CN111603567A - Cd44靶向多臂偶联物 - Google Patents
Cd44靶向多臂偶联物 Download PDFInfo
- Publication number
- CN111603567A CN111603567A CN201910131331.1A CN201910131331A CN111603567A CN 111603567 A CN111603567 A CN 111603567A CN 201910131331 A CN201910131331 A CN 201910131331A CN 111603567 A CN111603567 A CN 111603567A
- Authority
- CN
- China
- Prior art keywords
- compound
- cancer
- poly
- drug conjugate
- branched
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102100032912 CD44 antigen Human genes 0.000 title claims abstract description 28
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 title claims abstract description 28
- 150000001875 compounds Chemical class 0.000 claims abstract description 110
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 58
- 239000003814 drug Substances 0.000 claims abstract description 34
- 230000008685 targeting Effects 0.000 claims abstract description 34
- 229940079593 drug Drugs 0.000 claims abstract description 28
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 7
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 7
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims abstract description 7
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims abstract description 7
- 201000002528 pancreatic cancer Diseases 0.000 claims abstract description 7
- 208000008443 pancreatic carcinoma Diseases 0.000 claims abstract description 7
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 6
- 206010041067 Small cell lung cancer Diseases 0.000 claims abstract description 6
- 208000005718 Stomach Neoplasms Diseases 0.000 claims abstract description 6
- 206010017758 gastric cancer Diseases 0.000 claims abstract description 6
- 208000000587 small cell lung carcinoma Diseases 0.000 claims abstract description 6
- 201000011549 stomach cancer Diseases 0.000 claims abstract description 6
- 208000029742 colonic neoplasm Diseases 0.000 claims abstract description 5
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims abstract description 4
- 206010025323 Lymphomas Diseases 0.000 claims abstract description 4
- 208000005017 glioblastoma Diseases 0.000 claims abstract description 4
- 201000005249 lung adenocarcinoma Diseases 0.000 claims abstract description 4
- 208000008839 Kidney Neoplasms Diseases 0.000 claims abstract description 3
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 claims abstract description 3
- 206010061306 Nasopharyngeal cancer Diseases 0.000 claims abstract description 3
- 206010033128 Ovarian cancer Diseases 0.000 claims abstract description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 claims abstract description 3
- 206010038389 Renal cancer Diseases 0.000 claims abstract description 3
- 201000010536 head and neck cancer Diseases 0.000 claims abstract description 3
- 208000014829 head and neck neoplasm Diseases 0.000 claims abstract description 3
- 201000010982 kidney cancer Diseases 0.000 claims abstract description 3
- 201000007270 liver cancer Diseases 0.000 claims abstract description 3
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 3
- 201000011216 nasopharynx carcinoma Diseases 0.000 claims abstract description 3
- 238000002360 preparation method Methods 0.000 claims description 36
- 229920001184 polypeptide Polymers 0.000 claims description 28
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 28
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 28
- 229920000642 polymer Polymers 0.000 claims description 20
- 229960004768 irinotecan Drugs 0.000 claims description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 13
- 150000003839 salts Chemical class 0.000 claims description 12
- -1 amino, substituted amino, hydroxycarbonyl Chemical group 0.000 claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 229920001223 polyethylene glycol Polymers 0.000 claims description 6
- 239000002202 Polyethylene glycol Substances 0.000 claims description 5
- 239000013543 active substance Substances 0.000 claims description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 4
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims description 4
- 125000003342 alkenyl group Chemical group 0.000 claims description 4
- 125000000217 alkyl group Chemical group 0.000 claims description 4
- 125000000753 cycloalkyl group Chemical group 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 3
- XLPJNCYCZORXHG-UHFFFAOYSA-N 1-morpholin-4-ylprop-2-en-1-one Chemical compound C=CC(=O)N1CCOCC1 XLPJNCYCZORXHG-UHFFFAOYSA-N 0.000 claims description 2
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 claims description 2
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims description 2
- 229920002125 Sokalan® Polymers 0.000 claims description 2
- 125000002252 acyl group Chemical group 0.000 claims description 2
- 125000003545 alkoxy group Chemical group 0.000 claims description 2
- 125000004453 alkoxycarbonyl group Chemical group 0.000 claims description 2
- 125000005194 alkoxycarbonyloxy group Chemical group 0.000 claims description 2
- 125000005278 alkyl sulfonyloxy group Chemical group 0.000 claims description 2
- 125000000304 alkynyl group Chemical group 0.000 claims description 2
- 229940061720 alpha hydroxy acid Drugs 0.000 claims description 2
- 150000001280 alpha hydroxy acids Chemical class 0.000 claims description 2
- 125000003368 amide group Chemical group 0.000 claims description 2
- 125000005279 aryl sulfonyloxy group Chemical group 0.000 claims description 2
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 2
- PVEOYINWKBTPIZ-UHFFFAOYSA-N but-3-enoic acid Chemical compound OC(=O)CC=C PVEOYINWKBTPIZ-UHFFFAOYSA-N 0.000 claims description 2
- 229940127093 camptothecin Drugs 0.000 claims description 2
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 claims description 2
- 150000001720 carbohydrates Chemical class 0.000 claims description 2
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 2
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 claims description 2
- 125000001188 haloalkyl group Chemical group 0.000 claims description 2
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 150000002367 halogens Chemical class 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 125000002768 hydroxyalkyl group Chemical group 0.000 claims description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 2
- 229920000765 poly(2-oxazolines) Polymers 0.000 claims description 2
- 229920001390 poly(hydroxyalkylmethacrylate) Polymers 0.000 claims description 2
- 229920002627 poly(phosphazenes) Polymers 0.000 claims description 2
- 229920001451 polypropylene glycol Polymers 0.000 claims description 2
- 125000005415 substituted alkoxy group Chemical group 0.000 claims description 2
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 2
- 206010041823 squamous cell carcinoma Diseases 0.000 claims 2
- 210000000481 breast Anatomy 0.000 claims 1
- 210000001072 colon Anatomy 0.000 claims 1
- 230000002496 gastric effect Effects 0.000 claims 1
- 150000002431 hydrogen Chemical class 0.000 claims 1
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 claims 1
- 210000004185 liver Anatomy 0.000 claims 1
- 210000004072 lung Anatomy 0.000 claims 1
- 230000002611 ovarian Effects 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 10
- 210000004881 tumor cell Anatomy 0.000 abstract description 10
- 230000002829 reductive effect Effects 0.000 abstract description 7
- 230000001988 toxicity Effects 0.000 abstract description 3
- 231100000419 toxicity Toxicity 0.000 abstract description 3
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 abstract description 2
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 60
- 210000004027 cell Anatomy 0.000 description 53
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 51
- 239000000243 solution Substances 0.000 description 35
- 238000004128 high performance liquid chromatography Methods 0.000 description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 27
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 26
- 238000005406 washing Methods 0.000 description 21
- 238000011580 nude mouse model Methods 0.000 description 19
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 18
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 18
- 229960002685 biotin Drugs 0.000 description 18
- 239000011616 biotin Substances 0.000 description 18
- GURKHSYORGJETM-WAQYZQTGSA-N irinotecan hydrochloride (anhydrous) Chemical compound Cl.C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 GURKHSYORGJETM-WAQYZQTGSA-N 0.000 description 17
- 239000002904 solvent Substances 0.000 description 17
- 241001465754 Metazoa Species 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 238000003756 stirring Methods 0.000 description 16
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 15
- 238000001035 drying Methods 0.000 description 14
- 239000003153 chemical reaction reagent Substances 0.000 description 13
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 12
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 235000020958 biotin Nutrition 0.000 description 12
- 238000012544 monitoring process Methods 0.000 description 12
- 239000007787 solid Substances 0.000 description 12
- 230000005764 inhibitory process Effects 0.000 description 10
- SELCJVNOEBVTAC-UHFFFAOYSA-N nktr-102 Chemical compound C12=NC3=CC=C(OC(=O)N4CCC(CC4)N4CCCCC4)C=C3C(CC)=C2CN(C2=O)C1=CC1=C2COC(=O)C1(CC)OC(=O)CNC(=O)COCCOCC(COCCOCC(=O)NCC(=O)OC1(CC)C2=C(C(N3CC4=C(CC)C5=CC(OC(=O)N6CCC(CC6)N6CCCCC6)=CC=C5N=C4C3=C2)=O)COC1=O)(COCCOCC(=O)NCC(=O)OC1(CC)C2=C(C(N3CC4=C(CC)C5=CC(OC(=O)N6CCC(CC6)N6CCCCC6)=CC=C5N=C4C3=C2)=O)COC1=O)COCCOCC(=O)NCC(=O)OC1(CC)C(=O)OCC(C(N2CC3=C(CC)C4=C5)=O)=C1C=C2C3=NC4=CC=C5OC(=O)N(CC1)CCC1N1CCCCC1 SELCJVNOEBVTAC-UHFFFAOYSA-N 0.000 description 10
- 239000008213 purified water Substances 0.000 description 10
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 9
- 239000012091 fetal bovine serum Substances 0.000 description 9
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 9
- 238000002054 transplantation Methods 0.000 description 9
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 8
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 8
- 239000012043 crude product Substances 0.000 description 8
- 238000001914 filtration Methods 0.000 description 8
- 239000006166 lysate Substances 0.000 description 8
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 8
- 235000017557 sodium bicarbonate Nutrition 0.000 description 8
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 7
- 238000010171 animal model Methods 0.000 description 7
- 238000004108 freeze drying Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 239000012980 RPMI-1640 medium Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 230000003698 anagen phase Effects 0.000 description 5
- 239000002246 antineoplastic agent Substances 0.000 description 5
- 125000004429 atom Chemical group 0.000 description 5
- 239000006285 cell suspension Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000007920 subcutaneous administration Methods 0.000 description 5
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 description 4
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 description 4
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 description 4
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 4
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 4
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- 239000002033 PVDF binder Substances 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 230000000397 acetylating effect Effects 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 229940041181 antineoplastic drug Drugs 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 239000007810 chemical reaction solvent Substances 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 4
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 4
- 230000001376 precipitating effect Effects 0.000 description 4
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 3
- 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 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 229920002971 Heparan sulfate Polymers 0.000 description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 3
- 230000001093 anti-cancer Effects 0.000 description 3
- LUFPJJNWMYZRQE-UHFFFAOYSA-N benzylsulfanylmethylbenzene Chemical compound C=1C=CC=CC=1CSCC1=CC=CC=C1 LUFPJJNWMYZRQE-UHFFFAOYSA-N 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 239000012230 colorless oil Substances 0.000 description 3
- 230000009982 effect on human Effects 0.000 description 3
- 239000012065 filter cake Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 229920002674 hyaluronan Polymers 0.000 description 3
- 229960003160 hyaluronic acid Drugs 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 239000000600 sorbitol Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 description 2
- KJYAFJQCGPUXJY-UMSFTDKQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-(tritylamino)butanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KJYAFJQCGPUXJY-UMSFTDKQSA-N 0.000 description 2
- VRPJIFMKZZEXLR-UHFFFAOYSA-N 2-[(2-methylpropan-2-yl)oxycarbonylamino]acetic acid Chemical compound CC(C)(C)OC(=O)NCC(O)=O VRPJIFMKZZEXLR-UHFFFAOYSA-N 0.000 description 2
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 description 2
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 2
- 102000008102 Ankyrins Human genes 0.000 description 2
- 108010049777 Ankyrins Proteins 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- 108010085895 Laminin Proteins 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 102000004264 Osteopontin Human genes 0.000 description 2
- 108010081689 Osteopontin Proteins 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 150000001242 acetic acid derivatives Chemical class 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- KXKPYJOVDUMHGS-OSRGNVMNSA-N chondroitin sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](OS(O)(=O)=O)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](C(O)=O)O1 KXKPYJOVDUMHGS-OSRGNVMNSA-N 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 229940068840 d-biotin Drugs 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 210000003194 forelimb Anatomy 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 2
- 150000003840 hydrochlorides Chemical class 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 238000013392 nude mouse xenograft model Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 229940002612 prodrug Drugs 0.000 description 2
- 239000000651 prodrug Substances 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 238000004537 pulping Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 229920003169 water-soluble polymer Polymers 0.000 description 2
- 230000004580 weight loss Effects 0.000 description 2
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 description 1
- JDEYYCJOIRKTCS-UHFFFAOYSA-N 2,5-dioxopyrrolidine-3-sulfonic acid;octanedioic acid Chemical compound OS(=O)(=O)C1CC(=O)NC1=O.OS(=O)(=O)C1CC(=O)NC1=O.OC(=O)CCCCCCC(O)=O JDEYYCJOIRKTCS-UHFFFAOYSA-N 0.000 description 1
- JBIKZDFUXGHTHD-UHFFFAOYSA-N 2-chloro-2-fluoroacetic acid Chemical compound OC(=O)C(F)Cl JBIKZDFUXGHTHD-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- ZHGNHOOVYPHPNJ-UHFFFAOYSA-N Amigdalin Chemical compound FC(F)(F)C(=O)OCC1OC(OCC2OC(OC(C#N)C3=CC=CC=C3)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C2OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C1OC(=O)C(F)(F)F ZHGNHOOVYPHPNJ-UHFFFAOYSA-N 0.000 description 1
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- 206010055113 Breast cancer metastatic Diseases 0.000 description 1
- 102000003930 C-Type Lectins Human genes 0.000 description 1
- 108090000342 C-Type Lectins Proteins 0.000 description 1
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 1
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 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
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 108010022355 Fibroins Proteins 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 102000000802 Galectin 3 Human genes 0.000 description 1
- 108010001517 Galectin 3 Proteins 0.000 description 1
- 102000007563 Galectins Human genes 0.000 description 1
- 108010046569 Galectins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108010092694 L-Selectin Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 102100033467 L-selectin Human genes 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 102000010954 Link domains Human genes 0.000 description 1
- 108050001157 Link domains Proteins 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- 108010035766 P-Selectin Proteins 0.000 description 1
- 102100023472 P-selectin Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000005889 cellular cytotoxicity Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229940120124 dichloroacetate Drugs 0.000 description 1
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- AKRQHOWXVSDJEF-UHFFFAOYSA-N heptane-1-sulfonic acid Chemical compound CCCCCCCS(O)(=O)=O AKRQHOWXVSDJEF-UHFFFAOYSA-N 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229950010538 irinotecan hydrochloride trihydrate Drugs 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 102000015340 serglycin Human genes 0.000 description 1
- 108010050065 serglycin Proteins 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 229940066528 trichloroacetate Drugs 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical compound [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- 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
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
- C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
- C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
- C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Polymers & Plastics (AREA)
- Medicinal Preparation (AREA)
- Peptides Or Proteins (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
本发明公开了一类可以特异性靶向CD44的多支链药物偶联物。这类化合物与CD44发生了特异性结合,可以靶向高表达CD44的肿瘤细胞和组织,使得该偶联物在目标组织的浓度更高,提高其临床治疗效果,降低毒性。本发明化合物适用于治疗所有高表达CD44的肿瘤,包括但不限于胃癌、胰腺癌、小细胞肺癌、结肠癌、乳腺癌、肺腺癌、肝癌、鼻咽癌、恶性胶质瘤、淋巴瘤、肾癌、卵巢癌、头颈癌、鳞状细胞癌等。
Description
技术领域
本发明总体上涉及多臂靶向偶联物。具体地,本发明涉及CD44靶向分子与抗癌剂协同作用的多臂偶联物。
背景技术
恶性肿瘤的发病率与死亡率近些年来呈明显上升趋势,已经对人类的身体健康构成了严重的威胁。
CD44是一组分布较为广泛、分子形式多样的膜整合糖蛋白,包括胞外区、跨膜区和膜近侧区。CD44作为I型跨膜糖蛋白,首先结合透明质酸,引发细胞内信号途径,继而导致了一系列细胞脱落、转移、侵袭反应。CD44作为细胞黏附分子家族中的一员,在调节细胞与细胞间和细胞与细胞外基质之间的粘附和结合方面发挥重要作用,对维系组织结构整体性非常重要。CD44还可传导细胞内信号,调节肿瘤细胞的发生、活动等,如乳腺癌、结直肠癌等。当前大量研究认为CD44与肿瘤的发生、发展、浸润、转移关系密切,CD44的表达常出现在肿瘤发生的早期阶段,并且随着肿瘤发展,差异更加显著,像一些癌前病变,CD44就有所表达。
“CD44靶向多肽”是指能与CD44靶向结合的多肽。典型的“CD44靶向多肽”包括具有以下结构的多肽:Ac-KPSSPPEE-NH2和Ac-NASAPPEE-NH2。上述两个多肽与CD44的部分链接域具有序列同源性,可以与CD44靶向性结合。
其中,结构为Ac-KPSSPPEE-NH2的多肽也被称之为A6多肽,A6的结构如下:
Ac-NASAPPEE-NH2的多肽结构如下:
WO2005028539、WO2010019233、WO2011063156、WO2011063158公开了一种处于临床三期的药物nktr-102,该药物主要用于转移性乳腺癌,由NektarTherapeutics研发。该药物是一种水溶性的多支链聚合物药物前体,以提高药物的负载,结构如下:
该化合物是使用多臂PEG与伊立替康连接,以提高水溶性,增加载药量,在抗癌作用不变的情况下,降低副作用。但该药物仍具有缺点,比如,靶向性较差,不能作用于特定的癌细胞,在杀死癌细胞的同时,也会影响正常细胞的性能,而使不良反应发生率仍然比较高。
中国专利申请CN 108727583A公开了一种多臂的聚合靶向抗癌偶联物,该类化合物不仅呈现出高载荷能力,而且具有靶向性,使得该偶联物在目标组织的浓度更高。但由于恶性肿瘤细胞的结构、功能和代谢出现异常,细胞的***和增殖无休止并且无秩序,非常难以清除,需要药品研发人员不断开发疗效更好的抗癌药物。
发明内容
本发明公开了一种具有靶向性的多支链药物偶联物,该偶联物具有三个或更多的支链,该偶联物可表示为下式:
R为有机中心,POLY为聚合物,L为多价接头,T为靶向分子,D为活性剂,q为3-8之间的任一整数。
有机中心,“R”
在结构式(Ⅰ)中,“R”是一个1-100个原子的有机核心基团。比较好地是,R含有3-50个原子,更好地是,R含有大约3-30个原子。R可以为全部为碳原子的核心,也可选择性的含有1个或多个杂原子,例如,O、S、N、P等,依赖于所使用的特殊中心分子。R可以是直链、支链或环状的,发出至少3个独立的聚合物支链。结构式(Ⅰ)中,“q”与从“R”发出的聚合物支链的数量相对应。
在一些具体的方案中,“R”具有三个聚合物支链,“R”优选为:
在一些具体的方案中,“R”具有四个聚合物支链,“R”优选为:
在一些具体的方案中,“R”具有六个聚合物支链,“R”优选为:
在一些具体的方案中,“R”具有八个聚合物支链,“R”优选为:
聚合物,“POLY”
在结构式(Ⅰ)中,“POLY”为聚合物,优选的聚合物为水溶性的,任意的水溶性聚合物可以用于形成本发明的偶联物,本发明所指聚合物可以是任意几何形态或形式的。代表性的聚合物包括但并不限于:聚乙二醇、聚丙二醇、聚(乙烯吡咯烷酮)、聚(羟烷基甲基丙烯酸胺)、聚(羟烷基甲基丙烯酸盐)、聚(糖类)、聚(α-羟基酸)、聚(丙烯酸)、聚(乙烯乙酸)、聚膦嗪、聚唑啉、聚(N-丙烯酰吗啉)等。
在典型的化合物中,“POLY”为聚乙二醇,可为任意几何形态或形式,包括直链、支链、叉形链等,较好的,本发明优选的“POLY”为直线型的聚乙二醇,典型的结构为:
代表原子的连接处,标“&”号的氧原子为与有机中心“R”连接的原子。其中k的取值范围为大约5~500,最好为50~200,优选113。平均分子量约为1k~60kDa。本领域技术人员应该知晓,在高分子领域,k代表所述的聚合物的聚合度,即聚合物大分子链上所含重复单元数目的平均值,其取决于所述的聚合物的分子量,例如,当k为113时,是指平均值为113。
r为1-10之间的任一整数,更优选的,本发明“POLY”为:
本发明POLY还可为:
本发明优选的方案中,“POLY”为线状的聚乙二醇连接臂,即本发明偶联物优选下述类型的化合物:
在本发明中,多价接头L优选为:
符号“*”在这里代表多价接头L与靶向分子T的连接点,“#”代表多价接头L与活性剂D的连接点,“%”代表多价接头L与POLY的连接点。其中,m为1-20之间的任一整数,l、n分别为1-10之间的任一整数。最优选的。l为5,m为3,n为4,即本发明优选的多价接头L为
D为如下式所示的喜树碱类药物:
R1-R5相互独立地选自于以下基团:氢、卤素、酰基、烷基、取代烷基、烷氧基、取代烷氧基、烯基、炔基、环烷基、羟基、氰基、硝基、叠氮基、酰胺基、肼、胺基、取代胺基、羟基羰基、烷氧羰基、烷氧羰氧基、氨基甲酰氧基、芳基磺酰氧基、烷基磺酰氧基;R6为H或OR8,R8为烷基、烯基、环烷基、卤代烷基或羟烷基;R7为羟基、氨基、或硫醇。优选的,D为伊立替康或SN38。
其中,伊立替康结构如下:
SN38的结构如下:
T为靶向分子,其为可与CD44结合的“CD44靶向多肽”。典型的“CD44靶向多肽”包括具有以下序列的多肽:
Ac-KPSSPPEE-NH2;Ac-NASAPPEE-NH2;QETWFQNGWQGKNP;
KEKWFENEWQGKNP;KEQWFGNRWHEGYR;
QIRQQPRDPPTETLELEVSPDPAS。
还包括上述多肽的替代变体(substitution variants)、增加变体(additionvariants)和化学修饰衍生物。
T还可为WO99/05263所公开的多肽,即具有以下序列的多肽或者其替代变体(substitution variants)、增加变体(addition variants)和化学修饰衍生物:Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu(KPSSPPEE)、Asn-Ala-Ser-Ala-Pro-Pro-Glu-Glu(NASAPPEE)。此外,T还可为WO2007141533所公开的FKBP-L多肽。
靶向分子T还可为能与CD44结合的单抗或其天然配体。能与CD44结合的天然配体包括且不限于透明质酸(hyaluronic acid,HA)、骨桥蛋白(osteopontin,OPN)、丝甘蛋白(Serglycin)、胶原蛋白(Collagen)、纤维连接蛋白(Fibronectin)、层黏连蛋白(Laminin)、硫酸软骨素C(chondroitin sulfate,CSC)、硫酸肝素(heparansulfate,HS)、锚定蛋白(ankyrin)、半乳糖凝集素(Galectin-3)、L-选凝素、P-选凝素、C型凝集素和导向蛋白(addressin)等。
本发明优选的靶向分子T为Ac-KPSSPPEE-NH2和Ac-NASAPPEE-NH2。这两个多肽以封端的NH2端和多价接头L相连。
本发明优选的化合物为:
在化合物a中,D为伊立替康,T为A6,即Ac-KPSSPPEE-NH2。
在化合物b中,D为伊立替康,T为Ac-NASAPPEE-NH2。
在化合物c中,D为SN38,T为A6,即Ac-KPSSPPEE-NH2
在化合物d中,D为SN38,T为Ac-NASAPPEE-NH2。
本发明为一个多臂聚合物修饰的靶向抗癌偶联物,其中,水溶性的聚合物修饰可增强该偶联物的水溶性,从而提高载药量;靶向分子增加靶向性,使得该偶联物在目标组织的浓度更高;L为任意的连接接头,其作用是首先将靶向分子与抗癌药物连接起来,再将“靶向分子、抗癌药物与聚合物臂连接起来,使得整个偶联物形成一个有机的整体。本发明偶联物是典型的药物前体,通过水解作用或酶解作用,活性剂D被释放出来,与母体分离,发挥生理活性。相比未偶联的药物,本发明偶联物能够表现出更强的作用,在人体或其他动物体内组织更加富集。
本发明偶联物,药学上可接受的盐包括无机盐和有机盐,典型的盐包括硝酸盐、硫酸盐、磷酸盐、氢氟酸盐、盐酸盐、氢溴酸盐、氢碘酸盐、甲酸盐、乳酸盐、苯甲酸盐、醋酸盐、三氟乙酸盐、二氯乙酸盐、三氯乙酸盐、混合的氯氟乙酸盐、柠檬酸盐、草酸盐、磺酸盐、甲磺酸盐、三氟甲磺酸盐、庚烷磺酸盐等,其中优选盐酸盐和醋酸盐,可用药物化学领域的常规手段成盐。
化合物a、化合物b、化合物c、化合物d典型的盐酸盐为每个支链分别结合一分子或两分子盐酸的盐酸盐,该优选的偶联物为四或八分子盐酸盐。
化合物a、化合物b、化合物c、化合物d典型的醋酸盐为每个支链分别结合一分子或两分子醋酸的醋酸盐,该优选的偶联物为四或八分子醋酸盐。
本发明化合物与CD44发生了特异性结合,可以靶向高表达CD44的肿瘤细胞和组织,使得该偶联物在目标组织的浓度更高,提高其临床治疗效果,降低毒性。
本发明化合物适用于治疗所有高表达CD44的肿瘤,包括但不限于胃癌、胰腺癌、小细胞肺癌、结肠癌、乳腺癌、肺腺癌、肝癌、鼻咽癌、恶性胶质瘤、淋巴瘤、肾癌、卵巢癌、头颈癌、鳞状细胞癌等。
具体实施例
在下面将对本发明进行详细描述。然而,本发明可能具体体现为许多不同的形式,而且它不应该被局限于此处所描述的实施例中,提供这些实施例中的目的是使所披露内容更完整与全面。所用试剂和原料,除了提供制备方法的除外,其余均为市售。4armPEG20K-SCM购自北京键凯科技有限公司,分子量约为20kDa。除非另有定义,否则本文中所有科技术语具有的含义与权利要求主题所属技术领域人员通常理解的含义相同。
4arm-PEG20K-SCM结构如下:
名词解释
实施例1
1.BP103a的制备
向250mL三口瓶中加入18.20g化合物BP103a05,100ml EA,搅拌溶解后降温至0℃,加入150ml HCl/EA(3.5M),保温0℃,TLC监控反应完毕反应液浓缩至干,加入甲基叔丁基醚打浆,过滤,滤饼用TBME洗涤、真空干燥得白色固体BP103a 10.40g。
2.BP103a07的制备
向100mL烧瓶加入3.00g BP103a(1.0eq),4.02g(1.0eq)BP102c07,40mlDCM,4.0mlDIEA(2.0eq),室温搅拌,TLC监控反应完毕,蒸去有机溶剂,柱层析得6.4g油状物BP103a075.26g。
3.BP103a08的制备
向200mL三口瓶中加入9.00g BP103a07(1.0eq),3.96g HOSU(1.53eq),90ml DCM,6.60g EDC·HCl(1.53eq),室温反应2h,TLC监控反应完毕后,DCM稀释后用50mmol/L的磷酸二氢钾水溶液洗涤2次,饱和食盐水洗涤,无水硫酸钠干燥,浓缩得无色油状物5.93gBP103a08。
4.BP103a30的制备
向200mL烧瓶加入2.93g化合物NH2-Lys(Boc)-OH·HCl(1.0eq),60ml水,2.00gNaHCO3(2.0eq),搅拌滴加5.91g BP103a08(1.0eq)溶于60ml DME的溶液,补加60ml THF,搅拌过夜,TLC监控反应完毕,蒸去有机溶剂,用稀盐酸调节pH≈3,EA萃取,无水硫酸钠干燥,浓缩,得4.50g无色油状物BP103a30。5.BP103a31的制备
向100mL三口瓶中加入5.00g BP103a30(1.0eq),1.40g HOSU(1.53eq),50ml DCM,2.33g EDC·HCl(1.53eq),室温反应2h,TLC监控反应完毕后,DCM稀释后用50mmol/L的磷酸二氢钾水溶液洗涤2次,饱和食盐水洗涤,无水硫酸钠干燥,浓缩得无色油状物4.89gBP103a31。
实施例2中间体Ac-KPSSPPEEC-NH2的合成
Ac-KPSSPPEEC-NH2合成采用本专业人员熟知的Fmoc法固相合成,利用Rink-amideResin、采用20%的哌啶/DMF脱除Fmoc,偶联试剂采用HOBT/DIC,DMF为反应溶剂,反应监控采用茚三酮检测法,依次将下列保护氨基酸连接到树脂上:Fmoc-Cys(Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Lys(Boc)-OH,并用醋酸酐、吡啶乙酰化,然后DMF洗涤、甲醇洗涤、DCM洗涤后干燥,加入裂解试剂(TFA:苯甲硫醚:苯酚:TIS=85:5:5:5),反应2小时后用冰TBME沉淀、离心,得Ac-KPSSPPEEC-NH2粗品,经HPLC制备纯化后冻干得纯品。
实施例3中间体Ac-NASAPPEEC-NH2的合成
Ac-NASAPPEEC-NH2合成采用本专业人员熟知的Fmoc法固相合成,利用Rink-amideResin、采用20%的哌啶/DMF脱除Fmoc,偶联试剂采用HOBT/DIC,DMF为反应溶剂,反应监控采用茚三酮检测法,依次将下列保护氨基酸连接到树脂上:Fmoc-Cys(Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ala-OH、Fmoc-Asn(Trt)-OH,并用醋酸酐、吡啶乙酰化,然后DMF洗涤、甲醇洗涤、DCM洗涤后干燥,加入裂解试剂(TFA:苯甲硫醚:苯酚:TIS=85:5:5:5),反应2小时后用冰TBME沉淀、离心,得Ac-NASAPPEEC-NH2粗品,经HPLC制备纯化后冻干得纯品。
实施例4
1.BP143b02的制备
向250mL圆底烧瓶中加入3.50g盐酸伊立替康三水合物(1.0eq),52.5mlDMF,加热至60℃溶解,5-10min后减压蒸去DMF,加入300ml正庚烷减压蒸馏,重复三次,旋干后加入105mlDCM,1.08g Boc-Gly-OH(1.2eq),63mgDMAP(0.1eq),滴加1.59g DCC(1.5eq)溶于10mlDCM的溶液,20℃反应4小时,TLC监控反应完毕后,过滤,浓缩至剩余25%体积时加入120mlIPA,蒸去75%的溶剂,加入150ml正庚烷,室温搅拌1小时,过滤,正庚烷洗涤2次,干燥得淡黄色固体4.02g BP143b02。
2.BP143b03的制备
向100mL三口瓶中加入4.02g BP143b02,50ml DCM,搅拌溶解后滴加11.6mlTFA,室温反应2h,TLC监控反应完毕后加入150ml乙腈,减压蒸馏120ml溶剂后倒入320ml TBME溶液中,搅拌30min,过滤,滤饼用TBME洗涤得淡黄色固体BP143b03 4.00g。
3.BP143b04的制备
向200mL三口瓶中加入3.69g BP143b03,100ml DCM,3.21g(1.05eq)BP103a30,2.7ml DIEA(3.0eq),1.2ml DEPC(1.5eq),室温反应1h,TLC监控反应完毕后,DCM稀释后,水洗两次,饱和食盐水洗涤一次,干燥,浓缩,HPLC纯化后冻干得到淡黄色固体BP143b041.85g。
4.BP143b05的制备
向50mL圆底烧瓶中加入1.20g BP143b04,10ml的10%TFA/DCM,室温反应,TLC监控反应完毕后,倒入TBME中,离心,干燥得淡黄色固体化合物BP143b05 210mg。
5.BP143b06的制备
向10mL圆底烧瓶中加入51mg BP143b05(4.0eq),2ml DCM,11ul TEA(8.0eq),201mg 4armPEG20K-SCM(1.0eq),室温反应过夜后,浓缩,加入TBME中,离心,经HPLC制备纯化、冻干得黄绿色固体化合物BP143b06 85mg。
实施例5
1.BP149a01的制备
向100mL三口烧瓶中加入10.00g化合物SN38(1.0eq),110ml DMSO,溶清后加入5.56g(Boc)2O(1.0eq)、9.82g DIEA(3.0eq),反应2小时后TLC监控反应完毕,将反应液倒入纯化水中,乙酸乙酯萃取3次,合并有机相,用纯化水洗涤,饱和食盐水洗涤,无水硫酸钠干燥,得类白色固体,甲基叔丁基醚打浆后过滤,干燥后得类白色固体7.95g化合物BP149a01。
2.BP149a02的制备
向250mL圆底烧瓶中加入7.95g化合物BP149a01(1.0eq),2.83g Boc-Gly-OH(1.0eq),80ml二氯甲烷,390mg DMAP(0.2eq),滴加5.00gDCC(1.5eq)溶于20mlDCM的溶液,室温反应1小时,TLC监控反应完毕后,过滤,浓缩至无液体蒸出,加入30ml IPA,溶清后加入200ml正庚烷,搅拌打浆30分钟,过滤,正庚烷洗涤2次,干燥得淡黄色固体8.73g化合物BP149a02。
3.BP149a03的制备
向100mL三口瓶中加入8.73g化合物BP149a02,40ml DCM,搅拌溶解后降温至0℃,滴加40ml TFA,室温反应2h,TLC监控反应完毕后将反应液倒入800ml TBME溶液中,搅拌30min,过滤,滤饼用TBME洗涤,正真空干燥后得淡黄色固体BP149a03 4.95g。
4.BP149b01的制备
向200mL三口瓶中加入2.3g化合物BP149a03,45ml DCM,3.196g(1.05eq)化合物BP103a31,1.75ml TEA(3.0eq),室温反应4h,TLC监控反应完毕后,DCM稀释,稀盐酸洗涤1次,水洗两次,饱和食盐水洗涤一次,无水硫酸钠干燥,浓缩得粗品,粗品经HPLC制备纯化后DCM萃取,无水硫酸钠干燥、浓缩干燥得淡黄色固体BP149b01 2.20g。
5.BP149b02的制备
向200mL三口烧瓶中加入2.0g化合物BP149b01,40ml的10%TFA/DCM,室温反应,TLC监控反应完毕后,倒入TBME中,离心,干燥得淡黄色固体BP149b02 1.75g。
6.BP149b03的制备
向200mL三口烧瓶中加入4.08g化合物BP149b02(5.0eq),100ml DMF,1.96g DIEA(20eq),16.05g 4armPEG20K-SCM(1.0eq),室温反应2小时后,倒入1L甲基叔丁基醚中,搅拌30分钟后过滤,干燥得类白色固体20.50g粗品,粗品经HPLC制备纯化、冻干得BP149b0312.36g纯品。
实施例6
室温条件下向100ml三口反应瓶中加入2.00g(1.0eq)BP143b06,35ml pH=7.0的PBS溶液搅拌溶清,取0.45g(5.0eq)靶向多肽1(Ac-KPSSPPEEC-NH2)溶于30ml纯化水中,用碳酸氢钠溶液调节pH≈6.5后加入到反应瓶中,0.5小时后HPLC检测反应完毕,经HPLC制备纯化、冻干,得化合物a 1.65g纯品。
MALDI-TOF检测分子量为28810.73。
实施例7
室温条件下向100ml三口反应瓶中加入2.00g(1.0eq)BP143b06,35ml pH=7.0的PBS溶液搅拌溶清,取0.38g(5.0eq)靶向多肽2(Ac-NASAPPEEC-NH2)溶于30ml纯化水中,用碳酸氢钠溶液调节pH≈6.5后加入到反应瓶中,0.5小时后HPLC检测反应完毕,经HPLC制备纯化、冻干,得化合物b 1.42g纯品。
MALDI-TOF检测分子量为28931.52。
实施例8
室温条件下向100ml三口反应瓶中加入2.00g(1.0eq)BP149b03,35ml pH=7.0的PBS溶液搅拌溶清,取0.46g(5.0eq)靶向多肽1(Ac-KPSSPPEEC-NH2)溶于30ml纯化水中,用碳酸氢钠溶液调节pH≈6.5后加入到反应瓶中,0.5小时后HPLC检测反应完毕,经HPLC制备纯化、冻干,得化合物c 1.45g纯品。
MALDI-TOF检测分子量为28191.63。
实施例9
室温条件下向100ml三口反应瓶中加入2.00g(1.0eq)BP149b03,35ml pH=7.0的PBS溶液搅拌溶清,取0.39g(5.0eq)靶向多肽2(Ac-NASAPPEEC-NH2)溶于30ml纯化水中,用碳酸氢钠溶液调节pH≈6.5后加入到反应瓶中,0.5小时后HPLC检测反应完毕,经HPLC制备纯化、冻干,得化合物d 1.53g纯品。
MALDI-TOF检测分子量为27982.32。
实施例10合成生物素标记的靶向多肽3
生物素标记的靶向多肽3的合成采用本专业人员熟知的Fmoc法固相合成,利用Rink-amide Resin、采用20%的哌啶/DMF脱除Fmoc,偶联试剂采用HOBT/DIC,DMF为反应溶剂,反应监控采用茚三酮检测法,依次将下列保护氨基酸连接到树脂上:Fmoc-Cys(Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Lys(Dde)-OH,脱除Fmoc保护后用醋酸酐、吡啶乙酰化,加入水合肼的DMF溶液,脱除Dde保护基,依次连接Fmoc-PEG3-OH、生物素,然后DMF洗涤、甲醇洗涤、DCM洗涤后干燥,加入裂解试剂(TFA:苯甲硫醚:苯酚:EDT:水=82.5:5:5:2.5:5),反应2小时后用冰TBME沉淀、离心,得粗品,经HPLC制备纯化后冻干得纯品。
实施例11合成生物素标记的靶向多肽4
生物素标记的靶向多肽4采用本专业人员熟知的Fmoc法固相合成,利用Rink-amide Resin、采用20%的哌啶/DMF脱除Fmoc,偶联试剂采用HOBT/DIC,DMF为反应溶剂,反应监控采用茚三酮检测法,依次将下列保护氨基酸连接到树脂上:Fmoc-Cys(Trt)-OH、Fmoc-Lys(Dde)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ala-OH、Fmoc-Asn(Trt)-OH,脱除Fmoc后用醋酸酐、吡啶乙酰化,加入水合肼的DMF溶液,脱除Dde保护基,依次连接Fmoc-PEG3-OH、Biotin,然后DMF洗涤、甲醇洗涤、DCM洗涤后干燥,加入裂解试剂(TFA:苯甲硫醚:苯酚:EDT:水=82.5:5:5:2.5:5),反应2小时后用冰TBME沉淀、离心,得粗品,经HPLC制备纯化后冻干得纯品。
实施例12化合物a-生物素偶合物的制备
室温条件下向100ml三口反应瓶中加入2.00g(1.0eq)BP143b06,35ml pH=7.0的PBS溶液搅拌溶清,取0.57g(5.0eq)生物素标记的靶向多肽3溶于30ml纯化水中,用碳酸氢钠溶液调节pH≈6.5后加入到反应瓶中,0.5小时后HPLC检测反应完毕,经HPLC制备纯化、冻干,得1.24g纯品。MALDI-TOF检测分子量为30325.51。
实施例13化合物b-生物素偶合物的制备
室温条件下向100ml三口反应瓶中加入2.00g(1.0eq)BP143b06,35ml pH=7.0的PBS溶液搅拌溶清,取0.60g(5.0eq)生物素标记的靶向多肽4溶于30ml纯化水中,用碳酸氢钠溶液调节pH≈6.5后加入到反应瓶中,0.5小时后HPLC检测反应完毕,经HPLC制备纯化、冻干,得1.33g纯品。MALDI-TOF检测分子量为30596.56。
实施例14化合物c-生物素偶合物的制备
室温条件下向100ml三口反应瓶中加入2.00g(1.0eq)BP149b03,35ml pH=7.0的PBS溶液搅拌溶清,取0.55g(5.0eq)生物素标记的靶向多肽3溶于30ml纯化水中,用碳酸氢钠溶液调节pH≈6.5后加入到反应瓶中,0.5小时后HPLC检测反应完毕,经HPLC制备纯化、冻干,得1.26g纯品。MALDI-TOF检测分子量为30223.25。
实施例15化合物d-生物素偶合物的制备
室温条件下向100ml三口反应瓶中加入2.00g(1.0eq)BP149b03,35ml pH=7.0的PBS溶液搅拌溶清,取0.58g(5.0eq)生物素标记的靶向多肽4溶于30ml纯化水中,用碳酸氢钠溶液调节pH≈6.5后加入到反应瓶中,0.5小时后HPLC检测反应完毕,经HPLC制备纯化、冻干,得1.33g纯品。MALDI-TOF检测分子量为30491.18。
实施例16本发明化合物与CD44特异性结合
1.材料和方法
1.1本发明化合物用生物素标记的偶合物
化合物a-生物素偶合物、化合物b-生物素偶合物、化合物c-生物素偶合物、化合物d-生物素偶合物的结构及其制备方法见实施例10-15,在试验中分别被称之为偶合物a、偶合物b、偶合物c、偶合物d。
1.2细胞培养
SKOV3细胞在McCoy’s 5A培养液中培养。细胞培养液中添加了10%FBS,1000U/mL青霉素,100μg/mL链霉素和4mmol/L l-谷氨酰胺。
1.3偶合物与SKOV3细胞的结合
向偶合物a、偶合物b、偶合物c、偶合物d(分别溶于冰的PBS中,使其浓度为100μmol/L)中加入5mmol/L双(磺基丁二酰亚胺)辛二酸盐,使其与SKOV3细胞结合。加入TritonX-100细胞裂解液,细胞裂解后离心,转移到聚偏氟乙烯(PVDF)膜后进行免疫印迹分析。用与辣根过氧化物酶结合的链霉亲和素(HRP-SA)孵育PVDF膜,检测与偶合物交联的蛋白。通过抗CD44的单克隆抗体DF1485和过氧化物酶标记的驴抗小鼠免疫球蛋白印迹染色来检测CD44。用新鲜配制的化学发光试剂和Hypermax ECL胶片曝光进行检测。
1.4偶合物标记蛋白的免疫沉淀
与偶合物交联的SKOV3细胞通过Triton X-100裂解,与蛋白A琼脂糖在室温下孵育2小时进行预清洗,将100μg清洗过的裂解液与加了2μgDF1485(抗Cd44的抗体)或者鼠源IgG1的20μL蛋白A琼脂糖在室温下孵育过夜。用裂解缓冲液洗涤5次后,在SDS-PAGE上样缓冲液中煮沸后从蛋白A-琼脂糖中洗脱免疫沉淀蛋白。洗脱的物料和5μg原始物料被加入SDS-PAGE,转移到PVDF膜,再通过HRP-SA染色。
2.结果
SKOV3细胞与偶合物结合。用DF1485对以此细胞制备的裂解物进行免疫沉淀。对被洗脱的蛋白和起始的裂解物进行PAGE/blotting,分别以HRP-SA染色来检测生物素标签,以DF1485染色来检测CD44。
当反应体系中包括起始裂解物、偶合物和HRP-SA,在0-250kDa范围内出现很多非特异性条带;当反应体系中包括起始裂解物和HRP-SA,在0-250kDa范围内无条带,说明以HRP-SA染色被偶合物标记的蛋白具有特异性。当反应体系中包括免疫沉淀蛋白、偶合物和HRP-SA,在85kDa处出现特异性条带;当反应体系中包括免疫沉淀蛋白和HRP-SA,在0-250kDa范围内无条带,说明DF1485特异性地沉淀了被偶合物标记的约85kDa的蛋白质。与初始裂解物相比,该条带在免疫沉淀蛋白中的染色程度更明显,表明该蛋白通过抗CD44免疫沉淀得到了极大的富集。
当反应体系中包括起始裂解物、偶合物和DF1485,在85kDa处出现特异性条带;当反应体系中包括起始裂解物和DF1485,在85kDa处出现特异性条带。当反应体系中包括免疫沉淀蛋白、偶合物和DF1485,在85kDa处出现特异性条带;当反应体系中包括免疫沉淀蛋白和DF1485,在85kDa处出现特异性条带。说明该85kDa的条带就是CD44,且CD44存在于所有样品中。
以上结果表明,本发明化合物与CD44发生了特异性结合,可以靶向高表达CD44的肿瘤细胞和组织,使得该偶联物在目标组织的浓度更高,提高其临床治疗效果,降低毒性。
在实施例17-21中,所有动物实验操作严格遵守动物使用和管理规范。肿瘤相关参数的计算参考中国CFDA《细胞毒类抗肿瘤药物非临床研究技术指导原则》,根据中国SFDA《细胞毒类抗肿瘤药物非临床研究技术指导原则》(2006年11月),%T/C≤40%并经统计学分析P<0.05为有效。相对肿瘤增殖率越低,说明抑制肿瘤效果越良好
肿瘤体积(TV)计算公式如下:TV(mm3)=l×w2/2
其中,l表示肿瘤长径(mm);w表示肿瘤短径(mm)。
相对肿瘤体积(RTV)的计算公式为:RTV=TVt/TVinitial
其中,TVinitial为分组给药时测量到的肿瘤体积;TVt为给药期间每一次测量时的肿瘤体积。
相对肿瘤增殖率(%T/C)的计算公式为:%T/C=100%×(RTVT/RTVC)
其中,RTVT表示治疗组RTV;RTVC表示溶剂对照组RTV。
肿瘤生长抑制率TGI(%)的计算公式为:TGI=100%×[1-(TVt(T)-TVinitial(T))/(TVt(C)-TVinitial(C))]
其中,TVt(T)表示治疗组每次测量的肿瘤体积;TVinitial(T)表示分组给药时治疗组的肿瘤体积;TVt(C)表示溶剂对照组每次测量的肿瘤体积;TVinitial(C)表示分组给药时溶剂对照组的肿瘤体积。
动物体重下降率的计算公式为:动物体重下降率=100%×(BWinitial-BWt)/BWinitial
其中,BWt表示给药期间每次测量的动物体重;BWinitial表示分组给药时的动物体重。
瘤重抑瘤率IR(%)的计算公式为:IR(%)=100%×(WC-WT)/WC
其中,WC表示对照组瘤重;WT表示治疗组瘤重。
试验数据用Microsoft Office Excel 2007软件进行计算和相关统计学处理。数据除特别说明外,用均数±标准误(Mean±SE)表示,组间比较采用t-检验,P<0.05为显著性差异。
实验动物:雌性BALB/c裸小鼠(只数:100只;周龄:6~8周),正常喂养约1周后,经兽医检验,体征状况良好小鼠可入选本实验。分组前使用记号笔于动物尾根部进行标识,分组后每只动物均用耳部剪缺方式标识。
化合物M、化合物a、化合物b给药制剂配制:每次给药前,分别准确称量,加入2.5mL的生理盐水,涡旋振荡使药物完全溶解,溶液中伊立替康有效浓度为4.0mg·mL-1。
化合物c、化合物d给药制剂配制:每次给药前,分别准确称量,加入2.5mL的生理盐水,涡旋振荡使药物完全溶解,溶液中SN38有效浓度为4.0mg·mL-1。
随机分组:对动物接种肿瘤细胞后,当肿瘤体积达到100-300mm3时,将动物按随机区组法分为6组,每组6只小鼠,保证各组间肿瘤体积和小鼠体重均一。各组肿瘤体积的均值与所有实验动物肿瘤体积的均值差异不超过±10%。
实验开始后每周测量2次瘤径,计算肿瘤体积,同时称量动物体重并记录。
实施例17-20的动物给药为:于分组当天开始第一次给药。第一组为溶剂对照组,尾静脉注射给予生理盐水。第2-6组分别尾静脉注射给予受试样品化合物M、化合物a、化合物b、化合物c、化合物d。
实验结束后,称量体重、测量瘤径后动物安乐死(CO2)。剥取肿瘤组织并称重,计算RTV和%T/C。
实施例中所涉及到的对照供试品化合物M公开于中国专利申请CN108727583A,其结构如下:
实施例17本发明化合物对人乳腺癌MDA-MB-231裸鼠异种移植瘤的抗肿瘤药效实验
供试品:化合物M、化合物a、化合物b、化合物c、化合物d。
试剂:DMEM培养液,胎牛血清(FBS),胰蛋白酶,青-链双抗,生理盐水。
移植性肿瘤瘤株:人乳腺癌MDA-MB-231,来源于中国科学院典型培养物保藏委员会细胞库(CAS,本实验室液氮冻存)。
细胞培养:MDA-MB-231培养于DMEM培养基(DMEM,美国),含10%胎牛血清FBS(GIBCO,美国),培养于含5%CO2的37℃培养箱。
动物模型制备:细胞接种法建立肿瘤裸鼠皮下移植模型,收集对数生长期的肿瘤细胞,计数后重悬于1×PBS,调整细胞悬液浓度至1×107/ml。用1ml注射器(4号针头)在裸鼠右侧背部皮下接种肿瘤细胞,1×106/小鼠。
给药频次及剂量:各组均为每周给药一次,共给药三次,QW×3,给药剂量均为6mg·kg-1(以伊立替康或SN38计)。
结果见表1:
表1对肿瘤增殖率T/C(%)
*与空白溶剂、化合物M组的RTV相比,P<0.05
#与空白溶剂、化合物M组的%T/C相比,P<0.05
实验结果显示,本发明化合物对人乳腺癌MDA-MB-231裸鼠移植瘤有良好抑制作用,且优于化合物M。
实施例18本发明化合物对人胃癌NCI-N87细胞株裸鼠移植瘤模型肿瘤体内生长的抑制作用。
供试品:化合物M、化合物a、化合物b、化合物c、化合物d。
试剂:RPMI-1640培养液,胎牛血清(FBS),胰蛋白酶,青-链双抗,生理盐水。
移植性肿瘤瘤株:人胃癌细胞NCI-N87,来源于中国科学院典型培养物保藏委员会细胞库(CAS,本实验室液氮冻存)。
细胞培养:在5%CO2、37℃培养条件下,NCI-N87细胞在含10%胎牛血清RPMI-1640培养液中进行常规细胞培养;以0.25%胰酶消化传代;根据细胞生长情况,每周传代1到2次,传代比例为1:2到1:6。
动物模型制备:收取对数生长期NCI-N87细胞,细胞计数后重悬于无血清的RPMI-1640培养基中,调整细胞浓度至5×107细胞/mL;用移液器吹打细胞使其分散均匀后装入50mL离心管中,将离心管置于冰盒中;用1mL注射器吸取细胞悬液,注射到裸鼠前右肢腋窝皮下,每只动物接种100μL(5×106细胞/只),建立NCI-N87裸鼠移植瘤模型。
给药频次及剂量:各组均为每周给药一次,共给药四次,QW×4,给药剂量均为20mg·kg-1。
结果见表2:
表2对肿瘤增殖率T/C(%)
*与空白溶剂、化合物M组的RTV相比,P<0.05
#与空白溶剂、化合物M组的%T/C相比,P<0.05
实验结果显示,本发明化合物对人胃癌NCI-N87细胞株裸鼠移植瘤模型肿瘤生长有良好抑制作用,且优于化合物M。
实施例19HT-29裸鼠移植瘤模型体内药效评价
供试品:化合物M、化合物a、化合物b、化合物c、化合物d。
试剂:McCoy’s 5A培养液,胎牛血清(FBS),胰蛋白酶,青-链双抗,注射用水,生理盐水,乳酸,山梨糖醇。
移植性肿瘤瘤株:人结肠癌细胞HT-29,来源于中国科学院典型培养物保藏委员会细胞库(CAS,本实验室液氮冻存)。
细胞培养:在5%CO2、37℃培养条件下,HT-29细胞在含10%胎牛血清McCoy’s 5A培养液中进行常规细胞培养;以0.25%胰酶消化传代;根据细胞生长情况,每周传代2到3次,传代比例为1:4到1:6。
动物模型制备:收取对数生长期HT-29细胞,细胞计数后重悬于无血清McCoy’s 5A培养基中,调整细胞浓度至4×107细胞/mL;用移液器吹打细胞使其分散均匀后装入50mL离心管中,将离心管置于冰盒中;用1mL注射器吸取细胞悬液,注射到裸鼠前右肢腋窝皮下,每只动物接种100μL(4×106细胞/只),建立HT-29裸鼠移植瘤模型。
给药频次及剂量:各组均为每周给药一次,共给药四次,QW×4,给药剂量均为20mg·kg-1。
结果见表3:
表3对肿瘤增殖率T/C(%)
*与空白溶剂、化合物M组的RTV相比,P<0.05
#与空白溶剂、化合物M组的%T/C相比,P<0.05
实验结果显示,本发明化合物对人结肠癌HT-29裸鼠移植瘤模型肿瘤体内生长有良好抑制作用,且优于化合物M。
实施例20对人胰腺癌MIA Paca-2裸鼠异种移植模型的抑制作用
供试品:化合物M、化合物a、化合物b、化合物c、化合物d。
移植性肿瘤瘤株:人胰腺癌MIA Paca-2购于上海中科院细胞生物研究所。
细胞培养:MIA Paca-2细胞培养于DMEM,含10%胎牛血清FBS(GIBCO,美国)和2.5%HS。细胞放置于5%CO2培养箱37℃培养。
动物模型制备:细胞接种法建立人胰腺癌MIA Paca-2裸鼠皮下移植瘤模型:收集对数生长期的肿瘤细胞,计数后重悬于1×PBS,调整细胞悬液浓度至3×107/ml。用1ml注射器(4号针头)在裸鼠右侧背部皮下接种肿瘤细胞,3×106/0.1ml/鼠。
给药频次及剂量:各组均为每周给药一次,共给药三次,QW×3,给药剂量均为6mg·kg-1。
结果见表4:
表4对肿瘤增殖率T/C(%)
*与空白溶剂、化合物M组的RTV相比,P<0.05
#与空白溶剂、化合物M组的%T/C相比,P<0.05
实验结果显示,本发明化合物对人胰腺癌MIA Paca-2裸鼠移植瘤有良好抑制作用,且优于化合物M。
实施例21对人小细胞肺癌细胞NCI-H446裸鼠异种移植模型的抑制作用
供试品:伊立替康、nktr-102、化合物a、化合物b、化合物c、化合物d。
伊立替康(原料药)系购买所得。nktr-102的制备方法如下:
将实施例4中的BP143b03(829mg,4.5eq)添加到250mL的反应瓶中,加入DCM(50mL),三乙胺(221mg,9.0eq),溶解后加入4ARM-PEG20K-SCM(5.00g,1.0eq)添加到该反应瓶中。HPLC监控反应没有明显进展后,减压蒸馏出去约20mL DCM,将溶液倒入300mL TBME中搅拌沉淀,过滤,得到5.4g粗品,粗品经HPLC制备纯化,脱盐,用稀盐酸调节pH至5-6,冻干得到2.71g淡绿色粉末nktr-102。
伊立替康给药制剂配制:称取12.0mg的伊立替康,加入0.15mL的1%乳酸,涡旋振荡使药物完全溶解,再分别加入2.85mL的1%山梨糖醇水溶液,涡旋振荡混合均匀,溶液中1%乳酸、1%山梨糖醇水溶液的比例约为5:95(v/v)。溶液中伊立替康有效浓度为4.0mg·mL-1。
nktr-102给药制剂配制:每次给药前,准确称量101.5mg的nktr-102,加入2.5mL的生理盐水,涡旋振荡使药物完全溶解,溶液中伊立替康有效浓度为4.0mg·mL-1。
移植性肿瘤瘤株:人小细胞肺癌细胞NCI-H446来源于中国科学院典型培养物保藏委员会细胞库。
细胞培养:在5%CO2、37℃、饱和湿度条件下,NCI-H446细胞在含10%胎牛血清RPMI-1640培养液中进行常规细胞培养;根据细胞生长情况,以0.25%胰酶消化传代,每周传代2到4次,传代比例为1:3到1:4。
动物模型制备:细胞接种法建立肿瘤裸鼠皮下移植模型:收取对数生长期NCI-H446细胞,细胞计数后重悬于含50%的RPMI-1640基础培养基和50%的Matrigel中,调整细胞浓度至4×107细胞/mL;用移液器吹打细胞使其分散均匀后装入50-mL离心管中,将离心管置于冰盒中;用合适的注射器吸取细胞悬液,注射到裸鼠前右肢腋窝皮下,每只动物接种200μL(8×106细胞/只),建立NCI-H446裸鼠移植瘤模型。
动物给药:于分组当天开始第一次给药。第一组为溶剂对照组,尾静脉注射给予生理盐水。第2-7组分别尾静脉注射给予受试样品伊立替康、nktr102、化合物a、化合物b、化合物c、化合物d,以上各组均为每周给药一次,共给药三次,QW×3,给药剂量均为6mg·kg-1。
结果见表5:
表5对肿瘤增殖率T/C(%)
*与空白溶剂、伊立替康、nktr102组的RTV相比,P<0.05
#与空白溶剂、伊立替康、nktr102组的%T/C相比,P<0.05
实验结果显示,本发明化合物对人小细胞肺癌细胞NCI-H446裸鼠移植瘤有良好抑制作用,且优于伊立替康和nktr102。
Claims (14)
2.如权利要求1所述的多支链药物偶联物或其药学上可接受的盐,POLY为聚乙二醇、聚丙二醇、聚(乙烯吡咯烷酮)、聚(羟烷基甲基丙烯酸胺)、聚(羟烷基甲基丙烯酸盐)、聚(糖类)、聚(α-羟基酸)、聚(丙烯酸)、聚(乙烯乙酸)、聚膦嗪、聚唑啉或聚(N-丙烯酰吗啉)。
6.如权利要求5所述的多支链药物偶联物或其药学上可接受的盐,D为伊立替康或SN38。
7.如权利要求6所述的多支链药物偶联物或其药学上可接受的盐,T为Ac-KPSSPPEE-NH2或Ac-NASAPPEE-NH2,及其替代变体、增加变体和化学修饰衍生物。
9.如权利要求8所述的多支链药物偶联物或其药学上可接受的盐,其为:
化合物a、化合物b、化合物c、化合物d的每个支链分别结合一分子或两分子盐酸的盐酸盐以及每个支链分别结合一分子或两分子醋酸的醋酸盐。
10.如权利要求1-9任一所述的多支链药物偶联物在制备用于治疗CD44高表达肿瘤的药物中的应用。
11.如权利要求10所述的多支链药物偶联物在制备用于治疗胃癌、胰腺癌、小细胞肺癌、结肠癌、乳腺癌、肺腺癌、肝癌、鼻咽癌、恶性胶质瘤、淋巴瘤、肾癌、卵巢癌、头颈癌、鳞状细胞癌的应用。
12.一种药学上可接受的组合物,其包含权利要求1-9所述的多支链药物偶联物或其药学上可接受的盐,以及药学上可接受的赋形剂。
13.如权利要求12所述的组合物在制备用于治疗CD44高表达肿瘤的药物中的应用。
14.如权利要求13所述的组合物在制备用于治疗胃癌、胰腺癌、小细胞肺癌、结肠癌、乳腺癌、肺腺癌、肝癌、鼻咽癌、恶性胶质瘤、淋巴瘤、肾癌、卵巢癌、头颈癌、鳞状细胞癌的应用。
Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910131331.1A CN111603567A (zh) | 2019-02-22 | 2019-02-22 | Cd44靶向多臂偶联物 |
EP20760024.8A EP3928797A4 (en) | 2019-02-22 | 2020-02-17 | CD44-TARGETED MULTI-ARM CONJUGATE |
CA3126574A CA3126574A1 (en) | 2019-02-22 | 2020-02-17 | Cd44 targeted multi-arm conjugate |
KR1020217023336A KR20210107083A (ko) | 2019-02-22 | 2020-02-17 | Cd44 표적화된 멀티 아암 콘쥬게이트 |
US17/431,541 US20220096644A1 (en) | 2019-02-22 | 2020-02-17 | Cd44 targeted multi-arm conjugate |
PCT/CN2020/075578 WO2020169004A1 (zh) | 2019-02-22 | 2020-02-17 | Cd44靶向多臂偶联物 |
AU2020226475A AU2020226475B2 (en) | 2019-02-22 | 2020-02-17 | CD44 targeted multi-arm conjugate |
JP2021549200A JP7353378B2 (ja) | 2019-02-22 | 2020-02-17 | Cd44標的化マルチアームコンジュゲート |
TW109105629A TWI771652B (zh) | 2019-02-22 | 2020-02-21 | Cd44靶向多臂偶聯物 |
TW110128501A TWI792468B (zh) | 2019-02-22 | 2020-02-21 | Cd44靶向多臂偶聯物 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910131331.1A CN111603567A (zh) | 2019-02-22 | 2019-02-22 | Cd44靶向多臂偶联物 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111603567A true CN111603567A (zh) | 2020-09-01 |
Family
ID=72143902
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910131331.1A Pending CN111603567A (zh) | 2019-02-22 | 2019-02-22 | Cd44靶向多臂偶联物 |
Country Status (9)
Country | Link |
---|---|
US (1) | US20220096644A1 (zh) |
EP (1) | EP3928797A4 (zh) |
JP (1) | JP7353378B2 (zh) |
KR (1) | KR20210107083A (zh) |
CN (1) | CN111603567A (zh) |
AU (1) | AU2020226475B2 (zh) |
CA (1) | CA3126574A1 (zh) |
TW (2) | TWI792468B (zh) |
WO (1) | WO2020169004A1 (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20210072002A (ko) | 2018-09-17 | 2021-06-16 | 더 칠드런스 호스피탈 오브 필라델피아 | 폴리머 기반 거대분자 프로드러그 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015196944A1 (zh) * | 2014-06-26 | 2015-12-30 | 中国人民解放军军事医学科学院毒物药物研究所 | GnRH类似物-细胞毒分子缀合物、其制备方法及用途 |
CN108727583A (zh) * | 2017-04-21 | 2018-11-02 | 博瑞生物医药(苏州)股份有限公司 | 多臂靶向抗癌偶联物 |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5994309A (en) | 1997-07-25 | 1999-11-30 | Angstrom Pharmaceuticals, Inc. | Anti-invasive and anti-angiogenic compositions and methods |
US7744861B2 (en) | 2003-09-17 | 2010-06-29 | Nektar Therapeutics | Multi-arm polymer prodrugs |
GB0611405D0 (en) * | 2006-06-09 | 2006-07-19 | Univ Belfast | FKBP-L: A novel inhibitor of angiogenesis |
AU2009282413B2 (en) | 2008-08-11 | 2014-07-17 | Nektar Therapeutics | Multi-arm polymeric alkanoate conjugates |
JP2012519710A (ja) * | 2009-03-06 | 2012-08-30 | アングストロム プハルマセウトイカルス インコーポレイテッド | 細胞遊走を調整するための組成物、及び方法 |
WO2011063156A2 (en) | 2009-11-18 | 2011-05-26 | Nektar Therapeutics | Acid salt forms of polymer-drug conjugates and alkoxylation methods |
EP2542253A4 (en) * | 2010-03-05 | 2013-08-21 | Angstrom Pharmaceuticals Inc | MODULATION OF AN INTERCELLULAR SIGNALING |
EP3272766B1 (en) * | 2011-04-29 | 2019-02-20 | KCI Licensing, Inc. | Aptamer-modified polymeric materials for for use as a foam in a wound dressing |
US20150079626A1 (en) * | 2013-03-19 | 2015-03-19 | Lsip, Llc | Method of obtaining a cell population containing cancer stem cells |
US9636419B2 (en) * | 2013-10-11 | 2017-05-02 | The Universit of Kansas | Targeting multiple receptors on a cell surface for specific cell targeting |
CN104906076B (zh) * | 2015-07-03 | 2017-11-03 | 四川大学 | 程序化多重靶向的树状大分子组装体药物递送***及其制备方法和应用 |
JP6761993B2 (ja) * | 2016-01-29 | 2020-09-30 | 公立大学法人大阪 | プロテインキナーゼ阻害剤 |
WO2018192550A1 (zh) * | 2017-04-21 | 2018-10-25 | 博瑞生物医药(苏州)股份有限公司 | 多臂靶向抗癌偶联物 |
-
2019
- 2019-02-22 CN CN201910131331.1A patent/CN111603567A/zh active Pending
-
2020
- 2020-02-17 KR KR1020217023336A patent/KR20210107083A/ko not_active Application Discontinuation
- 2020-02-17 US US17/431,541 patent/US20220096644A1/en active Pending
- 2020-02-17 JP JP2021549200A patent/JP7353378B2/ja active Active
- 2020-02-17 CA CA3126574A patent/CA3126574A1/en active Granted
- 2020-02-17 AU AU2020226475A patent/AU2020226475B2/en active Active
- 2020-02-17 EP EP20760024.8A patent/EP3928797A4/en active Pending
- 2020-02-17 WO PCT/CN2020/075578 patent/WO2020169004A1/zh unknown
- 2020-02-21 TW TW110128501A patent/TWI792468B/zh active
- 2020-02-21 TW TW109105629A patent/TWI771652B/zh active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015196944A1 (zh) * | 2014-06-26 | 2015-12-30 | 中国人民解放军军事医学科学院毒物药物研究所 | GnRH类似物-细胞毒分子缀合物、其制备方法及用途 |
CN108727583A (zh) * | 2017-04-21 | 2018-11-02 | 博瑞生物医药(苏州)股份有限公司 | 多臂靶向抗癌偶联物 |
Also Published As
Publication number | Publication date |
---|---|
TW202142263A (zh) | 2021-11-16 |
JP2022521529A (ja) | 2022-04-08 |
US20220096644A1 (en) | 2022-03-31 |
AU2020226475B2 (en) | 2023-02-16 |
JP7353378B2 (ja) | 2023-09-29 |
EP3928797A4 (en) | 2022-04-13 |
TW202031296A (zh) | 2020-09-01 |
AU2020226475A1 (en) | 2021-08-05 |
TWI792468B (zh) | 2023-02-11 |
KR20210107083A (ko) | 2021-08-31 |
CA3126574A1 (en) | 2020-08-27 |
EP3928797A1 (en) | 2021-12-29 |
WO2020169004A1 (zh) | 2020-08-27 |
TWI771652B (zh) | 2022-07-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6880073B2 (ja) | マルチアーム重合標的抗がんコンジュゲート | |
JP2010526091A5 (zh) | ||
KR102279429B1 (ko) | 멀티 암 표적 항암 콘쥬게이트 | |
CN110746490B (zh) | 一种基于点击反应阻断免疫检查点的多肽组合物及其制备方法和应用 | |
CN108727583B (zh) | 多臂靶向抗癌偶联物 | |
CN107854693B (zh) | 整合素受体靶向的抗癌偶联物 | |
CN111603567A (zh) | Cd44靶向多臂偶联物 | |
CN109776787B (zh) | 多臂靶向偶联物 | |
CN106632613B (zh) | 与柯萨奇病毒腺病毒受体相关的亲和肽 | |
CN108727582B (zh) | 靶向抗癌偶联物 | |
CN109771658B (zh) | 靶向多臂偶联物 | |
CA3126574C (en) | Cd44 targeted multi-arm conjugate | |
CN108727584B (zh) | 抗癌偶联物 | |
CN109776788B (zh) | 叶酸受体靶向多臂偶联物 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
TA01 | Transfer of patent application right |
Effective date of registration: 20211022 Address after: 330115 room 111-42, unit 1, building 1, Xinghai Haoting, No. 666, Xinghai Hubei Road, Sanghai Economic Development Zone, Ganjiang new area, Nanchang City, Jiangxi Province Applicant after: Ganjiang New Area Borui innovative medicine Co.,Ltd. Address before: 215123 building C25, nanotechnology Park, 218 Xinghu street, Suzhou Industrial Park, Jiangsu Province Applicant before: BRIGHTGENE BIO-MEDICAL TECHNOLOGY Co.,Ltd. |
|
TA01 | Transfer of patent application right | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |