CN104231069A - Protein-polymer combination and preparation method thereof - Google Patents
Protein-polymer combination and preparation method thereof Download PDFInfo
- Publication number
- CN104231069A CN104231069A CN201410478802.3A CN201410478802A CN104231069A CN 104231069 A CN104231069 A CN 104231069A CN 201410478802 A CN201410478802 A CN 201410478802A CN 104231069 A CN104231069 A CN 104231069A
- Authority
- CN
- China
- Prior art keywords
- protein
- ifn
- polymer
- initiator
- polymer combination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229920000642 polymer Polymers 0.000 title claims abstract description 90
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 60
- 239000003999 initiator Substances 0.000 claims abstract description 57
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 53
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 50
- 102000014150 Interferons Human genes 0.000 claims abstract description 40
- 108010050904 Interferons Proteins 0.000 claims abstract description 40
- 229940079322 interferon Drugs 0.000 claims abstract description 40
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 6
- 229920002946 poly[2-(methacryloxy)ethyl phosphorylcholine] polymer Polymers 0.000 claims description 71
- 239000000178 monomer Substances 0.000 claims description 32
- 230000000694 effects Effects 0.000 claims description 27
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 24
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 14
- -1 2-ethyl Chemical group 0.000 claims description 13
- 125000004429 atom Chemical group 0.000 claims description 10
- 238000010526 radical polymerization reaction Methods 0.000 claims description 10
- 230000007704 transition Effects 0.000 claims description 10
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 9
- 238000013467 fragmentation Methods 0.000 claims description 9
- 238000006062 fragmentation reaction Methods 0.000 claims description 9
- 238000007152 ring opening metathesis polymerisation reaction Methods 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 9
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims description 8
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 230000002441 reversible effect Effects 0.000 claims description 8
- 150000001413 amino acids Chemical class 0.000 claims description 7
- 230000009471 action Effects 0.000 claims description 6
- 125000000217 alkyl group Chemical group 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 239000003054 catalyst Substances 0.000 claims description 6
- 238000006555 catalytic reaction Methods 0.000 claims description 6
- 238000013016 damping Methods 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 125000000524 functional group Chemical group 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 238000012711 chain transfer polymerization Methods 0.000 claims description 5
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 claims description 4
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 claims description 4
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 claims description 4
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 claims description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 4
- 230000008878 coupling Effects 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000003446 ligand Substances 0.000 claims description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
- 239000001294 propane Substances 0.000 claims description 4
- 238000007142 ring opening reaction Methods 0.000 claims description 4
- JHPBZFOKBAGZBL-UHFFFAOYSA-N (3-hydroxy-2,2,4-trimethylpentyl) 2-methylprop-2-enoate Chemical compound CC(C)C(O)C(C)(C)COC(=O)C(C)=C JHPBZFOKBAGZBL-UHFFFAOYSA-N 0.000 claims description 3
- DSSYKIVIOFKYAU-XCBNKYQSSA-N (R)-camphor Chemical compound C1C[C@@]2(C)C(=O)C[C@@H]1C2(C)C DSSYKIVIOFKYAU-XCBNKYQSSA-N 0.000 claims description 3
- 101800000224 Glucagon-like peptide 1 Proteins 0.000 claims description 3
- 108090000467 Interferon-beta Proteins 0.000 claims description 3
- 102000003996 Interferon-beta Human genes 0.000 claims description 3
- 108010074328 Interferon-gamma Proteins 0.000 claims description 3
- 102000008070 Interferon-gamma Human genes 0.000 claims description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 3
- 239000012298 atmosphere Substances 0.000 claims description 3
- 230000027455 binding Effects 0.000 claims description 3
- 229960000846 camphor Drugs 0.000 claims description 3
- 229940006607 hirudin Drugs 0.000 claims description 3
- 229960003130 interferon gamma Drugs 0.000 claims description 3
- 229960001388 interferon-beta Drugs 0.000 claims description 3
- 150000003254 radicals Chemical class 0.000 claims description 3
- 238000011160 research Methods 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 3
- XRUKRHLZDVJJSX-UHFFFAOYSA-N 4-cyanopentanoic acid Chemical compound N#CC(C)CCC(O)=O XRUKRHLZDVJJSX-UHFFFAOYSA-N 0.000 claims description 2
- RBRGWYHSVYKUQT-UHFFFAOYSA-N 5-oxabicyclo[2.2.1]hept-2-ene Chemical compound C1C2COC1C=C2 RBRGWYHSVYKUQT-UHFFFAOYSA-N 0.000 claims description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims description 2
- 235000016068 Berberis vulgaris Nutrition 0.000 claims description 2
- 241000335053 Beta vulgaris Species 0.000 claims description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 2
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 claims description 2
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims description 2
- 206010021143 Hypoxia Diseases 0.000 claims description 2
- 125000002009 alkene group Chemical group 0.000 claims description 2
- 150000001409 amidines Chemical class 0.000 claims description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 2
- 229960003237 betaine Drugs 0.000 claims description 2
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 2
- 229920000140 heteropolymer Polymers 0.000 claims description 2
- 229920001519 homopolymer Polymers 0.000 claims description 2
- 208000018875 hypoxemia Diseases 0.000 claims description 2
- 239000011261 inert gas Substances 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 claims description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 238000002715 modification method Methods 0.000 claims description 2
- OORPSDUNNZYIIB-UHFFFAOYSA-N n-(2-aminoethyl)-2-bromo-2-methylpropanamide Chemical compound CC(C)(Br)C(=O)NCCN OORPSDUNNZYIIB-UHFFFAOYSA-N 0.000 claims description 2
- DSQJGAPSYKWYQP-UHFFFAOYSA-N n-(2-aminoethyl)-2-chloro-2-methylpropanamide Chemical compound CC(C)(Cl)C(=O)NCCN DSQJGAPSYKWYQP-UHFFFAOYSA-N 0.000 claims description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 2
- 125000005574 norbornylene group Chemical group 0.000 claims description 2
- 229920000768 polyamine Polymers 0.000 claims description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- BDHFUVZGWQCTTF-UHFFFAOYSA-N sulfonic acid Chemical compound OS(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-N 0.000 claims description 2
- 229920001897 terpolymer Polymers 0.000 claims description 2
- 102000006992 Interferon-alpha Human genes 0.000 claims 1
- 108010047761 Interferon-alpha Proteins 0.000 claims 1
- 102100040918 Pro-glucagon Human genes 0.000 claims 1
- 238000001727 in vivo Methods 0.000 abstract description 14
- 230000000259 anti-tumor effect Effects 0.000 abstract description 12
- 230000009466 transformation Effects 0.000 abstract description 12
- 238000009826 distribution Methods 0.000 abstract description 10
- 229940079593 drug Drugs 0.000 abstract description 4
- 238000011065 in-situ storage Methods 0.000 abstract description 4
- 230000003389 potentiating effect Effects 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 2
- 230000000144 pharmacologic effect Effects 0.000 abstract description 2
- 230000004952 protein activity Effects 0.000 abstract 2
- 125000001246 bromo group Chemical group Br* 0.000 description 36
- 235000018102 proteins Nutrition 0.000 description 34
- 206010028980 Neoplasm Diseases 0.000 description 33
- 102100040019 Interferon alpha-1/13 Human genes 0.000 description 29
- 238000011580 nude mouse model Methods 0.000 description 24
- 241000699660 Mus musculus Species 0.000 description 20
- 238000010560 atom transfer radical polymerization reaction Methods 0.000 description 20
- 238000004458 analytical method Methods 0.000 description 19
- 239000000523 sample Substances 0.000 description 18
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 18
- 238000012360 testing method Methods 0.000 description 17
- 108090000279 Peptidyltransferases Proteins 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 13
- 108010078049 Interferon alpha-2 Proteins 0.000 description 12
- 238000002296 dynamic light scattering Methods 0.000 description 12
- 238000005227 gel permeation chromatography Methods 0.000 description 12
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 10
- 230000037396 body weight Effects 0.000 description 10
- 238000000746 purification Methods 0.000 description 10
- 238000005481 NMR spectroscopy Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 230000036571 hydration Effects 0.000 description 8
- 238000006703 hydration reaction Methods 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 238000012453 sprague-dawley rat model Methods 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- 238000001042 affinity chromatography Methods 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 150000002460 imidazoles Chemical class 0.000 description 5
- 229910052759 nickel Inorganic materials 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 239000003643 water by type Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 238000002983 circular dichroism Methods 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000011541 reaction mixture Substances 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 206010033128 Ovarian cancer Diseases 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- 238000005349 anion exchange Methods 0.000 description 3
- 238000001142 circular dichroism spectrum Methods 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000008030 elimination Effects 0.000 description 3
- 238000003379 elimination reaction Methods 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000005760 tumorsuppression Effects 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 2
- 102400000322 Glucagon-like peptide 1 Human genes 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- 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 2
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 230000001028 anti-proliverative effect Effects 0.000 description 2
- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000013622 capto Q Substances 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000036470 plasma concentration Effects 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 238000010926 purge Methods 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 238000011552 rat model Methods 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Natural products 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
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 210000000689 upper leg Anatomy 0.000 description 2
- YBARNBUFPMPLHH-UHFFFAOYSA-N 2-bromo-2-methylpentanoic acid Chemical compound CCCC(C)(Br)C(O)=O YBARNBUFPMPLHH-UHFFFAOYSA-N 0.000 description 1
- XBBVURRQGJPTHH-UHFFFAOYSA-N 2-hydroxyacetic acid;2-hydroxypropanoic acid Chemical compound OCC(O)=O.CC(O)C(O)=O XBBVURRQGJPTHH-UHFFFAOYSA-N 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- 101800001415 Bri23 peptide Proteins 0.000 description 1
- 101800000655 C-terminal peptide Proteins 0.000 description 1
- 102400000107 C-terminal peptide Human genes 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 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
- 238000002965 ELISA Methods 0.000 description 1
- 206010019799 Hepatitis viral 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
- 239000012741 Laemmli sample buffer Substances 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100498071 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cys-17 gene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000002902 bimodal effect Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940045803 cuprous chloride Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000009513 drug distribution Methods 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000007773 growth pattern Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000010550 living polymerization reaction Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 238000000074 matrix-assisted laser desorption--ionisation tandem time-of-flight detection Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- YHHSONZFOIEMCP-UHFFFAOYSA-O phosphocholine Chemical compound C[N+](C)(C)CCOP(O)(O)=O YHHSONZFOIEMCP-UHFFFAOYSA-O 0.000 description 1
- 229950004354 phosphorylcholine Drugs 0.000 description 1
- 238000013379 physicochemical characterization Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000012207 quantitative assay Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/57—IFN-gamma
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/565—IFN-beta
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses protein-polymer combination and a preparation method thereof. The polymer is combined to the protein through an initiator connected to the protein, and the initiator can be connected to the N- end or the C- end of the protein and any position which is far away from the protein activity site and/or where the protein activity is not influenced. The interferon-polymer combination which is prepared with the method and has the specific site not only reserves bioactivity in vitro better, but also greatly improves the half-life period, bio-distribution and anti-tumor effect of interferon in vivo, so that firm technological base of is established for clinical transformation of potent interferon; and besides, the in-situ fixed-point polymerization method can further be widely applied to other protein or small peptide drugs to improve the pharmacological characteristics.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to protein-polymer combination and preparation method thereof.
Background technology
Protein (such as antibody) has been widely used in multiple fields such as biological medicine development, targeted therapy and clinical diagnosis.Be used alone protein and there is the problems such as the transformation period is short, poorly soluble.Protein is connected with polymer and prepares protein-polymer combination, effectively can improve the solvability of protein, stability, pharmacokinetics and therapeutic efficiency and reduce its immunogenicity.The synthetic method of traditional protein-polymer combination is connected with protein by previously prepared good polymer, often also exists that conjugation sites is uncertain, efficiency is low, productive rate is poor, product is difficult to be separated, quality control is poor, activity is difficult to many difficult problems such as maintenance.Therefore, in the urgent need to designing a kind of general method, effectively to solve an above-mentioned difficult problem.
For solving the problem, proposing some protein modified modifying method, such as, designing Atrigel, as prepared liposome and microballoon, Albumin fusion, transgenation is introduced the insensitive amino acid of proteolytic enzyme etc., but all there are some problems.Liposome encapsulation is low, prominent release and discharge not exclusively produce untoward reaction, and at the protein denaturation inactivation that the strong physics of preparation process, chemical transformation cause.In addition, Albumin fusion need be expressed in eucaryon system, and productive rate is low, and cost is high.Introduce special acid and may reduce protein-active, and the emptying problem of kidney cannot be solved.
Interferon-' alpha ' 2 (IFN-α 2) is the potent inhibitor of virus replication and growth of tumour cell, has been employed successfully in disease such as treatment viral hepatitis and cancer etc.But IFN is very short through the systemic injection administration Posterior circle transformation period, needs frequent drug administration and just can reach the curative effect of expection in higher concentrations, thus causing some toxic side effect, bringing heavy economical load to patient simultaneously.Modify IFN with polyoxyethylene glycol (PEG), be the effective measure improved its pharmacokinetics and improve its curative effect, be called as long-acting interferon.But there is as low in reaction yield, binding site and the drawback such as conjugation chemistry metering is difficult to control, biological activity seriously reduces in current PEG-IFN.
Summary of the invention
In order to improve stability and the efficiency of protein and polymer coupling in protein-polymer combination, promote the separation of product, the control of quality and the maintenance of protein active, the invention provides a kind of method preparing protein-polymer combination, wherein, described polymer is attached on described protein by being connected to the initiator of described protein, wherein, described protein is selected from medicine, agricultural, the albumen that scientific research and other industrial circle are correlated with, little peptide and antibody, preferred granulocyte colony-stimulating factor, glucagon-like-peptide-1 and analogue thereof or r-hirudin.
Described polymer is connected to N-or the C-end of described protein and the site of other any activity away from described protein and/or does not disturb the site of activity of described protein.Such as, utilize the unique unpaired halfcystine Cys17 of granulocyte colony-stimulating factor with maleimide base group initiator specific reaction, by living polymerization growth in situ polymer.Introduce halfcystine or non-natural amino acid and initiator specific binding by genetic engineering means at glucagon-like peptide-1 analogs C end and grow polymer.
According to an embodiment, described method can comprise: a1) prepare the combination of protein-initiator; B1) described protein-initiator combination mixes with high polymer monomer in damping fluid, causes described high molecular monomer polymerization and prepare described protein-polymer combination under catalyst action.
According to another embodiment, described method can comprise: a2) described initiator causes the polymerization of described high polymer monomer under catalyst action, generates described polymer; B2) described polymer is by described initiator and described albumen coupling, prepares described protein-polymer combination.
According to an embodiment, by being selected from transgenation, introducing the binding site of the initiator that alpha-non-natural amino acid, redox, enzyme catalysis, the chemistry of orthogonal chemistry or biological modification method are introduced on described protein.
According to an embodiment, described polymer is generated by polyreaction by monomer, and described polyreaction can be selected from atom transition free radical polymerization reaction (ATRP), reversible addion-fragmentation chain transfer polymerization (RAFT), ring opening metathesis polymerization (ROMP), ring opening polyaddition and their combination.
According to an embodiment, the catalyzer causing described atom transition free radical polymerization reaction is selected from cupric ion, second bipyridine and derivative ligand thereof, π receptor derivative part, nitrogen-atoms chelating ligand and fat polyamine class part.The catalyzer causing described reversible addion-fragmentation chain transfer polyreaction is selected from 4,4 '-azo (4-cyanopentanoic acid), 2,2 '-azo [2-(2-tetrahydroglyoxaline-2-base) propane] dihydrochloride, 2,2 '-azo [2-(2-tetrahydroglyoxaline-2-y1) propane]-anhydrous pyrosulphate, 2, the water miscible radical initiator of 2 '-azo (2-ethyl third amidine) dihydrochloride.The catalyzer causing the polymerization of described ring opening metathesis is selected from water miscible Grubbs catalyzer.
According to an embodiment, described polyreaction can be carried out under hypoxemia or atmosphere of inert gases.The reaction times of described polyreaction can be 5 minutes to 24 hours, particularly, and 5,15,30,45,60 minutes, or 1,2,3,4,5,6,8,10,12,16,24 hour etc.The temperature of reaction of described polyreaction can be 0 ~ 80 DEG C, 0,4,10,15,20,25,30,35,40,50,60,70,80 DEG C etc. particularly.According to an embodiment, described initiator is selected from the initiator of atom transition free radical polymerization reaction, the initiator of reversible addion-fragmentation chain transfer polymerization, the initiator of ring opening metathesis polymerization and the initiator of ring opening polyaddition.The initiator of described atom transition free radical polymerization reaction can be 2-(2-(2-(3,4-bis-bromo maleimide-N-oxyethyl group) oxyethyl group) oxyethyl group) ethyl 2-bromo-2 Methylpropionic acid ester.The initiator of described atom transition free radical polymerization reaction can containing the functional group being selected from N-(2-aminoethyl)-2-bromo-2-methyl propanamide, N-(2-aminoethyl)-2-chloro-2-methyl propanamide, 2-bromo-N-(2-(2-diazanyl kharophen) ethyl)-2-methyl propanamide, 2-chloro-N-(2-(2-diazanyl kharophen) ethyl)-2-methyl propanamide; The initiator of described reversible addion-fragmentation chain transfer polymerization can contain functional group ZC (=S) SR, and wherein R group can be halfcystine, hydrazine, azanol, and Z group is selected from phenyl, alkyl, phthalimidomethyl; The initiator of described ring opening metathesis polymerization can contain A-B type functional group, and wherein A is halfcystine, hydrazine, azanol, and B is alkene.
According to an embodiment, described polymer is generated by polyreaction by monomer, and described monomer can be selected from least one in lactic acid, Epicholorohydrin, acrylate, methacrylic ester, acrylamide, Methacrylamide, norbornylene and oxanorbornene.
Described monomer has any one structure represented of chemical formula 1 to 4:
Wherein, the R group in chemical formula 1 ~ 4 be selected from alkyl, phenyl, benzyl, carboxylic beet base, sulphonic acid betaine base, oligomeric ethylene glycol, polyoxyethylene glycol,
preferably, described alkyl is selected from methyl, ethyl, propyl group, sec.-propyl, the tertiary butyl.
Described monomer can comprise two kinds of reactive groups, and these two kinds of reactive groups react each other and form described polymer.Described monomer can comprise one or more reactive group be embedded into when polymerization reaction take place in the skeleton of polymkeric substance further.Described monomer can be water-soluble or Biodegradable polymeric monomer.
Polymer in described protein-polymer combination can comprise homopolymer, many heteropolymers, block polymer, multipolymer, terpolymer.
Described high molecular side chain can comprise betaine side chain, carboxyl betaine side chain, sulfuryl betaine side chain, oligomeric ethylene glycol side chain, side-chain of polyelycol.
Described polymer can be POEGMA or PMPC etc.
Another aspect of the present invention provides a kind of protein-polymer combination prepared by aforesaid method.
Described protein can be Interferon, rabbit, described Interferon, rabbit optional self-interference element α, interferon beta, interferon-gamma, interferon lambda.Described initiator is connected to N-or the C-end of described Interferon, rabbit and other is away from interferon activity site and/or any position not affecting interferon activity.Preferably, described initiator can be connected to the C-end of described Interferon, rabbit.The present invention carries out modifying and original position efficient growth goes out site-specific and mono-modified Interferon, rabbit-polymer combination drastically increases the stability of Interferon, rabbit, pharmacokinetics, bio distribution and antitumor efficacy at the C-end of Interferon, rabbit by utilizing biological chemistry and macromolecular chemical technology.
The invention provides a kind of method of synthesizing potent Interferon, rabbit.By at the avtive spot away from IFN as C-or N-end, introduce halfcystine and alpha-non-natural amino acid etc. go out POEGMA (methoxypolyethylene glycol methacrylic ester) and PMPC (poly-2-methacryloxyethyl Phosphorylcholine) by atom transfer radical polymerization (ATRP) technology original position efficient growth by genetically engineered, produce site-specific and the protein of equivalent-polymer combination (i.e. IFN-POEGMA and IFN-PMPC).This site-specific Interferon, rabbit-polymer combination not only remains Bioactivity preferably, and significantly improve Interferon, rabbit transformation period in vivo, bio distribution and antitumous effect, thus lay solid technical foundation for potent Interferon, rabbit to clinical conversion.In addition, this original position fixed point polymerization also can be widely used in other albumen or little peptide medicine to improve its pharmacological characteristic.
Accompanying drawing explanation
Fig. 1 shows the schematic diagram of Interferon, rabbit-polymer combination synthetic route.
Fig. 2 shows the IFN-LPETGGH of structure
6plasmid schematic diagram.
Fig. 3 shows the expression and purification of IFN-LPETGGH6 and transpeptidase A.
Fig. 4 shows the structural formula of AEBM.
Fig. 5 shows ATRP initiator is connected to IFN C-end by transpeptidase A catalysis.
Fig. 6 shows IFN-Br synthesis and purge process analysis.
Fig. 7 shows AEBM specificity and modifies IFN-LPTEGGH6.
Fig. 8 shows ATRP method synthesis IFN-POEGMA schematic diagram.
Fig. 9 shows GPC and SDS-PAGE and analyzes IFN-POEGMA and IFN-PMPC.
Figure 10 shows and passes through
1h NMR analyzes IFN-POEGMA combination.
Figure 11 shows the controlled trial analyzed ATRP and synthesize IFN-POEGMA.
Figure 12 shows PEG typical curve.
Figure 13 shows the hydration radius that DLS analyzes IFN-POEGMA and IFN-PMPC.
Figure 14 shows the secondary structure of circular dichroism stratographic analysis IFN-Br, IFN-POEGMA and IFN-PMPC.
Figure 15 shows the Bioactivity that mtt assay measures IFN-POEGMA and IFN-PMPC.
Figure 16 shows the Plasma Concentration of IFN-POEGMA and IFN-PMPC in SD rat body over time.
Figure 17 shows the distribution situation of IFN-POEGMA and IFN-PMPC in tumour.
Figure 18 shows IFN-POEGMA and IFN-PMPC Tumor suppression growing state.
Figure 19 shows nude mice body weight situation over time after injectable drug.
Embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated, but the present invention is not limited to following embodiment.Described method is ordinary method if no special instructions, and described reactant all can obtain from open commercial sources if no special instructions.
The invention provides a kind of method preparing protein-polymer combination, wherein, described polymer is attached on described protein by being connected to the initiator of described protein, wherein, described protein is selected from medicine, agricultural, scientific research and other industrial circle relevant albumen, little peptide and antibody, such as granulocyte colony-stimulating factor, glucagon-like-peptide-1 and analogue thereof or r-hirudin.
According to an embodiment, described method can comprise: a1) prepare the combination of protein-initiator; B1) described protein-initiator combination mixes with high polymer monomer in damping fluid, causes described high molecular monomer polymerization and prepare described protein-polymer combination under catalyst action.
According to another embodiment, described method can comprise: a2) described initiator causes the polymerization of described high polymer monomer under catalyst action, generates described polymer; B2) described polymer is by described initiator and described albumen coupling, prepares described protein-polymer combination.
Described protein can be Interferon, rabbit, described Interferon, rabbit optional self-interference element α, interferon beta, interferon-gamma, interferon lambda.Described initiator is connected to N-or the C-end of described Interferon, rabbit and other is away from interferon activity site and/or any position not affecting interferon activity.Preferably, described initiator can be connected to the C-end of described Interferon, rabbit.Particularly, as shown in Figure 1, method concrete steps of the present invention are: (1) builds the plasmid of the sequence containing IFN, transpeptidase A transpeptidase recognition sequence LPETG and His6 tag fusion protein by recombination engineering, then by molecular cloning method by plasmid transfection to expression in escherichia coli fusion rotein, obtained the subject fusion proteins IFN-LPETGGH6 of purifying by the protein purification method such as affinity chromatography; (2) utilize transpeptidase A while excision Histidine purification tag, ATRP initiator is connected to the C-end of IFN-α 2, uses the technology separation purifying such as ion exchange chromatography to obtain IFN-Br; (3) in aqueous phase system, ATRP technology is adopted to go out water-soluble polymer POEGMA and PMPC at the C-end growth in situ of IFN-α 2.
In an embodiment, utilize gel permeation chromatography (GPC), the analysis means such as dynamic light scattering (DLS) characterize the physical and chemical performance such as molecular weight, hydration radius of IFN-POEGMA and IFN-PMPC combination; Select rational clone, test the Bioactivity of IFN-α 2, IFN-POEGMA and IFN-PMPC, be i.e. the ability of its extracorporeal anti-tumor cell proliferation; Use rat model, test I FN-α 2, IFN-POEGMA and IFN-PMPC pharmacokinetics in vivo; And set up nude mouse tumor model, the antitumous effect of test I FN-α 2, IFN-POEGMA and IFN-PMPC and drug distribution.
Detection means involved in the present invention is as follows:
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) carries out initial analysis to sample.
Gel permeation chromatography (GPC).Gpc analysis uses high performance liquid chromatography (HPLC) analytical system of Waters company.Use UV-detector (Waters 2489) to be 280nm, use chromatographic column to be used in conjunction or GS-520HQ (being connected with guard column) for GS-520HQ and GS-320HQ.Moving phase is 50mM TrisHCl (150mM NaCl, pH=7.4), and temperature is 25 DEG C, and flow velocity is 0.5mL/min.
Use dynamic light scattering (DLS) working sample particle diameter.DLS test uses Malvern Zetasizer Nano-zs90.Sample before testing will by the filter membrane of 0.22 μm, and the concentration of sample is 15 μMs.Data processing uses software Zetasizer software 6.32.
Use the nuclear magnetic resonance analyser of Japanese JEOL company working sample at 25 DEG C
1h NMR.Sample, after lyophilize, is dissolved in D
2o, JEOL ECX-400 400MHz nuclear magnetic resonance spectrometer is analyzed.
Measure protein concentration by bicinchoninic acid method (BCA), the bovine serum albumin of concentration known is standard.
MTT method is adopted to measure antiproliferation.
Adopt male Sprague Dawley (SD) rat test drug metabolism kinetics, utilize DAS 3.0 pharmacokinetic analyses computed in software to go out pharmacokinetic parameter.
Set up nude mice model test Interferon, rabbit-polymer combination anti-tumor in vivo activity and bio distribution.
Embodiment 1: build IFN-LPETGGH6 fusion rotein plasmid and express transpeptidase Sortase A-H6 plasmid and at expression in escherichia coli.
IFN-LPETGGH6 sequences Design following (sequence 1 see sequence table):
GSGGGGS
LP ETGGHHHHHH
Wherein, italic is IFN sequence, and LPETGGH6 indicates underscore and connects IFN by GSGGGGS.This gene order is synthesized by giving birth to work biotechnology (Shanghai, China) and inserts
in carrier.Utilize round pcr, from
increase in carrier IFN-LPETGGH6 encoding sequence, and be inserted into (as shown in Figure 2) in pET-25b (+) carrier by Nde I and Eco RI restriction enzyme site, primer is as follows:
Upstream primer (sequence 2 see sequence table):
5’TTCCCCCATATGTGTGATCTGCCTCAGACTCATT?3’
Downstream primer (sequence 3 see sequence table):
5’TTCCCCGAATTCTTATCAATGATGATGATGATGGTGGCCACC?3’
Express in intestinal bacteria (Rosetta-gami (DE3) pLysS, Novagen) subsequently.Before large-scale expression, be first seeded in (containing 100 μ g/mL penbritins) in 50mL TB substratum by transforming the mono-clonal bacterium obtained, 37 DEG C, shake overnight incubation under 250rpm condition.The fresh TB substratum of 1L central (be contained in the shaking flask of 2L, ampicillin concentration is 100 μ g/mL) of transferring again for second day carries out large scale culturing and abduction delivering.Concrete steps are as follows: first at 37 DEG C, and under 200rpm condition, 5h is cultivated in concussion, and subsequently culture temperature is set to 18 DEG C, adds isopropyl-β-D-thiogalactoside(IPTG) (IPTG), final concentration is 0.4mM, collects thalline after cultivating 16h.
Transpeptidase Sortase A-H6 sequence (sequence 4 see sequence table) is designed to:
GSS
HHHHHHSSG
Wherein, italic is Sortase A-H6 sequence, is synthesized by raw work biotechnology (Shanghai, China) and is inserted
in carrier.Same method, utilize round pcr amplification Sortase A-H6 encoding gene, be inserted in pET-25b (+) carrier by Nde I and Eco RI restriction enzyme site, verify the gene order of target protein by sequenator and express in E.coli BL21 (DE3) pLysS (Novagen).
Embodiment 2: by nickel affinity chromatography column purification IFN-LPETGGH
6albumen
1L E. coli broth is collected in centrifugal bottle, collects thalline with 3000 × g centrifugal force, remove culture supernatants.With the resuspended thalline of the ice-cold PBS of 30mL, then use ultrasonic apparatus smudge cells under 4 DEG C of conditions, then by intestinal bacteria breakdown products at 4 DEG C, under 14000 × g centrifugal force centrifugal 15 minutes.Collect supernatant liquor in add 2mL polymine (PEI, 10%), recentrifuge 15 minutes, object be removing cell pyrolysis liquid in nucleic acid and other electronegative materials.The supernatant liquor obtained is after 0.45 μm of membrane filtration, at AKTA protein purification system (AKTA Purifier 10, GE) purify through nickel affinity chromatography post (HisTrap HP 5mL) on, level pad is 10mM PBS, 500mM NaCl, 5% glycerine, 10mM imidazoles, elutriant is that above-mentioned solution adds 500mM imidazoles.The albumen that wash-out obtains removes imidazoles through desalting column (HiPrep 26/10 Desalting), replaces in 50mM TrisHCl damping fluid simultaneously.Purification of samples sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) tests purity, and determines the concentration of albumen with spectrophotometer method (NanoDrop 2000).SDS-PAGE analytic sample is prepared by the Laemmli sample buffer containing 5% beta-mercaptoethanol, concentration is 1mg/mL, after heating 5min at 95 DEG C, 10 μ L samples are loaded in prefabricated 10%SDS-PAGE gel, run 90min (electrophoresis liquid is: 25mM Tris, 250mM Glycine, 0.1%SDS) under vertical electrophoresis 80 ~ 100V voltage.Pillar location is observed after gel Coomassie brilliant blue G-250 dyeing process.
Fig. 3 shows the expression and purification of IFN-LPETGGH6 and transpeptidase A, and wherein, A is that SDS-PAGE analyzes IFN-LPETGGH6 protein expression and nickel affinity chromatography column purification situation, and B is that SDS-PAGE analyzes transpeptidase A protein expression and ni-sepharose purification situation.Cell pyrolysis liquid before being followed successively by loading from left to right, nickel post stream wears liquid, 50mM imidazoles washes post, 500mM imidazoles wash-out target protein and standard protein sample.The IFN-α 2 and the transpeptidase A that express, nickel affinity chromatography column purification obtains purity > 95% is carried out by intestinal bacteria.
Embodiment 3: introduce ATRP initiator at the C-end of IFN by transpeptidase A enzyme catalysis
Containing IFN-LPETGGH
65mM 2-(2-(2-(2-glycyl amido) acetamido) acetamido) ethyl 2 bromo 2 methyl propionic acid ester (initiator A EBM as shown in Figure 4) is added, and containing 100 μMs of transpeptidase A and 20mM CaCl in the 50mM TrisHCl solution (10mL) of (200 μMs)
2trisHCl solution (10mL) mixing, room temperature reaction spends the night.Fig. 5 shows ATRP initiator is connected to IFN C-end by transpeptidase A catalysis, and in Fig. 5, the halfcystine (Cys) of transpeptidase A is attacked as nucleophilic group, acts on IFN-LPETGGH
6peptide bond between the Threonine of upper recognition sequence LPETG and glycine, makes it disconnect (acylation), and then produces an acyl enzyme intermediate, the subsequently amino nucleophillic attack acyl enzyme of triglycine, AEBM covalent attachment on IFN-α 2.React rear anion-exchange chromatography (HiTrap Capto Q 5mL) purifying to obtain IFN-Br (level pad is 20mM TrisHCl, pH 7.5; Elution buffer is that above-mentioned solution adds 1M NaCl), and remove small molecular weight impurity with PBS further through desalting column.
Utilize transpeptidase A transpeptidation to obtain IFN-Br and use anion-exchange column (AEX) purifying.Reaction process and purification effect SDS-PAGE have carried out check analysis.Fig. 6 shows IFN-Br synthesis and purge process analysis, and wherein, A:AEX is separated the mixture of transpeptidase A catalysis IFN-LPETGGH6 and AEBM reaction, and UV monitors linear elution and obtains target product IFN-Br (75 ~ 100mL); B:SDS-PAGE analyzes being separated when reaction mixture before and after IFN-LPETGGH6 and AEBM reaction.From left to right be followed successively by SrtA-H6, IFN-LPETGGH6, reaction mixture, eluted protein IFN-Br, stream wears sample SrtA-H6 and standard protein sample.
The molecular weight of purified product measures with MALDI _ TOFMS (MALDI-TOF), and instrument is 4800Plus MALDI-TOF/TOF
tManalyser (AB SCIEX).With High Performance Liquid Chromatography-Electrospray Ionization Tandem Mass method (LC-MS/ESI) analysis verification IFN-LPETGGH
6modify with the aminoacid sequence of IFN-Br and the specificity of C-end, instrument is Q-Exactive LC-MS mass spectrograph (Thermo Scientific), and table 1 shows the rear peptide section aminoacid sequence composition of IFN-Br trypsinase decomposition that LC-MS/ESI analyzes.Fig. 7 shows AEBM specificity and modifies IFN-LPTEGGH6, and wherein, A:MALDI-TOF analyzes IFN-LPTEGGH6 and IFN-Br.The C-terminal peptide fragment EGSGGGGSLPETGGG (-Br) isotopic distribution of B:IFN-Br, left figure is LC-MS/ESI experimental value; Right figure is theoretical expectation values.Fig. 7 and table 1 shows that site-directed quantitative obtains macromole evocating agent IFN-Br.
Table 1
Embodiment 4: synthesize POEGMA and PMPC at the C-end original position ATRP of IFN
Contain at 2.5mL in the PBS solution of 40 μMs of IFN-Br and add a certain amount of OEGMA and MPC (for obtaining corresponding POEGMA and PMPC, the mol ratio of monomer and initiator is respectively 500 and 1000), with 110 μ L vitamin c solutions (40mg/mL), after logical nitrogen 15min, add the cuprous chloride/N of 10 μ L, N, N ', N ", N "-PMDETA (PMDTA) (concentration is respectively 100mM and 300mM) mixture liquid storage, 1h is reacted under air tight condition, then termination reaction is in atmosphere exposed, Fig. 8 shows ATRP method synthesis IFN-POEGMA schematic diagram.Reaction mixture goes out IFN-POEGMA through anion-exchange column (HiTrap Capto Q column, GE Healthcare) separation and purification, and follows the tracks of ATRP process and the composition of analytical reaction product and refining effect with SDS-PAGE and GPC.
Fig. 9 shows GPC and SDS-PAGE and analyzes IFN-POEGMA and IFN-PMPC, wherein, A:GPC and SDS-PAGE analyzes IFN-POEGMA, left figure is gpc analysis IFN-POEGMA, right figure is that SDS-PAGE analysis of anions exchanges purifying IFN-POEGMA, is respectively from left to right: obtain unreacted IFN-Br before standard protein sample, IFN-POEGMA purifying, after IFN-POEGMA purifying, after separation; B:GPC and SDS-PAGE analyzes IFN-PMPC, left figure is gpc analysis IFN-PMPC, right figure is that SDS-PAGE analysis of anions exchanges purifying IFN-PMPC, be followed successively by from left to right, before standard protein sample, IFN-Br, IFN-PMPC purifying, after IFN-PMPC purifying, after separation, obtain unreacted IFN-Br.Result shows that anion exchange methods effectively can be separated IFN-POEGMA and IFN-PMPC combination from ATRP reaction mixture.
In order to verify that POEGMA synthesizes at the C-end of IFN-Br, being connected on IFN-α 2 with the triglycine not containing initiator, then having carried out controlled trial by above-mentioned identical condition.Figure 11 shows the controlled trial analyzed ATRP and synthesize IFN-POEGMA, and wherein, A:GPC analyzes IFN-Br and synthesizes IFN-POEGMA and IFN-GGG controlled trial.B:SDS-PAGE analyzes, and is followed successively by standard protein sample, IFN-LPETGGH6, IFN-Br, IFN-POEGMA and IFN-GGG controlled trial from left to right.Controlled trial adopts the reaction conditions identical with IFN-Br to be used for carrying out ATRP reaction to IFN-GGG, display IFN-GGG does not grow POEGMA, illustrate that this polyreaction is only carried out at the C-end of the IFN-Br being connected to ATRP initiator, and there is not the side reaction on other reactive groups of albumen.
The Physico-Chemical Characterization of embodiment 5:IFN-POEGMA and IFN-PMPC
The molecular weight of IFN-POEGMA and IFN-PMPC and polydispersity coefficient (PDI) GPC measure, and instrument is that Waters HPLC/GPC system connects UV detector (Waters 2489) and differential refraction detector (Waters 2414).Chromatographic column is Asahipak GS-520HQ and GS-320HQ or GS-520HQ series connection, moving phase is 50mM TrisHCl damping fluid (pH 7.4), testing conditions is 25 DEG C, and the typical curve that the narrow ditribution PEG standard of flow velocity 0.5mL/min. different molecular weight generates is used for calculating molecular weight and PDI.
The IFN-POEGMA combination (being designated as IFN-POEGMA 500 and IFN-POEGMA 1000 respectively) that monomers/initiator ratio 500 and 1000 has synthesized two kinds of different molecular weights is corresponded in the present invention.Be used to generate molecular weight and the PDI that typical curve calculates IFN-POEGMA and IFN-PMPC combination by the PEG standard substance of known molecular amount.Figure 12 shows PEG typical curve.The molecular weight of corresponding IFN-POEGMA 500, IFN-POEGMA 1000 and IFN-PMPC is respectively 60.0kDa, 112.6kDa and 67.0kDa, and PDI is respectively 1.28,1.33 and 1.30.Also prove can by the molecular weight of regulation and control monomers/initiator ratio control protein-polymer combination simultaneously.
Malvern Zetasizer Nano-zs90 measures by dynamic light scattering (DLS) method the hydration radius of IFN-POEGMA and IFN-PMPC.Diluted sample is in PBS damping fluid, and test is front through 0.22 μm of aperture membrane filtration process.Through DLS test, the hydration radius of IFN-Br is 2.4nm, and the IFN-POEGMA500 of synthesis and the hydration radius of 1000 combinations and IFN-PMPC are respectively 10.6nm, 15.2nm and 16.3nm.Figure 13 shows the hydration radius that DLS analyzes IFN-POEGMA and IFN-PMPC, and wherein, A:DLS analyzes the hydration radius of IFN-POEGMA.B:DLS analyzes the hydration radius of IFN-PMPC.
With nuclear magnetic resonance analyser (
1h NMR) profiling protein matter-high molecular chemical structure.IFN-POEGMA and IFN-PMPC sample, after lyophilize, is dissolved in D2O, and JEOL ECX-400400MHz nuclear magnetic resonance spectrometer is analyzed.Pass through
1h NMR spectrogram confirms and protein molecular successfully synthesizes POEGMA and PMPC.Figure 10 shows and passes through
1h NMR analyzes IFN-POEGMA combination.
The secondary structure circular dichroism spectrum analysis of IFN-POEGMA and IFN-PMPC records.Samples with water solution dilution, to 0.18mg/mL (1.2 μMs), carries out UV scanning analysis with Pistar π-180 (Applied Photophysics company limited) in 200-250nm wavelength region.Figure 14 shows and the secondary structure of the stratographic analysis of opinion circular dichroism IFN-Br, IFN-POEGMA and IFN-PMPC, wherein, A:IFN-Br and IFN-POEGMA circular dichroism spectrogram, the circular dichroism spectrogram of B:IFN-Br and IFN-PMPC.Circular dichroism spectrum analysis has been done to IFN-Br and IFN-POEGMA and IFN-PMPC combination, circular dichroism spectrum all in 200-260nm wavelength region all presents same 209/219nm bimodal curve, and good with IFN-Br curves overlapped, show that on protein, grow the secondary structure of original position polymer to protein molecule does not have a significant effect.
Measure the protein concentration in IFN-POEGMA and IFN-PMPC by bicinchoninic acid method (BCA), the bovine serum albumin of concentration known is standard, and concrete steps to specifications.
The Bioactivity of embodiment 6:IFN-POEGMA and IFN-PMPC is surveyed
In the present invention, the antiproliferation of IFN-POEGMA and IFN-PMPC adopts MTT method to measure.We have selected people Burkitt ' s B lymphoma cell (Daudi B), because this cell has higher sensitivity to IFN-α 2.After Daudi B cell cultivates for some time in the RMPI-1640 containing 15%FBS, 50U/mL penicillin and 50 μ g/mL Streptomycin sulphates, in 96 orifice plates, inoculate certain density cell suspending liquid (50 μ L/ holes, 10
4individual cell), by IFN-α 2, IFN-POEGMA or IFN-PMPC combination sample series dilution after IFN-Br reference substance and purifying, each 50 μ L add in 96 well culture plates, if negative control (not containing IFN-α 2) and blank (only containing nutrient solution), 37 DEG C, 5%CO
2cultivate 72 ~ 96h, add MTT lysate 20 μ L/ hole, after 3h, measure the absorption value of each hole 490nm wavelength by microplate reader, the degree of cell proliferation after more different sample preparation.Figure 15 shows the Bioactivity that MTT measures IFN-POEGMA and IFN-PMPC, and wherein, A:MTT measures the Bioactivity of IFN-POEGMA500 and IFN-POEGMA1000, and B:MTT measures the Bioactivity of IFN-PMPC.Table 2 shows the Bioactivity of IFN-Br and IFN-POEGMA.In summary, the IC of IFN-POEGMA500, IFN-POEGMA1000, IFN-PMPC and IFN-Br
50be respectively 21.9pg/mL, 44.7pg/mL and 18.3pg/mL, active conservation rate is respectively 42%, 21% and 51%, far away higher than the active conservation rate (< 10%) of the PEGization IFN reported in other document.Figure 15 and table 2 shows that original position ATRP does not seriously reduce the activity of IFN, for anti-tumor in vivo active testing provides foundation.
Table 2
| Sample | IC 50(pg/mL) | Relative reactivity (%) |
| IFN | 9.4 | 100 |
| IFN-POEGMA?500 | 21.9 | 42 |
| IFN-POEGMA?1000 | 44.7 | 21 |
| IFN-PMPC | 18.3 | 51 |
The pharmacokinetics test of embodiment 7:IFN-POEGMA and IFN-PMPC
All experimentation on animalies all complete under Tsing-Hua University instructs about zooperal every regulation below.Utilize SD rat model in the present invention after tail vein injection IFN, IFN-POEGMA or IFN-PMPC, determine the situation of Interferon, rabbit concentration changes with time in blood, and utilize DAS software to carry out data analysis.Before the drug treating phase, 12 8 week age body weight be, after male Sprague Dawley (SD) rat of about 250g observes for some time, be divided into 3 groups at random.With 125 μ g/kg body weight dose tail vein injection IFN-POEGMA or IFN-PMPC and unmodified IFN-Br contrast, then setting time point isoflurane rat is anaesthetized after through intraocular corner of the eyes venous blood sampling 0.3-0.4mL, room temperature leaves standstill 1h, 4 DEG C, collected by centrifugation upper serum under 3000 × g, be stored in-80 DEG C of cryogenic refrigerators.With humanIFN-α 2ELISA test kit (PBL interferon source) according to IFN-α 2 content in specification sheets mensuration serum.DAS 3.0 pharmacokinetic analyses computed in software is utilized to go out pharmacokinetic parameter.Two chambers in DAS software are utilized to eliminate the pharmacokinetic parameter of model analysis IFN-POEGMA and IFN, the IFN initial transformation period (t1/2 α) eliminates the transformation period (t1/2 β) and is respectively 0.11h and 2.15h, after administration several minutes, in blood, namely the concentration of Interferon, rabbit quickly fall to 0.1% of the not enough starting point concentration of residual concentration after less than 50%, 24h of predose.And IFN-POEGMA concentration in vivo reduces gradually, its initial transformation period and elimination transformation period are respectively 2.15h and 45.3h, are about 20 times of IFN.After administration 72h, still have more than 10% residual.The area under the drug-time curve (AUC0-∞) of IFN-POEGMA is 37 times of IFN.A compartment model in DAS software is utilized to analyze IFN-PMPC pharmacokinetic parameter, IFN-PMPC concentration in vivo reduces gradually, it eliminates the transformation period is 25.1h, it is 73 times that IFN eliminates the transformation period, in its body, average retention time (MRT0-∞) and area under the drug-time curve (AUC0-∞) are 14 times and 48 times of IFN respectively, and elimination factor is decline more obvious than IFN also.Until after administration 72h, IFN-PMPC still have more than 10% residual.Above result shows, compared with the IFN of unmodified, in the elimination transformation period of IFN-POEGMA and IFN-PMPC combination and body, average residence time obviously extends, and Drug-time curve area significantly increases, and clearance rate significantly reduces.Table 3 and table 4 respectively illustrate the pharmacokinetic data analysis of IFN-POEGMA and IFN-PMPC in SD rat body.
Table 3
| Parameter | IFN | IFN-POEGMA |
| t 1/2α(h) | 0.11±0.02 | 2.15±1.91 |
| t 1/2β(h) | 2.22±0.74 | 45.4±7.3 |
| AUC 0-t(10 6pg/mL·h) | 0.83±0.42 | 25.0±1.3 |
| AUC 0-∞(10 6pg/mL·h) | 0.98±0.51 | 35.9±2.8 |
| CL 1(L/h/kg) | 0.268±0.114 | 0.004±0.000 |
| CL 2(L/h/kg) | 0.300±0.061 | 0.052±0.060 |
| V 1(L/kg) | 0.192±0.154 | 0.151±0.015 |
| V 2(L/kg) | 0.409±0.103 | 0.114±0.022 |
| K 10 | 1.83 | 0.028 |
| K 12 | 3.47 | 0.367 |
| K 21 | 1.55 | 0.528 |
Table 4
| Parameter | IFN | IFN-PMPC |
| t 1/2(h) | 0.35±0.10 | 25.1±9.9 |
| AUC 0-∞(10 6pg/mL·h) | 0.76±0.20 | 36.0±12.6 |
| MRT 0-∞(h) | 2.67±0.60 | 38.0±12.6 |
| CL(L/h/kg) | 0.233±0.130 | 0.165±0.012 |
| V(L/kg) | 0.128±0.108 | 5.97±2.41 |
| K e | 2.10 | 0.030 |
Embodiment 8:IFN-POEGMA and IFN-PMPC tumour distribution tests
The concentration of the Interferon, rabbit remained in tumour after utilizing the nude mice having transplanted ovarian cancer cell to determine administration 24h in the present invention.9 female athymic (Nude) nude mices are divided into 3 groups, IFN-POEGMA group, IFN-PMPC group and IFN-Br control group, Proliferation of Human Ovarian Cell (OVCAR-3) is containing 10%FBS, after cultivating for some time in the RMPI-1640 substratum of 50U/mL penicillin and 50 μ g/mL Streptomycin sulphates, peel off with tryptic digestion, wash through PBS, be resuspended in not containing above-mentioned additive RMPI-1640 substratum and with BD Matrigel Matrix equal-volume mixture, 0.2mL single cell suspension (5 × 10
6individual cell) be inoculated in nude mice left hind femur place dorsal sc, cultivate and form 300mm after 30 days
3the solid tumor lump of size.IFN-POEGMA, IFN-PMPC, IFN-Br squeeze in nude mouse with tail vein injection, and dosage is 10 μ g/20g body weight.Put to death nude mice after administration 24h, collect blood and tumour.(PBS is containing 1mM EDTA with Extraction buffer for tumour, 0.5%Triton X-100,0.5% Sodium desoxycholate, 1mM PMSF, protease inhibitor cocktail and inhibitors of phosphatases mixture (Sigma-Aldrich) by 1: 100 dilution proportion) after fragmentation, centrifuging and taking supernatant extracting solution.The concentration ELISA method quantitative assay of the IFN in serum and tumor extraction.
Figure 16 shows the Plasma Concentration of IFN-POEGMA and IFN-PMPC in SD rat body over time.After injected sample 24h, in IFN-POEGMA and IFN-PMPC group tumour, the concentration of Interferon, rabbit is 152 and 157 times of distributed density in IFN group tumour.The Interferon, rabbit that this result sufficient proof POEGMA and PMPC combines effectively can utilize and strengthens penetrating and be detained (EPR) effect, enable albumen in blood circulating half-life more assembled in tumour while extending, thus raising Interferon, rabbit bioavailability in vivo and antitumor efficacy.
Embodiment 9: nude mice model test I FN-POEGMA and IFN-PMPC anti-tumor in vivo activity
The in vivo bioactivity of IFN-POEGMA and IFN-PMPC measures with animal transplanting tumor laboratory method.As stated above, OVCAR-3 cell is inoculated in nude mice left hind femur place dorsal sc, cultivates and forms solid tumor lump afterwards in 4 ~ 6 days, thus set up nude mouse tumor model.32 nude mices are divided into 4 groups, (IFN-Br is positive control for IFN-POEGMA or IFN-PMPC and reference substance, physiological saline is negative control) squeeze in nude mouse with tail vein injection, dosage is 300 μ g/20g body weight, observe nude mice survival condition and tumor growth situation weekly, dynamic measurement nude mice body weight and gross tumor volume are over time.
Evaluate the anti-tumor in vivo activity of IFN-POEGMA and IFN-PMPC combination at nude mice by subcutaneous tumor model with ovarian cancer cell in the present invention.Figure 17 shows the distribution situation of IFN-POEGMA and IFN-PMPC in tumour, wherein, and the distribution situation of A:IFN-POEGMA in tumour.The distribution situation of B:IFN-PMPC in tumour.Figure 18 shows IFN-POEGMA and IFN-PMPC Tumor suppression growing state, wherein, and A: tumor size is growth pattern in time.Left figure is IFN-POEGMA group, and right figure is IFN-PMPC group; B: administration after 60 days in body tumour pictorial diagram.Upper figure is IFN-POEGMA group, and figure below is IFN-PMPC group.
Inoculate ovarian cancer cell after 4 days, to nude mice through tail intravenous single dosage injecting normal saline, 300 μ g IFN, IFN-POEGMA or IFN-PMPC respectively.In upon administration two weeks, tumor size by inject tumour cell time size reduce gradually, subsequently, the tumour of physiological saline and IFN two groups of nude mices starts to increase gradually, and during to 60 days, mean tumour volume all grows to 500mm
3above.On the contrary, the tumour of IFN-POEGMA and IFN-PMPC group nude mice fades away, and namely gross tumor volume contrasted with other two groups from 36 days exists notable difference (p < 0.05), until after 60 days, 6 nude mices all do not find tumour.This illustrates, single dose injection, can not effectively Tumor suppression growth in conjunction with high molecular Interferon, rabbit.On the contrary, IFN-POEGMA and the IFN-PMPC combination of ATRP fixed point fabricated in situ can restrained effectively the growth of tumour, has extraordinary anti-tumor in vivo active.In the present invention, the concentration of IFN-POEGMA and IFN-PMPC group in tumour is concentration 37 times and 152 times in the IFN group tumour of unmodified respectively.IFN-POEGMA and IFN-PMPC combination effectively can utilize and strengthens penetrating and be detained (EPR) effect, enable albumen in blood circulating half-life assembled in tumour more while extending, thus improve Interferon, rabbit bioavailability in vivo and antitumor efficacy.
Figure 19 shows nude mice body weight situation over time after injectable drug, wherein A is nude mice body weight situation over time after injection IFN-POEGMA, B is nude mice body weight situation over time after injection IFN-PMPC, in Figure 19, nude mice body weight within the test period slightly increases, and shows that IFN-POEGMA and IFN-PMPC does not have obvious side effect.
Interferon, rabbit-polymer combination prepared by method of the present invention has higher biological activity storage rate, the transformation period of significant prolongation and effective tumor inhibition effect, show more excellent effect compared with the In vitro and in vivo activity of PEGization IFN-α 2 in bibliographical information.Therefore, ATRP synthetic technology be expected to replace PEG change into for modified protein medicine improving medicine stability, improve the novel method of pharmacokinetics and enhancing therapeutic efficiency.
Claims (23)
1. prepare the method for protein-polymer combination for one kind, wherein, described polymer is attached on described protein by being connected to the initiator of described protein, wherein, described polymer is connected to N-or the C-end of described protein and the site of other any activity away from described protein and/or does not disturb the site of activity of described protein, described protein is selected from medicine, agricultural, scientific research and other industrial circle relevant albumen, little peptide and antibody, preferred granulocyte colony-stimulating factor, glucagon-like-peptide-1 and analogue thereof or r-hirudin.
2. prepare protein-polymer combination method as claimed in claim 1, wherein, described protein is Interferon, rabbit, and described Interferon, rabbit is selected from interferon alpha, interferon beta, interferon-gamma, interferon lambda.
3. prepare the method for protein-polymer combination as claimed in claim 2, wherein, described initiator is connected to the C-end of described Interferon, rabbit.
4. the method preparing protein-polymer combination as described in any one of claims 1 to 3, described method comprises:
A1) combination of protein-initiator is prepared;
B1) described protein-initiator combination mixes with high polymer monomer in damping fluid, causes the polymerization of described high polymer monomer and prepare described protein-polymer combination under catalyst action.
5. the method preparing protein-polymer combination as described in any one of claims 1 to 3, described method comprises:
A2) described initiator causes described high molecular monomer polymerization under catalyst action, generates described polymer;
B2) described polymer is by described initiator and described albumen coupling, prepares described protein-polymer combination.
6. prepare the method for protein-polymer combination as claimed in claim 1, wherein, the binding site of described initiator by being selected from transgenation, introduce alpha-non-natural amino acid, redox, enzyme catalysis, the chemistry of orthogonal chemistry or biological modification method and introduce on described protein.
7. prepare the method for protein-polymer combination as claimed in claim 1, wherein, described polymer is generated by polyreaction by monomer, and described polyreaction is selected from the one in atom transition free radical polymerization reaction, reversible addion-fragmentation chain transfer polymerization, ring opening metathesis polymerization, ring opening polyaddition.
8. prepare the method for protein-polymer combination as claimed in claim 7, wherein, the catalyzer causing described atom transition free radical polymerization reaction is selected from cupric ion, second bipyridine and derivative ligand thereof, π receptor derivative part, nitrogen-atoms chelating ligand and fat polyamine class part.
9. prepare the method for protein-polymer combination as claimed in claim 7, wherein, the catalyzer causing described reversible addion-fragmentation chain transfer polyreaction is selected from water miscible radical initiator, preferably, described water miscible radical initiator is selected from 4,4 '-azo (4-cyanopentanoic acid), 2,2 '-azo [2-(2-tetrahydroglyoxaline-2-base) propane] dihydrochloride, 2,2 '-azo [2-(2-tetrahydroglyoxaline-2-base) propane]-anhydrous pyrosulphate, 2, at least one of 2 '-azo (2-ethyl third amidine) dihydrochloride.
10. prepare the method for protein-polymer combination as claimed in claim 7, wherein, the catalyzer causing the polymerization of described ring opening metathesis is selected from water miscible Grubbs catalyzer.
11. methods preparing protein-polymer combination as claimed in claim 7, wherein, described polyreaction is carried out under hypoxemia or atmosphere of inert gases, and the reaction times is 5 minutes to 24 hours, and temperature of reaction is 0 ~ 80 DEG C.
12. methods preparing protein-polymer combination as claimed in claim 1, wherein, described initiator is selected from the initiator of atom transition free radical polymerization reaction, the initiator of reversible addion-fragmentation chain transfer polymerization, the initiator of ring opening metathesis polymerization and the initiator of ring opening polyaddition.
13. methods preparing protein-polymer combination as claimed in claim 12, wherein, the initiator of described atom transition free radical polymerization reaction is 2-(2-(2-(3,4-bis-bromo maleimide-N-oxyethyl group) oxyethyl group) oxyethyl group) ethyl 2-bromo-2 Methylpropionic acid ester.
14. methods preparing protein-polymer combination as claimed in claim 12, wherein, the initiator of described atom transition free radical polymerization reaction contains the functional group being selected from N-(2-aminoethyl)-2-bromo-2-methyl propanamide, N-(2-aminoethyl)-2-chloro-2-methyl propanamide, 2-bromo-N-(2-(2-diazanyl kharophen) ethyl)-2-methyl propanamide, 2-chloro-N-(2-(2-diazanyl kharophen) ethyl)-2-methyl propanamide; The initiator of described reversible addion-fragmentation chain transfer polyreaction contains ZC (=S) SR functional group, and wherein R group can be halfcystine, hydrazine, azanol, and Z group is selected from phenyl, alkyl, phthalimidomethyl; The initiator of described ring opening metathesis polymerization contains A-B type functional group, and wherein A is selected from halfcystine, hydrazine or azanol, and B is alkene.
15. methods preparing protein-polymer combination as claimed in claim 1, wherein, described polymer is generated by polyreaction by monomer, and described monomer is selected from least one in lactic acid, Epicholorohydrin, acrylate, methacrylic ester, acrylamide, Methacrylamide, norbornylene and oxanorbornene.
The method of 16. protein-polymer combinations as claimed in claim 1, wherein, described polymer is generated by polyreaction by monomer, and described monomer has any one structure represented of chemical formula 1 to 4:
Wherein, the R group in chemical formula 1 ~ 4 be selected from alkyl, phenyl, benzyl, carboxylic beet base, sulphonic acid betaine base, oligomeric ethylene glycol, polyoxyethylene glycol,
preferably, described alkyl is selected from methyl, ethyl, propyl group, sec.-propyl, the tertiary butyl.
17. methods preparing protein-polymer combination as claimed in claim 1, wherein, the polymer in described protein-polymer combination comprises homopolymer, many heteropolymers, block polymer, multipolymer, terpolymer.
18. methods preparing protein-polymer combination as claimed in claim 1, wherein, described polymer is generated by polyreaction by monomer, and described monomer comprises two kinds of reactive groups, and described two kinds of reactive groups react formation polymer each other.
The method of 19. protein-polymer combinations as claimed in claim 1, wherein, described polymer is generated by polyreaction by monomer, and described monomer comprises one or more reactive group be embedded into when polymerization reaction take place in described high molecular skeleton further.
20. methods preparing protein-polymer combination as claimed in claim 1, wherein, described polymer is generated by polyreaction by monomer, and described monomer is water-soluble or Biodegradable polymeric monomer.
21. methods preparing protein-polymer combination as claimed in claim 1, wherein, described high molecular side chain comprises betaine side chain, carboxyl betaine side chain, sulfuryl betaine side chain, oligomeric ethylene glycol side chain, side-chain of polyelycol.
The method of 22. preparation site-specific protein-polymer combinations as claimed in claim 1, wherein, described polymer is selected from POEGMA or PMPC.
23. 1 kinds of protein-polymer combinations, described protein-polymer combination is prepared by the method described in any one of claim 1 to 22.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410478802.3A CN104231069B (en) | 2014-09-18 | 2014-09-18 | Protein high molecular combination and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410478802.3A CN104231069B (en) | 2014-09-18 | 2014-09-18 | Protein high molecular combination and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104231069A true CN104231069A (en) | 2014-12-24 |
| CN104231069B CN104231069B (en) | 2017-09-22 |
Family
ID=52220054
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410478802.3A Active CN104231069B (en) | 2014-09-18 | 2014-09-18 | Protein high molecular combination and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104231069B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106046384A (en) * | 2016-06-08 | 2016-10-26 | 北京市理化分析测试中心 | Protein grafted copolymer and preparation method thereof |
| WO2017075866A1 (en) * | 2015-11-04 | 2017-05-11 | 清华大学 | Method for preparing interferon-polymer conjugate ifn-poegma |
| CN107226858A (en) * | 2016-03-23 | 2017-10-03 | 清华大学 | Interferon macromolecule combination IFN-PMPC preparation and its application |
| WO2018121129A1 (en) * | 2016-12-30 | 2018-07-05 | 江南大学 | Carbonyl reductase oligomer and use thereof in synthesis of chiral alcohol |
| CN108822267A (en) * | 2018-03-23 | 2018-11-16 | 清华大学 | The preparation and its application of two parent of pH responsiveness albumen macromolecule |
| CN109265512A (en) * | 2018-09-25 | 2019-01-25 | 清华大学 | The preparation method of protein conjugate based on pyridine dicarbaldehyde |
| CN111848720A (en) * | 2020-08-10 | 2020-10-30 | 福州大学 | Method for performing macromolecule functional modification on glycosylated protein |
| CN114685699A (en) * | 2020-12-25 | 2022-07-01 | 江苏百赛飞生物科技有限公司 | Protein initiator and preparation method and application thereof |
| CN116077674A (en) * | 2022-12-26 | 2023-05-09 | 北京大学 | Biomolecule-polynitrogen oxide conjugate, preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8497356B2 (en) * | 2009-02-17 | 2013-07-30 | Duke University | Biomolecule polymer conjugates and methods for making the same |
-
2014
- 2014-09-18 CN CN201410478802.3A patent/CN104231069B/en active Active
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017075866A1 (en) * | 2015-11-04 | 2017-05-11 | 清华大学 | Method for preparing interferon-polymer conjugate ifn-poegma |
| CN106674325A (en) * | 2015-11-04 | 2017-05-17 | 清华大学 | Method of preparing interferon macromolecular combination IFN-POEGMA |
| CN107226858A (en) * | 2016-03-23 | 2017-10-03 | 清华大学 | Interferon macromolecule combination IFN-PMPC preparation and its application |
| CN107226858B (en) * | 2016-03-23 | 2020-12-29 | 清华大学 | Preparation and application of interferon high-molecular conjugate IFN-PMPC |
| CN106046384A (en) * | 2016-06-08 | 2016-10-26 | 北京市理化分析测试中心 | Protein grafted copolymer and preparation method thereof |
| WO2018121129A1 (en) * | 2016-12-30 | 2018-07-05 | 江南大学 | Carbonyl reductase oligomer and use thereof in synthesis of chiral alcohol |
| CN115260412A (en) * | 2018-03-23 | 2022-11-01 | 清华大学 | preparation and application of pH-responsive protein macromolecule amphiphile |
| CN108822267A (en) * | 2018-03-23 | 2018-11-16 | 清华大学 | The preparation and its application of two parent of pH responsiveness albumen macromolecule |
| CN108822267B (en) * | 2018-03-23 | 2022-12-16 | 清华大学 | preparation and application of pH-responsive protein macromolecule amphiphile |
| CN109265512A (en) * | 2018-09-25 | 2019-01-25 | 清华大学 | The preparation method of protein conjugate based on pyridine dicarbaldehyde |
| CN111848720A (en) * | 2020-08-10 | 2020-10-30 | 福州大学 | Method for performing macromolecule functional modification on glycosylated protein |
| CN114685699A (en) * | 2020-12-25 | 2022-07-01 | 江苏百赛飞生物科技有限公司 | Protein initiator and preparation method and application thereof |
| CN116077674A (en) * | 2022-12-26 | 2023-05-09 | 北京大学 | Biomolecule-polynitrogen oxide conjugate, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104231069B (en) | 2017-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104231069A (en) | Protein-polymer combination and preparation method thereof | |
| US20220202949A1 (en) | Ligand-targeted cell conjugate (ltcc)-based anti-tumor immune cell, as well as preparation method and use thereof | |
| CN106632682A (en) | Fusion protein IFN-ELP and application thereof | |
| CN106924752B (en) | Method for preparing protein-polyamino acid conjugate | |
| CN109453187B (en) | Antibody nucleic acid drug conjugate with dual enzyme sensitivity characteristics and preparation method and application thereof | |
| CN108853515B (en) | Preparation method and application of short peptide hydrogel and pharmaceutical composition | |
| KR20190007483A (en) | Multi-cancer Polymer Targeted Anticancer Conjugate | |
| CN109265512B (en) | Preparation method of protein conjugate based on pyridine dicarbaldehyde | |
| KR102279429B1 (en) | Multi-cancer target anti-cancer conjugate | |
| CN101163716B (en) | Interleukin-6 polyethylene glycol conjugate and its preparing method and use | |
| CN101491682A (en) | PEG-IFN omega conjugate and preparation technique thereof | |
| CN107226858B (en) | Preparation and application of interferon high-molecular conjugate IFN-PMPC | |
| CN106674325A (en) | Method of preparing interferon macromolecular combination IFN-POEGMA | |
| CN105085658B (en) | Interleukin 29 mutant and polyethylene glycol derivative | |
| CN108727583A (en) | Multi-arm target anticancer conjugate | |
| WO2014093688A1 (en) | Compositions and methods for functional nucleic acid delivery | |
| TW201002350A (en) | Polyethylenenized erythropoietin conjugate, preparation thereof and its use | |
| CN101172161A (en) | Water-soluble polymers decorated G-CSF conjugates | |
| CN106749608B (en) | Interferon alpha conjugates | |
| WO2021070920A1 (en) | Cyclic peptide | |
| CN110551179B (en) | Modified anti-HIV polypeptide and preparation method and application thereof | |
| CN103012579B (en) | A kind of long-acting human interferon and preparation method thereof | |
| CN112424239A (en) | Polymer compound and intracellular compound introduction promoter using same | |
| WO2024104444A1 (en) | Il-2 mutant and use thereof | |
| CN102949732A (en) | Medicine fusion specifically bound with human angiopoietin-2 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| OL01 | Intention to license declared | ||
| OL01 | Intention to license declared |