed and stored in a refrigerator at the temperature of 20 ℃ below zero for later use.
Oncolytic peptide fluorescent probe wavelength scanning experiment
The Flexstation instrument was started and warmed, and WKW-385 solution was prepared at 300 μ M concentration using triple distilled water, and 200 μ L per well was added to a black 96-well plate. Reading in a flexstation of a 96-hole plate, firstly scanning emission wavelength, setting the excitation wavelength to be 550 nanometers, setting the scanning emission wavelength range to be 560-650 nanometers, and setting the scanning interval to be 1 nanometer. And scanning the excitation wavelength, wherein the emission wavelength is set to be 600 nanometers, the scanning excitation wavelength range is 500-580 nanometers, and the scanning interval is 1 nanometer.
As shown in FIG. 6, the maximum excitation wavelength of the oncolytic peptide fluorescent probes WKW-385 is 560 nm and the maximum emission wavelength is 590 nm.
Experiment of relationship between fluorescence intensity and concentration of oncolytic peptide fluorescent probe
The flexstation master was turned on and warmed up, and WKW-385 solutions at concentrations of 50. mu.M, 75. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, 300. mu.M, 400. mu.M, 500. mu.M, 700. mu.M, 800. mu.M, 900. mu.M and 1000. mu.M were prepared with a triple-distilled water gradient, 200. mu.L per well was added to a black 96-well plate and three wells were set. The 96-well plate was read in flexstation, setting the excitation wavelength at 560 nm and the emission wavelength at 590 nm.
As a result, as shown in FIG. 7, the fluorescence intensity of the novel oncolytic peptide probes WKW-385 shows a concentration-dependent pattern, and the fluorescence intensity increases with increasing concentration.
Distribution experiment of oncolytic peptide fluorescent probe in adherent tumor cells
Liver cancer cell HepG2(derived from the center for cell resources of the institute of basic medicine of Chinese academy of medical sciences) in MEM medium containing 10% fetal bovine serum. Good-state HepG2Adherent cells were trypsinized, collected, diluted with medium, and plated well in 6-well plates (500. mu.L/well). After overnight cell culture, 1 mL of WKW-385 solution (final concentration 30. mu.M) was prepared with medium, which also contained 2. mu.g/mL Hoechst 33258 (nuclear blue fluorescent probe) and 1. mu.M Mito-Tracker Green (mitochondrial Green fluorescent probe). After adding to the cells, the 6-well plate was returned to the incubator for incubation, taken out at 30 minutes, the supernatant was discarded, washed three times with phenol red-free DMEM, observed with a fluorescence microscope and photographed.
As shown in FIG. 8, WKW-385 fluoresced red in the cells and, as seen from the fused image, there was good co-localization of red and blue fluorescence, indicating that WKW-385 mainly concentrated in the nucleus. The above experiments show that fluorescent probes such as WKW-385 can be used as tool molecules to search the action mechanism of oncolytic peptide.
In vivo imaging experiment of oncolytic peptide fluorescent probe in mouse
In the fluorescence probe in-vivo imaging experiment, CB6F1 mice loaded with B16-F10 transplantation tumor are selected, after the trunk of the mice is unhaired, the experimental group is injected with 50 mu L of 10mg/mL WKW-385 solution (in normal saline) in the transplantation tumor under the right axilla, and the control group is injected with 50 mu L of 10mg/mL LTX-315 solution (in normal saline). The mice were subjected to in vivo imaging at 0 hours, 1 hour, 6 hours, 12 hours, 24 hours and 48 hours, and were anesthetized with chloral hydrate prior to imaging. After the imaging was completed for 24 hours, the mice were sacrificed, and the organs thereof were dissected and subjected to fluorescent imaging of the organs. Imaging conditions are as follows: excitation wavelength 570 nm and emission wavelength 620 nm.
As shown in FIG. 9, the right mice showed significant aggregation of the novel oncolytic probes WKW-385, which were able to aggregate in tumor regions for a long time, with significant distribution in tumor tissues and no significant distribution in other organs within 48 hours, while the left control group showed no fluorescence.
