NL2035226A - Preparation method and application of novel artemisinin derivatives and liposomes - Google Patents
Preparation method and application of novel artemisinin derivatives and liposomes Download PDFInfo
- Publication number
- NL2035226A NL2035226A NL2035226A NL2035226A NL2035226A NL 2035226 A NL2035226 A NL 2035226A NL 2035226 A NL2035226 A NL 2035226A NL 2035226 A NL2035226 A NL 2035226A NL 2035226 A NL2035226 A NL 2035226A
- Authority
- NL
- Netherlands
- Prior art keywords
- ats
- tpp
- liposomes
- tumour
- organic solvent
- Prior art date
Links
- 239000002502 liposome Substances 0.000 title claims abstract description 50
- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical class C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 29
- 230000008685 targeting Effects 0.000 claims abstract description 21
- 230000002438 mitochondrial effect Effects 0.000 claims abstract description 18
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 16
- 239000006185 dispersion Substances 0.000 claims abstract description 9
- 229960003180 glutathione Drugs 0.000 claims abstract description 8
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 6
- 108010024636 Glutathione Proteins 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 42
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000003960 organic solvent Substances 0.000 claims description 18
- -1 alkyl glycosides Chemical class 0.000 claims description 17
- 239000002245 particle Substances 0.000 claims description 17
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- FIHJKUPKCHIPAT-AHIGJZGOSA-N artesunate Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2O[C@@H](OC(=O)CCC(O)=O)[C@@H]4C FIHJKUPKCHIPAT-AHIGJZGOSA-N 0.000 claims description 14
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 14
- 229960004991 artesunate Drugs 0.000 claims description 13
- 150000003904 phospholipids Chemical class 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- 239000000741 silica gel Substances 0.000 claims description 12
- 229910002027 silica gel Inorganic materials 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 239000008213 purified water Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- KYNFOMQIXZUKRK-UHFFFAOYSA-N 2,2'-dithiodiethanol Chemical compound OCCSSCCO KYNFOMQIXZUKRK-UHFFFAOYSA-N 0.000 claims description 9
- 238000009826 distribution Methods 0.000 claims description 9
- 239000012634 fragment Substances 0.000 claims description 9
- 229930182478 glucoside Natural products 0.000 claims description 9
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims description 7
- 235000012000 cholesterol Nutrition 0.000 claims description 7
- 239000003208 petroleum Substances 0.000 claims description 7
- 230000002829 reductive effect Effects 0.000 claims description 7
- SRLOHQKOADWDBV-NRONOFSHSA-M sodium;[(2r)-2,3-di(octadecanoyloxy)propyl] 2-(2-methoxyethoxycarbonylamino)ethyl phosphate Chemical compound [Na+].CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCCNC(=O)OCCOC)OC(=O)CCCCCCCCCCCCCCCCC SRLOHQKOADWDBV-NRONOFSHSA-M 0.000 claims description 7
- 229930182470 glycoside Natural products 0.000 claims description 6
- 230000005540 biological transmission Effects 0.000 claims description 5
- 229940041181 antineoplastic drug Drugs 0.000 claims description 4
- 239000012982 microporous membrane Substances 0.000 claims description 4
- 229940083466 soybean lecithin Drugs 0.000 claims description 4
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 claims description 3
- 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 claims description 3
- 239000012043 crude product Substances 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 3
- 230000008020 evaporation Effects 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229940099578 hydrogenated soybean lecithin Drugs 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000002246 antineoplastic agent Substances 0.000 claims description 2
- 230000000887 hydrating effect Effects 0.000 claims description 2
- 238000005374 membrane filtration Methods 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 claims 2
- 101100459438 Caenorhabditis elegans nac-1 gene Proteins 0.000 claims 1
- GJYVZUKSNFSLCL-UHFFFAOYSA-N dichloromethanol Chemical compound OC(Cl)Cl GJYVZUKSNFSLCL-UHFFFAOYSA-N 0.000 claims 1
- 238000004090 dissolution Methods 0.000 claims 1
- 229930195733 hydrocarbon Natural products 0.000 claims 1
- 238000002525 ultrasonication Methods 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 32
- 239000003814 drug Substances 0.000 abstract description 23
- 229940079593 drug Drugs 0.000 abstract description 21
- 230000000259 anti-tumor effect Effects 0.000 abstract description 15
- 229960004191 artemisinin Drugs 0.000 abstract description 13
- 229930101531 artemisinin Natural products 0.000 abstract description 13
- 210000003470 mitochondria Anatomy 0.000 abstract description 9
- 210000001519 tissue Anatomy 0.000 abstract description 9
- 210000004881 tumor cell Anatomy 0.000 abstract description 7
- 238000012377 drug delivery Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 30
- 241000699670 Mus sp. Species 0.000 description 26
- 238000002474 experimental method Methods 0.000 description 15
- 239000000243 solution Substances 0.000 description 11
- 206010006187 Breast cancer Diseases 0.000 description 10
- 208000026310 Breast neoplasm Diseases 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 210000004185 liver Anatomy 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- 238000011156 evaluation Methods 0.000 description 9
- CMSYDJVRTHCWFP-UHFFFAOYSA-N triphenylphosphane;hydrobromide Chemical compound Br.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 CMSYDJVRTHCWFP-UHFFFAOYSA-N 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 8
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 8
- 238000005538 encapsulation Methods 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 210000002216 heart Anatomy 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 4
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000003907 kidney function Effects 0.000 description 4
- 230000003908 liver function Effects 0.000 description 4
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 238000010827 pathological analysis Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- MLOSJPZSZWUDSK-UHFFFAOYSA-N 4-carboxybutyl(triphenyl)phosphanium;bromide Chemical compound [Br-].C=1C=CC=CC=1[P+](C=1C=CC=CC=1)(CCCCC(=O)O)C1=CC=CC=C1 MLOSJPZSZWUDSK-UHFFFAOYSA-N 0.000 description 3
- 108010087230 Sincalide Proteins 0.000 description 3
- 238000012742 biochemical analysis Methods 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000002489 hematologic effect Effects 0.000 description 3
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 3
- 230000036571 hydration Effects 0.000 description 3
- 238000006703 hydration reaction Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 2
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000000113 differential scanning calorimetry Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 2
- 229960005277 gemcitabine Drugs 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000005534 hematocrit Methods 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 230000002107 myocardial effect Effects 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 1
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 238000001061 Dunnett's test Methods 0.000 description 1
- 206010015548 Euthanasia Diseases 0.000 description 1
- 102000058063 Glucose Transporter Type 1 Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000000037 Prosopis spicigera Species 0.000 description 1
- 235000006629 Prosopis spicigera Nutrition 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 108091006296 SLC2A1 Proteins 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000001434 glomerular Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000004898 mitochondrial function Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000010494 opalescence Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000001839 systemic circulation Effects 0.000 description 1
- GYTROFMCUJZKNA-UHFFFAOYSA-N triethyl triethoxysilyl silicate Chemical compound CCO[Si](OCC)(OCC)O[Si](OCC)(OCC)OCC GYTROFMCUJZKNA-UHFFFAOYSA-N 0.000 description 1
- 210000004926 tubular epithelial cell Anatomy 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Preparation (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Disclosed is a preparation method and application of the novel artemisinin derivative and its 5 liposomes. The method comprises the following steps: firstly, synthesizing artemisinin derivative (TPP-SS—ATS) which is sensitive to glutathione (GSH) and has mitochondrial targeting function; then the novel artemisinin derivative liposomes (TPP-SS—ATS—LS) were prepared by film dispersion method. The novel artemisinin derivative liposomes (TPP-SS—ATS—LS) prepared by the invention can realize dual-target drug delivery to tumour tissues and tumour cell mitochondria, 10 and remarkably improve the anti-tumour efficacy of artemisinin drugs.
Description
Preparation method and application of novel artemisinin derivatives and liposomes
The invention belongs to the field of traditional Chinese medicine preparations, and particularly relates to a novel artemisinin derivative, a preparation method of liposomes and an anti-tumour effect.
In anti-tumour application, the mechanism of artemisinin and its derivatives is quite different from other drugs. Some chemotherapy drugs exert anti-tumour effects by interfering with nucleic acid biosynthesis of tumour cells and destroying DNA function (references: Fang
Miao, Wang Degang, Liu Peiging. Research Progress on Anti-tumour Mechanism of Natural
Products [J]. Food and Drug, 2022,24 (2): 167-171; Wei Qiuhong, Liu Xiaoyue, Wang Pan,
Zhang Wenrui. Research Progress in Classification and Pharmacodynamics of Anti-Tumour
Drugs [J]. Medical Recapitulate, 2020,26 (18): 3707-3711,3716; Xu Jiao, Meng Linghua, Qing
Chen. The clinical application and development of traditional antitumor drugs [J]. Acta
Pharmaceutica Sinica, 2021,56 (6): 1551-1561); Artemisinin drugs are anti-tumour drugs by releasing oxygen free radical ROS, starting mitochondrial apoptosis program and combining with oxidative damage of cell model structure (references: Zhou Y, Li W, Xiao Y. Profiling of
Multiple Targets of Artemisinin Activated by Chemincancer Cell Proteome [J]. ACS Chembiol, 2016, 11: 882-888; Liu Qingging, Yang Zhenhua. Progress of anti-tumour activities of artemisinin and its derivatives [J]. Chinese Bulletin of Life Sciences, 2020,32(1).62-69).
Because of the unique anti-tumour mechanism of artemisinin, these drugs have become attractive anti-tumour candidates, which have attracted widespread attention in the world.
However, artemisinin drugs have some problems, such as poor solubility, low bioavailability and no tumour targeting, which restrict their anti-tumour efficacy. To sum up, improving the solubility and targeting of artemisinin drugs is the key to increase its anti-tumour efficacy.
According to the invention, tumour targeting liposomes is constructed by taking tumour cell membrane transporter GLUT1 as a target, so that tumour targeting delivery of artemisinin drugs is realized; Based on the mitochondrial targeted migration of triphenylphosphine, artemisinin- based mitochondrial targeted prodrugs were synthesized to further realize mitochondrial targeted delivery of liposomes. The invention improves that anti-tumour efficacy of artemisinin drugs through the delivery of tumour and tumour cell mitochondria double target drugs.
Aiming at the problems existing in the prior art, the invention aims to provide a preparation method and application of novel artemisinin derivatives and liposomes. After artemisinin derivatives enter tumour cells, their common structural peroxy bridge groups will break,
producing a large number of free radicals, causing oxidative stress and mitochondrial function damage in cancer cells. Based on this, the invention designed and prepared the novel artemisinin derivative liposomes (TPP-SS-ATS-LS) with dual targeting of tumour cells and their mitochondria.
The technical scheme of the invention is as follows:
A novel artemisinin derivative is characterized in that the structure of the novel artemisinin derivative is shown in the following formula:
Ee 3 \ oS oo Na, 5 5 Er gg Send PN Ne A
A preparation method of the novel artemisinin derivative liposomes comprises the following steps: firstly, synthesizing an artemisinin derivative (TPP-SS-ATS) which is sensitive to glutathione (GSH) and has mitochondrial targeting function; then the novel artemisinin derivative liposomes (TPP-SS-ATS-LS) was prepared by film dispersion method.
Furthermore, the particle size of the novel artemisinin derivative liposomes is in a normal distribution, and it is a regular spherical shape observed by transmission electron microscope.
Further, the method for synthesizing TPP-SS-ATS comprises the following steps: (1) dissolving artesunate and 2,2-dithiodiethanol in an organic solvent, wherein the dosage ratio of artesunate and 2,2-dithiodiethanol is 1:50 to 50:1, reacting at -20°C to 40°C for 0.1 to 96 hours, and recovering the organic solvent at 20 to 100 °C by using a rotary evaporator after the reaction, so as to obtain crude intermediate (SS-ATS). Using petroleum ether: ethyl acetate solution as mobile phase, the crude SS-ATS was purified by silica gel column to obtain SS-ATS. (2) weighing and dissolving that SS-ATS and the {(4-Carboxybutyl)triphenylphosphonium bromide (TPP) in an organic solvent, wherein the dosage ratio of the intermediate SS-ATS and the TPP is 1:50 to 50:1, react for 0.1 to 96 hours at the temperature of -20°C to 40°C, and recovering the organic solvent at the temperature of 20 to 100 °C by using a rotary evaporator after the reaction, so as to obtain the product. Using dichloromethane: methanol solution as mobile phase, the crude product was purified by silica gel column, and a novel artemisinin derivative (TPP-SS-ATS) was obtained.
Further, the specific method for preparing novel artemisinin derivative liposomes (TPP-SS-
ATS-LS) by film dispersion method is as follows: phospholipid, alkyl glycoside, TPP-SS-ATS, cholesterol and distearoyl phosphoethanolamine-PEG2000(DSPE-mPEG2000) are dissolved in an organic solvent; then transfer the obtained solution to an evaporation bottle of a rotary evaporator, concentrate under reduced pressure at 20°C-100°C until the organic solvent is completely volatilized, add purified water into the rotary evaporator, hydrate at 20°C-80°C for 0.1-3.0 hours, and then sequentially perform ultrasonic vibration and microporous membrane filtration to prepare TPP-SS-ATS-LS.
Further, the weight percentage of phospholipid is 0.2-5.0%, cholesterol is 0.01-0.5%, alkyl glycoside is 0.2-3.0%, DSPE-mPEG2000 is 0.02-5.0%, TPP-SS-ATS is 0.02-3.0% and purified water is 73.5-99.0%.
Further, the phospholipid is one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin and PEGylated phospholipid.
Further, the alkyl glucoside is one or more alkyl glucosides containing 2-30 alkyl carbons and 1-10 glucose segments per molecule of alkyl glucoside.
Furthermore, the ratio of petroleum ether to ethyl acetate ranges from 10: 1 to 2: 1; the ratio of dichloromethane to methanol ranges from 500: 1 to 50: 1; Purification was carried out by silica gel column with 50-500 meshes.
Application of a novel artemisinin derivative liposomes in preparation of anti-tumour drugs.
In the invention, artesunate is selected as a raw material to synthesize TPP-SS-ATS which is sensitive to GSH and has a mitochondrial targeting function, and after its structure is confirmed by nuclear magnetic resonance, mass spectrometry and other methods, TPP-SS-
ATS-LS are prepared by a film dispersion method.
A preparation method of TPP-SS-ATS-LS comprises the following steps: firstly, synthesizing TPP-SS-ATS which is sensitive to GSH and has mitochondrial targeting function; then, TPP-SS-ATS-LS are prepared by film dispersion method.
The synthesis method of TPP-SS-ATS, a novel artemisinin derivative, comprises the following steps: (1) dissolving artesunate and 2,2- dithiodiethanol in an organic solvent, wherein the dosage ratio of artesunate and 2,2- dithiodiethanol is 1:50 to 50:1, reacting at -20°C to 40°C for 0.1 to 96 hours, and using a rotary evaporator after the reaction. Using petroleum ether: ethyl acetate solution as mobile phase, the crude SS-ATS was purified by silica gel column, and the SS-ATS was obtained. (2) weigh and dissolving that intermediate SS-ATS and the TPP in an organic solvent, wherein the dosage ratio of the intermediate SS-ATS and the TPP is 1:50 to 50:1, react for 0.1 to 96 hours at the temperature of -20°C to 40°C, and recovering the organic solvent at the temperature of 20°C to 100°C by using a rotary evaporator after the reaction, so as to obtain the product. Using dichloromethane: methanol solution as mobile phase, the crude product was purified by silica gel column, and TPP-SS-ATS is obtained.
In the synthesis step (1) of TPP-SS-ATS, a novel artemisinin derivative, the particle size of silica gel for column chromatography used to purify SS-ATS is between 50 and 500 meshes, and the ratio of petroleum ether to ethyl acetate is between 10: 1 and 2: 1.
In the synthesis step (2) of TPP-SS-ATS, a novel artemisinin derivative, the particle size of silica gel for column chromatography used to purify SS-ATS is between 100 and 500 meshes, and the ratio of dichloromethane: methanol is 500: 1-50: 1.
The specific method of preparing TPP-SS-ATS-LS by film dispersion method is: (1) dissolving phospholipid, alkyl glycoside, TPP-SS-ATS, cholesterol and DSPE-mPEG2000 in organic solvent; (2) Then transfer the obtained solution to an evaporation bottle of a rotary evaporator, concentrate under reduced pressure until the organic solvent is completely volatilized, add purified water into the rotary evaporation bottle, and then it is sequentially processed through hydration, ultrasound, and filtration with microporous membrane to obtain
TPP-SS-ATS-LS.
The dosage of auxiliary materials used in the steps (1) and (2) of preparing TPP-SS-ATS-
LS is (in percentage by weight) as follows: 0.2-5.0% of phospholipid, 0.01-0.5% of cholesterol, 0.2-3.0% of alkyl glycoside, 0.02-5.0% of DSPE-mPEG2000, 0.02-3.0% TPP-SS-ATS and 73.5- 99.0% of purified water.
The phospholipids used in step (1) of preparing TPP-SS-ATS-LS are one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin and PEGylated phospholipids.
The alkyl glucoside used in step (2) of preparing TPP-SS-ATS-LS is that each molecule of alkyl glucoside contains one or more alkyl glucosides with 2-30 alkyl carbons and 1-10 glucose molecular fragments.
In the step (2) of preparing TPP-SS-ATS-LS, the temperature for removing the organic solvent under reduced pressure by using a rotary evaporator is 20°C-100°C; the hydration temperature of liposomes is 20-80°C and the hydration time is 0.1-3.0 hours.
The invention has the following advantages. (1) TPP-SS-ATS, a novel artemisinin derivative synthesized in this invention, has a triphenylphosphine structural fragment in its structure. This derivative can use the mitochondrial targeting of triphenylphosphine to realize the mitochondrial targeted delivery of drugs, improve the mitochondrial killing ability, and thus enhance the anti-tumour performance of artemisinin drugs. (2) TPP-SS-ATS-LS prepared by the invention can realize dual-target drug delivery to tumour tissues and tumour cell mitochondria, and remarkably improve the antitumor efficacy of artemisinin drugs. (3) The invention adopts two-step condensation esterification reaction to synthesize TPP-SS-
ATS, without heating, with fast reaction speed, low raw material cost and mature synthesis process. (4) Conventional auxiliary materials are selected to prepare TPP-SS-ATS-LS, so that the process is simple and the energy consumption is low, and the production cost can be effectively controlled. (5) TPP-SS-ATS-LS prepared by the invention has excellent pharmaceutical characteristics and high biological safety.
Fig. 1: is the synthesis roadmap of TPP-SS-ATS.
Fig. 2: shows the chemical structure of SS-ATS.
Fig. 3: Mass spectrum of SS-ATS. 5 Fig. 4: is the NMR spectrum of SS-ATS.
Fig. 5: is the chemical structure diagram of TPP-SS-ATS.
Fig. 8: is the mass spectrum of TPP-SS-ATS.
Fig. 7: shows the "H NMR spectrum of TPP-SS-ATS.
Fig. 8: is the 13C NMR spectrum of TPP-SS-ATS.
Fig. 9: is the HSQC spectrum of TPP-SS-ATS.
Fig. 10: is the chemical structure and HMBC spectrum of TPP-SS-ATS. (a) The chemical structure of TPP-SS-ATS. (b) HMBC spectrum of TPP-SS-ATS.
Fig. 11: shows the particle size distribution of liposomes (TPP-SS-ATS-LS).
Fig. 12: is a transmission electron microscope image of TPP-SS-ATS-LS.
Fig. 13: is a differential scanning calorimetry graph of TPP-SS-ATS-LS and blank liposome.
Fig. 14: shows the stability data of TPP-SS-ATS-LS. (a) Particle size and polydispersity coefficient of TPP-SS-ATS-LS. (b) The encapsulation efficiency and zeta potential of TPP-SS-ATS-LS.
Fig. 15: is the mitochondrial targeting imaging of TPP-SS-ATS-LS.
Fig. 16: is a tumour-targeted in vivo imaging image of TPP-SS-ATS-LS.
Fig. 17: shows the results of in vitro evaluation of anticancer activity of TPP-SS-ATS-LS on three breast cancer cells.
Fig. 18: is a schematic diagram of the administration scheme of in vivo anti-tumour experiment.
Fig. 19: is a diagram showing the anti-breast cancer effect of drugs (A: tumour appearance of mice in each administration group; B: the effect of drugs on tumour weight; C: body weight changes in each group during the experiment).
Fig. 20: is a pathological analysis (H&E staining) diagram of the organs and tissues of mice after administration.
Fig. 21: shows the results of haematological data and liver and kidney function biochemical analysis of mice after administration (A: haematological data of mice; B: blood biochemical indexes of liver and kidney function).
The present invention will be described in further detail with reference to the attached drawings, and the examples given are only for explaining the present invention, not for limiting the scope of the present invention. . Synthesis and structural identification of artemisinin derivatives
1. Synthesis of Intermediate (SS-ATS)
Weigh 5 g artesunate and 3.6 g N,N-dicyclohexylcarbodiimide (DCC), dissolve them in 500ml dichloromethane, and stir them for 1h at room temperature of 350 rpm to make them dissolve completely; 3 g of 2,2-dithiodiethanol (HEDS) and 0.5 g of 4-dimethylaminopyridine (DMAP) were added to dichloromethane solution, and the reaction was stirred at room temperature of 350 rpm for 3 hours (Fig. 1); after the reaction, dichloromethane was recovered by rotary evaporator at 35°C, and the intermediate (SS-ATS) was obtained. Using petroleum ether: ethyl acetate (10:1-2:1) as mobile phase, the intermediate (SS-ATS) was purified by silica gel (200-300 mesh) column. Its structure was determined by MS and NMR. 2. Synthesis of a novel artemisinin derivative (TPP-SS-ATS). 1.6 g of SS-ATS and 3.2 g of 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDCI) were weighed and dissolved in 50 ml of dichloromethane, and stirred at room temperature of 350 rpm for 1 hour. After completely dissolving to obtain a clear solution, 3.2 g of
TPP and 0.6 g of DMAP were added to the dichloromethane solution, stirred at room temperature of 350 rpm, followed by reaction for 12 hours (Fig. 1). After the reaction, dichloromethane was recovered under reduced pressure at 35°C by rotary evaporator to obtain crude TPP-SS-ATS. Using dichloromethane: methanol (500:1-50:1) as mobile phase and silica gel (200-300 mesh) column for purification, TPP-SS-ATS was obtained. The one-dimensional and two-dimensional NMR spectra and molecular weight data of TPP-SS-ATS were used to determine its chemical structure. 3. Structural identification of SS-ATS and TPP-SS-ATS. (1) Structural identification of SS-ATS.
SS-ATS is an oil, and its quasi-molecular ion peak is m/z:559.1[M+K]*. Combined with
NMR spectrum analysis, the molecular formula of the compound is C23H3sO9S: (Figs. 2-3). 'H
NMR(600MHz,CDCls) shows the characteristic proton signal of SS-ATS: ò42.37(1H,brtJ=13.5Hz,H-40), 2.03(1H,brd,J=14.5H-4B), 1.43-1.52(1H,m,H-50), 1.88- 1.94(1H,overlapped,H-5B), 1.27-1.30(1H,m,H-5a), 1.34-1.36(1H,m,H-6), 0.96-1.05(1H,m,H-7 qa), 1.72(1H,overlapped,H-7B), 1.36-1.38(1H,overlapped, H-80), 1.78(1H,brd,J=13.3Hz,H-8p), 1.62(1H,brd,J=13.9Hz,H-8a), 2.53-2.59(1H,m,H-9), 5.78(1H,d,J=9.8Hz H-10), 5.44(1H,s,H-12), 1.43(3H,s,H-14), 0.96(3H,d,J=5.0Hz,H-15), 0.86(3H,d,J=6.6Hz,H-16). The above ’H NMR and
MS spectra indicated that SS-ATS was successfully synthesized (Fig. 4). (2) Structural identification of TPP-SS-ATS.
TPP-SS-ATS is a white amorphous solid, and its quasi-molecular ion peak is m/z:851.4[M-
Br]*. Combined with NMR spectrum analysis, the molecular formula of the compound is determined to be C4sH56BrO10PS2 (Figs. 5-6). *H NMR(600MHz,CDCl3) and HSQC spectra (Figs. 7-9) show the characteristic proton signals of artesunate structural fragment: ò42.37(1H, td, J=14.0,3.8Hz,H-40),2.03(1H,dt, J=14.5,3.8Hz,H-468), 1.43-1.52(1H,m,H-50),1.87- 1.92(1H,m,H-58),1.27-1.30(1H,m,H-5a),1.33-1.34(1H,m,H-6},0.96-1.07(1H,m,H-
7a),1.72(1H,dd,J=13.4,3.0Hz,H-7B),1.37(1H,dd,J=13.5,3.3Hz H- 8a),1.78(1H,dd,J=13.6,3.6Hz,H-8B),1.62(1H,dt,J=13.7,4.4Hz,H-8a),2.54-2.56(1H,m,H- 9),5.78(1H,d,J=9.8Hz,H-10),5.31(1H,s,H-12),1.41(3H,s,H-14),0.96(3H,d,J=6.1Hz, H- 15),0.85(3H,d,J=7.1Hz, H-16); Characteristic proton signals of structural fragments of 2, 2- dithiodiethanol: ò44.32(4H,dd, J=15.9,6.6Hz,H-2'and7'jand 2.90-2.91(4H,m,H-3'and6’); the characteristic proton signals of the structural fragment of (4-Carboxybutyl)triphenylphosphonium bromide (TPP): òu2.92-2.94(2H,m,H-b}, 1.93-1.99(2H,m,H-c), 4.00-4.05(2H,m,H-d) and 7.70- 7.99 (15H,m,H-TPP aromatic proton).
The characteristic signals of *C NMR(150MHz,CDCls) spectrum (Fig. 8) are as follows: 6¢171.1(C-17, ester carbonyl of artesunate structural fragment), 171.9(C-20, ester carbonyl of artesunate structural fragment), 62.5 and 62.6 (C-2' or C-7. In the HMBC spectrum (Fig. 10), the remote correlation signals of 644.32(H-2") and òc171.9(C-20) confirmed that (4- carboxybutyl}triphenylphosphonium bromide (TPP) fragment was linked to C-20 of artesunate fragment. The remote correlation signals of 6n4.32(H-7") and òc172.9(C-a), ò41.93-1.99{H-c) and òc172.9(C-a) confirmed that TPP fragment was linked to C-7' of 2,2-dithiodiethanol fragment. The above spectral characteristics show that TPP-SS-ATS has been successfully synthesized.
II. Preparation of liposomes 1. Preparation of liposomes (TPP-SS-ATS-LS)
TPP-SS-ATS-LS of the present invention and its preparation method are shown in Fig. 1.
TPP-SS-ATS-LS are prepared by a film dispersion method. The specific method is as follows (the total weight of each auxiliary material (including purified water) and medicine is 100%): 1.43% by weight of soybean lecithin, 0.14% by weight of n-octyl-B-D-glucoside, 0.19% by weight of TPP-SS-ATS, 0.07% by weight of cholesterol and 0.17% by weight of DSPE- mPEG2000 were dissolved in 20ml of dichloromethane, the solution was transferred to a rotary evaporator bottle, and the solution was concentrated under reduced pressure at 40°C until dichloromethane completely volatilized, and 98% by weight of purified water was added to the rotary evaporator bottle. TPP-SS-ATS-LS were prepared by hydrating at 40°C and 150 rpm for 25 min, then ultrasonically vibrating at 250 W for 3min, and filtering with 0.22 um microporous membrane. Its appearance was translucent liquid with light blue opalescence. 2. Preparation of blank liposomes
The preparation method of blank liposomes is basically the same as that of TPP-SS-ATS-
LS. The only difference between them is that the blank liposomes (Blank LS) prescription does not contain TPP-SS-ATS.
III. Pharmacological evaluation of TPP-SS-ATS-LS 1. Determination of particle size, zeta potential distribution and polydiseperse index(PDI) of
TPP-SS-ATS-LS. (1) Experimental methods
Appropriate samples of TPP-SS-ATS-LS were placed in a test dish, and the particle size, zeta potential distribution and PDI of liposomes were determined by particle size and zeta potential analyser (Malvern Zetasizer Nano ZS). Determination method: The test temperature is 25°C, and the test times are 3 times, and the average value of the data is taken to analyse the results. (2) Experimental results
The particle size distribution of TPP-SS-ATS-LS is regular spherical (Fig. 11). The particle size, zeta potential and PDI of TPP-SS-ATS-LS are 87.60+1.65 nm, 31.4+1.7 mv and 0.241+0.013, respectively. The particle size, zeta potential and PDI of the blank liposomes are 94.8320.71 nm, -38.9+1.2 mv and 0.200+0.008, respectively (Table 1).
Table 1 shows zeta potential, particle size, PDI and encapsulation efficiency of liposomes “Liposomes Particle size(nm) Zeta potential (mv) PDI Encapsulation rate(%) ~ TPP-SS- 87.60%165 31.4%17 024120013 952%03
ATS-LS
Blank 94.83+0.71 -36.9+1.2 0.200+0.008 liposomes 2. Determination of encapsulation efficiency (EE) of TPP-SS-ATS-LS. (1) Experimental methods 1) Determination of the content of TPP-SS-ATS in liposomes (TPP-SS-ATS-LS)(M1): Add a proper amount of TPP-SS-ATS-LS into a volumetric flask, extract it by ultrasound at room temperature for 15 min, and determine the content of TPP-SS-ATS (M1) by HPLC. 2) Determination of free TPP-SS-ATS (M2): Add a proper amount of TPP-SS-ATS-LS into a4 ml ultrafiltration tube (molecular weight cut-off: 10kDa), centrifuge at 4°C and 5000 rpm for 120 min, collect ultrafiltrate, and determine the content of free TPP-SS-ATS (M2) by high performance liquid chromatography. The average value of three parallel experimental data was used to analyse the results. Calculate the entrapment efficiency (EE) of artemisinin derivative liposomes according to the following formula:
BEC Bnd HES
Ad: (2) Experimental results
The encapsulation efficiency of TPP-SS-ATS-LS is 95.2+0.3%, which indicates that the liposomes prepared by the invention can efficiently encapsulate TPP-SS-ATS. 3. TEM observation of TPP-SS-ATS-LS. (1) Experimental methods
TPP-SS-ATS-LS were diluted 5 times with purified water, then dropped on a 200-mesh copper grid of the electron microscope and the excess sample was removed by filter paper.
After natural drying, the microscopic morphology of TPP-SS-ATS-LS was observed by H7650 transmission electron microscope. (2) Experimental results
Transmission electron microscope observation showed TPP-SS-ATS-LS was a regular spherical shape (Fig. 12). 4. Differential scanning calorimetry (DSC) analysis of TPP-SS-ATS-LS. (1) Experimental method:
The invention adopts NANODSC differential scanning calorimeter to detect the phase transition temperature of TPP-SS-ATS-LS and blank liposomes; the temperature scanning range of the sample is 20°C-100°C, and the heating rate is set at 1°C/min. During the experiment, the pressure of the sample cell was 3 atmospheres, and the reference solution was purified water.. (2) Experimental results
The phase transition temperature of liposomes before and after drug loading decreased from 67.29°C to 57.75°C (Fig. 13), suggesting that the loading of TPP-SS-ATS, a novel artemisinin derivative, into the lipid membrane structure of liposomes had an impact on the phase transition temperature of liposomes membrane structure. This structural feature of TPP-
SS-ATS-LS is beneficial to be recognized by mitochondria in triphenylphosphine structural fragment (TPP), thus pulling liposomes to migrate to mitochondria and realizing mitochondrial targeting of liposomes. 5. Stability evaluation of TPP-SS-ATS-LS. (1) Experimental methods
TPP-SS-ATS-LS were sealed at 4°C after preparation. The particle size, entrapment efficiency, Zeta potential and PDI of liposomes were measured at 0, 7, 14, 28 and 42 days after preparation. (2) Experimental results
The RSD% of the particle size and encapsulation efficiency of liposomes (TPP-SS-ATS-
LS) were 1.60% and 0.96% on the 0, 7, 14, 28 and 42 days after sample preparation, respectively. And in the stability study, the zeta potential of TPP-SS-ATS-LS is always greater than 26mv, which shows that TPP-SS-ATS-LS has good stability at 4°C (Fig. 14, A: particle size, PDI ; B: encapsulation efficiency, zeta potential).
IV. Targeting evaluation of TPP-SS-ATS-LS 1. Mitochondrial targeting evaluation (1) Experimental methods
MDA-MB-231 cells were inoculated into a 96-well Petri dish with a cell density of 2000 cells/well and cultured for 12 hours. Subsequently, mitochondria were stained with Mito Tracker
Red CMXros, and nuclei were stained with 2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi- 1H- benzimidazole trinydrate (Hoechst33342 blue fluorescent probe). After washing with PBS,
FITC-TPP-SS-ATS-LS (green fluorescence) labeled with fluorescein PEG phospholipid was added. The cells were continuously monitored for 48 hours by PE PerkinElmer Operetta CLS™ (Perkinlemer, USA) to observe the mitochondrial targeting. (2) Experimental results
The mitochondrial targeting of TPP-SS-ATS-LS was monitored by high connotation imaging system. During the experiment, fluorescently labeled TPP-SS-ATS-LS was found in mitochondria at 6 hours after administration (Fig. 15), and then fluorescently labeledTPP-SS-
ATS-LS gradually accumulated in mitochondria. The results show that TPP-SS-ATS-LS have mitochondrial targeting. 2. In vivo tumour targeting evaluation (1) Experimental methods
Axillary tumour containing 4T1 cells was extracted and diluted in normal saline with 1x10’ cells/ml., and 0.2ml of cell suspension was inoculated to the mice. After the tumour model was established, the TPP-SS-ATS-LS labeled with 1,1-octadecyl-3,3,3-tetramethyl indocyanine iodide DiR was injected intraperitoneally. Mice were anesthetized with isoflurane at the 4, 8" 12 24M 36" 48M 60 72% 96M 120, 144” and 1868!" hours after drug injection, an /VIS
Lumina Ill imaging system (Perkinlemer, USA) was used for determining the fluorescence distribution in vivo. (2) Experimental results
The results of in vivo imaging show that TPP-SS-ATS-LS have good tumour targeting character, as shown in Fig. 16. In this study, the TPP-SS-ATS-LS were injected into the tumour model animals by intraperitoneal injection, and the fluorescence intensity of the tumour site gradually increased with time, while the fluorescence intensity of the administration site {abdominal cavity) gradually weakened (Fig. 16). The fluorescence signal intensity of tumour site reached the highest level 24-60 hours after administration, and then gradually decreased.
By intraperitoneal injection, the possible process of targeting tumour tissue with TPP-SS-ATS-
LS is as follows: after administration, liposomes enter the systemic circulation through capillaries and lymphatic vessels in peritoneum, and then liposomes are gradually captured by tumour tissue to achieve the effect of tumour targeted delivery.
V. Evaluation of efficacy and safety of TPP-SS-ATS-LS 1.CCK-8 method was used to detect the cytotoxicity of drugs on various breast cancer cells. (1) Experimental methods 1) Paving: observing the cell state, digesting when the cell adhesion length reaches 90%, using a counting plate to count the cells, inoculating the cells in a 96-well plate at a cell density of 3000-5000/ well, and culturing in a 5% CO: incubator at 37°C for 24 hours. 2) Dosing: when the cell density was observed to be about 60%-70% under the microscope, the administration was started. The groups were blank liposomes group, TPP-SS-
ATS-LS group, TPP-SS-ATS group and artesunate (original drug, ATS) group. The dosing concentration range from 2.5 uM to 160 uM. 3) CCK-8 was added: after 12 hours, 24 hours and 48 hours, the cell state was observed respectively, and CCK-8 was added to the 96-well plate and placed in the incubator for 2 hours. 4) Detection: the OD value was measured at 450 nm using EnVision microplate reader (PerkinElmer, USA), and the survival rate was calculated. The survival rate was calculated by the following formula:
Survival rate (%) __ODiest x 100%
ODcontrol (2) Experimental results
The effect of drugs on the viability of breast cancer cells is shown in Fig. 17. TPP-SS-ATS-
LS, TPP-SS-ATS and ATS can inhibit the viability of breast cancer cells in a dose-dependent manner after 12 hours, 24 hours and 48 hours respectively. Liposomes TPP-SS-ATS-LS and
TPP-SS-ATS have similar inhibitory effects, both of which can obviously inhibit the growth of different types of breast cancer cells. Compared with the original drug ATS, TPP-SS-ATS-LS has more obvious anti-tumour effect at the same concentration, starting from 20 HM. The IC50 value of TPP-SS-ATS-LS was 17.69 uM (4T1 cells, 48h), -67.48 uM (MCF-7 cells, 12h). The
IC50 value of ATS ranged from 33.50 uM (4T1 cells, 48h) to 797.5uM (MCF-7 cells, 12h). 2. Evaluation of antitumor activity of liposomes in vivo. (1) Experimental methods
The mice xenotransplant model of 4T1 cells of were washed with normal saline and resuspended, and the density was adjusted to 1x 107 cells/ml, and 0.2 ml of cell suspension was inoculated to mice. Tumour-bearing mice were randomly divided into six groups: control(model)group, gemcitabine(GEM) positive control group, ATS low-dose group and high- dose group, TPP-SS-ATS-LS low-dose group and high-dose group. The dosage of drugs in low- dose and high-dose groups was 15 mg/kg or 30 mg/kg, and intraperitoneal injection was started from the fourth day of inoculation, once every two days; the dose of GEM group was 30mg/kg, once every 4 days. Mice in the control group were given normal saline. The administration scheme is shown in Fig. 18.
Sampling and tumour inhibition rate calculation: weigh the mice before sampling, fix them onthe rat plate in supine position after euthanasia, dissect and take the heart, liver, kidney, spleen, brain and tumour tissues, and record the weight; Calculate the tumour growth inhibition rate TGI of breast cancer mice according to the following formula:
TGI (%) = He x 100%
Where "T" represents the average weight of tumour in the experimental group and "C" represents the average weight of tumour in the control group. (2) Experimental results
According to the invention, the anti-cancer effect of TPP-SS-ATS-LS on tumour-bearing mice with 4T1 breast cancer is evaluated by recording the change of tumour weight. The results showed that compared with the model group, the tumour weight of breast cancer tissue in the
ATS low and high dose groups was significantly reduced. However, the tumour weight of TPP-
SS-ATS-LS low and high dose groups decreased more. At the same dosage of 30 mg/kg, the tumour growth inhibition rate (TGI) of artesunate was 37.7%, while that of TPP-SS-ATS-LS was nearly 56.4%. Compared with the model group, the tumour weight of positive (gemcitabine) group was the lowest, and the high dose group of TPP-SS-ATS-LS was close to it. The weight of tumour-bearing mice was not affected by each administration group (Fig. 19). 3. Safety evaluation of novel liposomes (TPP-SS-ATS-LS)
The invention further studies the organic toxicity of TPP-SS-ATS-LS to other non-tumour organs. After drug administration, the heart, liver, kidney, spleen and brain tissues of mice are taken for pathological analysis, and at the same time, the blood routine and biochemical indexes of liver and kidney functions of mice are detected. (1) Pathological analysis 1) Experimental methods
In order to evaluate the safety of TPP-SS-ATS-LS, the invention carries out pathological analysis on the main organs (heart, liver, spleen, kidney and brain) of mice after administration.
The main steps of making pathological sections are as follows: 10% formalin tissue fixation, paraffin embedding, sectioning and H&E staining (hematoxylin-eosin staining method); After the pathological sections were made, they were observed under a microscope and photographed for preservation. 2) Experimental results
Histopathological observation showed that the tissues of heart, liver, spleen, kidney and brain of mice in the TPP-SS-ATS-LS treatment group were similar to those in the normal saline group, and there are no pathological changes caused by liposomes. The results were as shown in Fig. 20: (1) Heart: the morphology and structure of myocardial cells in the blank normal control group, the model group and each administration group were healthy, and the myocardial fibre cells were arranged regularly, with clear horizontal lines, complete cell membranes and even staining. (2) Liver: the vacuolar degeneration of liver cells in GEM group is obvious, and the pathological changes of liver tissue in the other groups are similar to those in the model group, indicating that TPP-SS-ATS-LS and ATS will not aggravate the liver damage of model mice. (3) Spleen: the junction of spleen cortex and medulla was obvious in blank group, model group and each administration group, and there is a dense distribution of lymphocytes in the white pulp and red pulp. (4) Kidney: The renal tubular epithelial cells of the mice in the blank group, model group and each administration group are arranged neatly, the renal capsule cavity is normal, and the glomerular structure is clear and uniform in size. (5) Brain: neurons and glial cells in brain tissue of mice in each group were tightly arranged without cell swelling. All the above results suggest that the TPP-SS-ATS-LS have no obvious toxicity to various organs of mice. (2) Blood biochemical analysis 1) Experimental methods
The collected blood was allowed to stand overnight at 4°C, centrifuged at 3500 rpm for 15min, and then the serum was isolated. The standard haematological markers including the white blood cells (WBC), red blood cells (RBC), platelets (PLT), haemoglobin (HGB), haematocrit (HCT), mean platelet volume (MPV), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), and platelet distribution width (PDW) were analysed by automatic biochemical analyser. Blood biochemical analysis were carried out and the parameters about the functions of the liver and kidney of mice including alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine (CR) were examined 2) Experimental results
The results of routine blood tests are shown in Fig. 20. Compared with the model group, all parameters in the treated groups appeared to be normal and there were no statistically significant differences between both groups (P > 0.05), suggesting that the administration of
TPP-SS-ATS-LS have not caused obvious damage and inflammation in mice. Compared with the control group, no meaningful difference was detected from the treated groups. Hence, the treatment did not affect the blood chemistry of mice. These blood biochemical results demonstrated that TPP-SS-ATS-LS treatment induced no obvious hepatic and kidney toxicity in mice (P > 0.05), indicating that TPP-SS-ATS-LS have no obvious effect on blood, liver and kidney function of mice (Fig. 21). It shows that TPP-SS-ATS-LS are safety and low toxicity.
VI. Statistical analysis of data
The experimental results were expressed by mean + standard deviation (SD), and compared and analysed using Graphpad Prism software and one-way ANOVA followed by
Dunnett's test was performed. P < 0.05 indicates that the difference is statistically significant.
The anti-breast cancer effect of the novel artemisinin derivative liposomes is shown in Figs. 18-21.
Although specific embodiments of the present invention have been disclosed for illustrative purposes, the purpose is to help people understand the content of the present invention and implement it accordingly, those skilled in the art can understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to what is disclosed in the best embodiment, and the scope of the present invention is subject to the scope defined by the claims.
Claims (10)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210792714.5A CN115340571B (en) | 2022-07-05 | 2022-07-05 | Preparation method and application of novel artemisinin derivative and liposome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| NL2035226A true NL2035226A (en) | 2024-01-19 |
| NL2035226B1 NL2035226B1 (en) | 2024-01-26 |
Family
ID=83947697
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NL2035226A NL2035226B1 (en) | 2022-07-05 | 2023-06-29 | Preparation method and application of novel artemisinin derivatives and liposomes |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN115340571B (en) |
| NL (1) | NL2035226B1 (en) |
| ZA (1) | ZA202306630B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109675048A (en) * | 2019-01-07 | 2019-04-26 | 中国科学院化学研究所 | A kind of anticancer prodrug liposome and artemisine liposome Nano medication |
| CN110354270A (en) * | 2019-08-30 | 2019-10-22 | 中国中医科学院中药研究所 | A kind of Artesunate polyethyleneglycol derivative and its preparation method and application, a kind of drug for preventing and treating pneumonia |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111939127A (en) * | 2019-05-17 | 2020-11-17 | 沈阳药科大学 | Artesunate liposome and preparation and application thereof |
| CN110179993B (en) * | 2019-06-19 | 2022-06-21 | 北京林业大学 | Ganoderma lucidum polysaccharide-based conjugate drug-loaded nanoparticle with pH/redox dual response and preparation method thereof |
| CN114014872B (en) * | 2021-11-29 | 2023-05-23 | 桂林医学院 | Artesunate derivative and preparation method and application thereof |
| CN114380864B (en) * | 2021-12-28 | 2023-06-23 | 湖南师范大学 | Dihydroartemisinin derivative, preparation method, pharmaceutical composition and application thereof in preparation of antitumor drugs |
-
2022
- 2022-07-05 CN CN202210792714.5A patent/CN115340571B/en active Active
-
2023
- 2023-06-28 ZA ZA2023/06630A patent/ZA202306630B/en unknown
- 2023-06-29 NL NL2035226A patent/NL2035226B1/en active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109675048A (en) * | 2019-01-07 | 2019-04-26 | 中国科学院化学研究所 | A kind of anticancer prodrug liposome and artemisine liposome Nano medication |
| CN110354270A (en) * | 2019-08-30 | 2019-10-22 | 中国中医科学院中药研究所 | A kind of Artesunate polyethyleneglycol derivative and its preparation method and application, a kind of drug for preventing and treating pneumonia |
Non-Patent Citations (7)
| Title |
|---|
| FANG MIAOWANG DEGANGLIU PEIQING: "Research Progress on Anti-tumour Mechanism of Natural Products", FOOD AND DRUG, vol. 24, no. 2, 2022, pages 167 - 171 |
| GU LIWEI ET AL: "Development of artesunate intelligent prodrug liposomes based on mitochondrial targeting strategy", JOURNAL OF NANOBIOTECHNOLOGY, vol. 20, no. 1, 24 March 2022 (2022-03-24), XP093105701, DOI: 10.1186/s12951-022-01569-5 * |
| KAILI WANG ET AL: "Novel Dual Mitochondrial and CD44 Receptor Targeting Nanoparticles for Redox Stimuli-Triggered Release", NANOSCALE RESEARCH LETTERS, SPRINGER, US, vol. 13, no. 1, 2 February 2018 (2018-02-02), pages 1 - 10, XP021253164, ISSN: 1931-7573, DOI: 10.1186/S11671-018-2445-1 * |
| LIU QINGQINGYANG ZHENHUA: "Progress of anti-tumour activities of artemisinin and its derivatives", CHINESE BULLETIN OF LIFE SCIENCES, vol. 32, no. 1, 2020, pages 62 - 69 |
| WEI QIUHONGLIU XIAOYUEWANG PANZHANG WENRUI: "Research Progress in Classification and Pharmacodynamics of Anti-Tumour Drugs", MEDICAL RECAPITULATE, vol. 26, no. 18, 2020, pages 3707 - 3711 |
| XU JIAOMENG LINGHUAQING CHEN: "The clinical application and development of traditional antitumor drugs", ACTA PHARMACEUTICA SINICA, vol. 56, no. 6, 2021, pages 1551 - 1561 |
| ZHOU YLI WXIAO Y: "Profiling of Multiple Targets of Artemisinin Activated by Chemincancer Cell Proteome", ACS CHEMBIOL, vol. 11, 2016, pages 882 - 888 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115340571B (en) | 2024-09-27 |
| ZA202306630B (en) | 2024-01-31 |
| NL2035226B1 (en) | 2024-01-26 |
| CN115340571A (en) | 2022-11-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102268191B (en) | Heptamethine indocyanine dye, synthetic method thereof and applications thereof | |
| KR102237234B1 (en) | Integrated nanosystem for co-transporting genes/drugs with liver targeting and method for manufacturing the same | |
| CN105622673B (en) | Glycosylation tetravalence platinum-like compounds with active anticancer, preparation method and application | |
| CN102977360B (en) | Triphenylphosphine-polyethyleneglycol 1000 vitamin E succinate (TPGS 1000-TPP) conjugated compound, and preparation method and application thereof | |
| CN107936058B (en) | Docetaxel derivative and preparation method and application thereof | |
| Xie et al. | Hybrid-cell membrane-coated nanocomplex-loaded chikusetsusaponin IVa methyl ester for a combinational therapy against breast cancer assisted by Ce6 | |
| CN102614198B (en) | Application of (4-substituted benzene formyl) fluorobenzene salicylamide compound in preparation of anti-lung-cancer medicines | |
| CN110433292A (en) | A kind of double targeting materials and its application in drug delivery | |
| CN106800650B (en) | Function targeting vector material distearoylphosphatidylethanolamine-polyethylene glycol-phenylglucopyranoside and the preparation method and application thereof | |
| CN110054660A (en) | A kind of preparation and application of the breast cancer targeting lipids material of fructose modification | |
| CN109350598A (en) | Sugar-polyethylene glycol-DSPE coupling compound and its preparation method and application | |
| CN114853810A (en) | Curcumin derivative and preparation method and application thereof | |
| US8853194B2 (en) | Sterol derivatives and their synthesis and use | |
| CN111249234A (en) | Glycosyl combined cell penetrating peptide modified brain targeting nanoliposome and preparation method and application thereof | |
| NL2035226B1 (en) | Preparation method and application of novel artemisinin derivatives and liposomes | |
| CN105131277A (en) | Polymer material containing cholic acid and liposome modified by same | |
| CN102731442B (en) | Preparation method and application of water-soluble docetaxel compounds | |
| CN103254263B (en) | Cardiac glycoside compound and preparation method as well as application thereof | |
| CN103319710A (en) | Poly(2-ethyl-2-oxazoline)-lipid derivatives and preparation method thereof | |
| CN113004301B (en) | Artesunate-based-diphenylurea derivative ARS-DPU as well as preparation method and application thereof | |
| CN106377528B (en) | A kind of pharmaceutical composition of hydroxycamptothecin glutaric acid monoester | |
| Yang et al. | 16-Tigloyl linked barrigenol-like triterpenoid from Semen Aesculi and its anti-tumor activity in vivo and in vitro | |
| Yousefi et al. | Synthesis and in vitro bioactivity evaluation of new glucose and xylitol ester derivatives of 5-aminosalicylic acid | |
| CN108553476B (en) | Preparation method of hinokiflavone derivative and application of hinokiflavone derivative in resisting breast cancer | |
| WO2016034126A1 (en) | Anti-tumor compound and medical use thereof |