Experiment for inhibiting proliferation of adherent tumor cells by using oncolytic peptide fluorescent probe
Good-state HepG2Cells were trypsinized, collected, diluted with medium, and plated well in 96-well plates (100. mu.L/well). After overnight cell culture, the drug solution was prepared and 30mM of WKW-212, WKW-217, WKW-223, WKW-385 and LTX-315 stock solutions were diluted with medium in 2-fold gradient to the indicated concentrations (100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M), and 3 wells (150. mu.L) were repeated for each concentration. After the drug solution was added to the wells, the 96-well plate was returned to the incubator and incubated for 24 hours. After 24 hours, 15. mu.L of MTT working solution (5mg/mL) was added to each well, and after 4 hours of reaction, the supernatant was discarded, 150. mu.L of DMSO was added thereto, and after standing for 1 hour, the OD value was read with a microplate reader at a wavelength of 490 nm.
As shown in FIG. 10, the novel oncolytic peptides kill tumor cells in a concentration-dependent manner with 24-hour IC50About 15 μ M, LTX-315 IC50The molecular weight of the fluorescent probe is about 150 mu M, and the experiment shows that the fluorescent probe shows stronger anti-adherent tumor cell activity which is ten times that of the classical oncolytic peptide LTX-315.
Morphological experiment for influence of oncolytic peptide fluorescent probe on tumor cells
The liver cancer adherent cells HepG with good state2Cells were trypsinized, collected, diluted with medium, and plated well in 6-well plates (500. mu.L/well). After overnight cell culture, 30mM WKW-385 stock was diluted to 30. mu.M with medium, added to the cells, and the 6-well plate was returned to the incubator for incubation, removed at 0 min, 15 min, 30 min and 60 min, observed with a microscope and photographed.
As shown in FIG. 11, cytomorphology experiments show that oncolytic peptide fluorescent probes WKW-385 induce adherent tumor cell morphology change, cell atrophy and fragmentation, and the number of dead cells gradually increases with time, and shows a time-dependent characteristic.
Cell stability assay for oncolytic peptide fluorescent probes
The liver cancer adherent cells HepG with good state2After trypsinization, the cells were collected, diluted with medium and plated well in 96-well plates (100. mu.L/well). After overnight cell culture, the drug solution was prepared and 30mM of WKW-212, WKW-217, WKW-223, WKW-385 and LTX-315 stock solutions were diluted to 30. mu.M with medium and 3 wells (150. mu.L) were repeated for each concentration. After the liquid medicine was added to the wells, the 96-well plate was returned to the incubator for incubation, and MTT experiments were performed at 4 hours, 12 hours, 24 hours, 36 hours, 48 hours, and 72 hours, respectively. mu.L of MTT working solution (5mg/mL) was added to each well, the reaction was carried out for 4 hours, the supernatant was discarded, 150. mu.L of DMSO was added, the mixture was allowed to stand for 1 hour, and then OD was read with a microplate reader at 490nm, and the inhibition ratio was calculated from the OD at each time point.
As shown in fig. 12, the antitumor activity of the oncolytic peptide fluorescent probe gradually increased with time, and reached a peak at 36 hours. The anti-tumor activity of the oncolytic peptide fluorescent probe at each time point is far higher than that of LTX-315, and the activity can be kept for more than 72 hours. The antitumor activity of LTX-315 peaked at 12 hours and then began to decline, by 48 hours the antitumor activity was substantially lost. The above results indicate that the oncolytic peptide fluorescent probe has higher anti-tumor activity and anti-tumor stability.
Finally, it should be noted that the above mentioned embodiments are only preferred embodiments of the present invention, and not intended to limit the present invention, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that the technical solutions described in the foregoing embodiments may be modified or partially replaced with equivalents. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. A high-stability oncolytic peptide fluorescent probe, which is characterized by comprising the following amino acid residue sequence:
WKW-212 rhodamine B-5-amino-3-oxopentanoic acid-KKWWKKW (Dip) K-NH2
WKW-217 rhodamine B-GABA-KKWWKKW (Dip) K-NH2
WKW-223 rhodamine B-11-amino-3, 6, 9-trioxaundecanoic acid-KKWWKKW (Dip) K-NH2
WKW-385 rhodamine B-AEEA-KKWWKKW (Dip) K-NH2。
2. A nucleotide encoding the high-stability oncolytic peptide fluorescent probe according to claim 1, comprising any one of the following groups:
(a) a nucleotide encoding a polypeptide having the amino acid sequence;
(b) a nucleotide complementary to the nucleotide of (a).
3. The method for synthesizing an oncolytic peptide fluorescent probe according to claim 1, wherein the method comprises the following steps: synthesizing polypeptide by solid phase polypeptide synthesis method; and carrying out condensation reaction connection on the polypeptide, a connecting group and a fluorescent group.
4. The method of claim 3, wherein the polypeptide is synthesized by a solid phase polypeptide synthesis method based on 9-fluorenylmethyloxycarbonyl.
5. The method of synthesizing according to claim 3 wherein the method of synthesizing the oncolytic peptide fluorescent probe comprises:
s1, synthesizing the polypeptide by a solid-phase polypeptide synthesis method based on 9-fluorenylmethyloxycarbonyl;
s2, carrying out condensation reaction on the polypeptide prepared in the step S1, a connecting group and a fluorescent group rhodamine B;
s3, adding a peptide cutting reagent into the condensation product prepared in the step S2 for peptide cutting treatment, and separating and purifying to obtain the oncolytic peptide fluorescent probe.
6. Use of the oncolytic peptide fluorescent probe according to claim 1 for the preparation of a medicament for preventing and/or treating tumor-related diseases;
preferably, the tumor comprises a solid tumor and a non-solid tumor.
7. A pharmaceutical composition comprising the oncolytic peptide fluorescent probe of claim 1.
8. A pharmaceutical formulation comprising an oncolytic peptide fluorescent probe according to claim 1 and a pharmaceutically acceptable carrier;
preferably, the carrier includes diluents, fillers, disintegrants, surfactants, suspending agents, binders, lubricants, colorants and flavoring agents.
9. A method for preventing and/or treating a tumor, the method comprising: comprising administering to a subject a therapeutically effective dose of the high stability oncolytic peptide fluorescent probe of claim 1, the pharmaceutical composition of claim 7 or the pharmaceutical formulation of claim 8.
10. Use of the high stability oncolytic peptide fluorescent probe according to claim 1 as a tumor cell inhibitor and/or tracer for non-therapeutic purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110910097.XA CN113604214B (en) | 2021-08-09 | 2021-08-09 | High-stability oncolytic peptide fluorescent probe and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110910097.XA CN113604214B (en) | 2021-08-09 | 2021-08-09 | High-stability oncolytic peptide fluorescent probe and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113604214A true CN113604214A (en) | 2021-11-05 |
| CN113604214B CN113604214B (en) | 2023-07-14 |
Family
ID=78307764
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110910097.XA Active CN113604214B (en) | 2021-08-09 | 2021-08-09 | High-stability oncolytic peptide fluorescent probe and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113604214B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114668843A (en) * | 2022-01-18 | 2022-06-28 | 中国人民解放军总医院第一医学中心 | Nano self-assembled glycopeptide BIVA-PK and application thereof in renal fibrosis caused by ischemia-reperfusion injury |
| WO2023197129A1 (en) * | 2022-04-12 | 2023-10-19 | Rise Biopharmaceuticals Inc. | Oncolytic peptide and use thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190328887A1 (en) * | 2016-06-16 | 2019-10-31 | Université De Strasbourg | Metabolically stable peptide analogs |
| WO2020002668A1 (en) * | 2018-06-28 | 2020-01-02 | Norce Norwegian Research Centre As | Peptides and cancer treatment |
| US20200113829A1 (en) * | 2018-10-12 | 2020-04-16 | Synergene Therapeutics, Inc. | Methods for reducing side effects of immunotherapy |
| CN111150833A (en) * | 2020-03-16 | 2020-05-15 | 中国科学院微生物研究所 | Application of LTX-315 in preparation of products for inhibiting coronavirus |
| US20210122814A1 (en) * | 2018-07-11 | 2021-04-29 | Scholar Rock, Inc. | HIGH-AFFINITY, ISOFORM-SELECTIVE TGFß1 INHIBITORS AND USE THEREOF |
-
2021
- 2021-08-09 CN CN202110910097.XA patent/CN113604214B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190328887A1 (en) * | 2016-06-16 | 2019-10-31 | Université De Strasbourg | Metabolically stable peptide analogs |
| WO2020002668A1 (en) * | 2018-06-28 | 2020-01-02 | Norce Norwegian Research Centre As | Peptides and cancer treatment |
| US20210122814A1 (en) * | 2018-07-11 | 2021-04-29 | Scholar Rock, Inc. | HIGH-AFFINITY, ISOFORM-SELECTIVE TGFß1 INHIBITORS AND USE THEREOF |
| US20200113829A1 (en) * | 2018-10-12 | 2020-04-16 | Synergene Therapeutics, Inc. | Methods for reducing side effects of immunotherapy |
| CN111150833A (en) * | 2020-03-16 | 2020-05-15 | 中国科学院微生物研究所 | Application of LTX-315 in preparation of products for inhibiting coronavirus |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114668843A (en) * | 2022-01-18 | 2022-06-28 | 中国人民解放军总医院第一医学中心 | Nano self-assembled glycopeptide BIVA-PK and application thereof in renal fibrosis caused by ischemia-reperfusion injury |
| WO2023197129A1 (en) * | 2022-04-12 | 2023-10-19 | Rise Biopharmaceuticals Inc. | Oncolytic peptide and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113604214B (en) | 2023-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101424306B1 (en) | Metastin derivatives and use thereof | |
| JP2021130672A (en) | Mt1-mmp specific bicyclic peptide ligands | |
| WO2020156513A1 (en) | Bi-ligand drug conjugate and use thereof | |
| CN113604214B (en) | High-stability oncolytic peptide fluorescent probe and preparation method and application thereof | |
| JP2016534147A (en) | Chlorotoxin conjugate and method of use thereof | |
| CN102725305A (en) | Melanocortin-1 receptor-specific cyclic peptides | |
| US11827687B2 (en) | Synthetic somatostatin receptor ligands | |
| AU2018363807A1 (en) | A double-labeled probe for molecular imaging and use thereof | |
| WO2017152756A1 (en) | Crgd-erlotinib conjugate and preparation method thereof | |
| JP2022525416A (en) | Peptidomimetic macrocyclic molecules and their use | |
| WO2015196944A1 (en) | Gnrh analog-cytotoxic molecule conjugate and preparation method and use thereof | |
| ES2361670T3 (en) | PEPTIDES MARKED WITH FLUORESCEINE. | |
| US20190083663A1 (en) | Carbonic anhydrase ix targeting agents and methods | |
| JPWO2010013762A1 (en) | Metastin derivatives and uses thereof | |
| US10500286B2 (en) | CCK2R-drug conjugates | |
| CN114456152A (en) | Golgi-targeted photo-thermal reagent covalently bound to protein and preparation method and application thereof | |
| US20230257420A1 (en) | Compounds for drug delivery across blood-brain barrier | |
| CN113024635B (en) | Application of stapling peptide compound and pharmaceutical composition thereof | |
| CN108314741A (en) | A kind of tumor vascular targeting anticancer peptide NKL-DOTA and preparation method thereof | |
| WO2023246703A1 (en) | Peptide inhibitor and use thereof | |
| US20240083949A1 (en) | Cell penetrating peptide compositions and methods thereof | |
| WO2023088236A1 (en) | Bicyclic peptide ligand of mt1-mmp and conjugate thereof | |
| RU2454425C2 (en) | Metastin derivatives and use thereof | |
| WO2019096096A1 (en) | Multi-arm targeting conjugate | |
| CN114437174A (en) | Aza-stable peptide for anti-estrogen receptor alpha, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |