TW200819468A - Method for the production of conjugates of insulin-like growth factor I and poly(ethylene glycol) - Google Patents
Method for the production of conjugates of insulin-like growth factor I and poly(ethylene glycol) Download PDFInfo
- Publication number
- TW200819468A TW200819468A TW096132101A TW96132101A TW200819468A TW 200819468 A TW200819468 A TW 200819468A TW 096132101 A TW096132101 A TW 096132101A TW 96132101 A TW96132101 A TW 96132101A TW 200819468 A TW200819468 A TW 200819468A
- Authority
- TW
- Taiwan
- Prior art keywords
- pro
- igf
- variant
- pegylated
- arg
- Prior art date
Links
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 title claims abstract description 113
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 title claims abstract description 112
- 238000000034 method Methods 0.000 title claims abstract description 46
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 12
- 229920001223 polyethylene glycol Polymers 0.000 title claims description 91
- -1 poly(ethylene glycol) Polymers 0.000 title claims description 13
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 54
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 52
- 150000001413 amino acids Chemical class 0.000 claims abstract description 40
- 108010060035 arginylproline Proteins 0.000 claims abstract description 22
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 claims abstract description 21
- 108010077515 glycylproline Proteins 0.000 claims abstract description 20
- KZNQNBZMBZJQJO-YFKPBYRVSA-N glyclproline Chemical compound NCC(=O)N1CCC[C@H]1C(O)=O KZNQNBZMBZJQJO-YFKPBYRVSA-N 0.000 claims abstract description 19
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000004472 Lysine Substances 0.000 claims abstract description 15
- ICTZKEXYDDZZFP-SRVKXCTJSA-N Pro-Arg-Pro Chemical compound N([C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(O)=O)C(=O)[C@@H]1CCCN1 ICTZKEXYDDZZFP-SRVKXCTJSA-N 0.000 claims abstract description 15
- 108010087924 alanylproline Proteins 0.000 claims abstract description 12
- LQJAALCCPOTJGB-YUMQZZPRSA-N Arg-Pro Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O LQJAALCCPOTJGB-YUMQZZPRSA-N 0.000 claims abstract description 11
- FELJDCNGZFDUNR-WDSKDSINSA-N Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 FELJDCNGZFDUNR-WDSKDSINSA-N 0.000 claims abstract description 11
- QOLYAJSZHIJCTO-VQVTYTSYSA-N Thr-Pro Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(O)=O QOLYAJSZHIJCTO-VQVTYTSYSA-N 0.000 claims abstract description 9
- WPWUFUBLGADILS-WDSKDSINSA-N Ala-Pro Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(O)=O WPWUFUBLGADILS-WDSKDSINSA-N 0.000 claims abstract description 7
- RNKSNIBMTUYWSH-YFKPBYRVSA-N L-prolylglycine Chemical compound [O-]C(=O)CNC(=O)[C@@H]1CCC[NH2+]1 RNKSNIBMTUYWSH-YFKPBYRVSA-N 0.000 claims abstract description 7
- GVUVRRPYYDHHGK-VQVTYTSYSA-N Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 GVUVRRPYYDHHGK-VQVTYTSYSA-N 0.000 claims abstract description 7
- 108010029020 prolylglycine Proteins 0.000 claims abstract description 7
- RWCOTTLHDJWHRS-YUMQZZPRSA-N Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 RWCOTTLHDJWHRS-YUMQZZPRSA-N 0.000 claims abstract description 5
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 4
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 4
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 4
- 235000001014 amino acid Nutrition 0.000 claims description 45
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 22
- 238000003776 cleavage reaction Methods 0.000 claims description 21
- 230000007017 scission Effects 0.000 claims description 21
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical group OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 19
- 239000002253 acid Substances 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 13
- 125000000539 amino acid group Chemical group 0.000 claims description 12
- 239000004475 Arginine Substances 0.000 claims description 10
- 150000001412 amines Chemical class 0.000 claims description 10
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 10
- 239000008194 pharmaceutical composition Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 8
- 235000003704 aspartic acid Nutrition 0.000 claims description 8
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 8
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 7
- 201000010099 disease Diseases 0.000 claims description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 7
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 7
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- 229930182817 methionine Natural products 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 5
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 5
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 3
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical group SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 2
- 150000003573 thiols Chemical group 0.000 claims 1
- 108010002231 IgA-specific serine endopeptidase Proteins 0.000 abstract description 17
- 208000024827 Alzheimer disease Diseases 0.000 abstract description 15
- 239000013604 expression vector Substances 0.000 abstract description 4
- 208000015122 neurodegenerative disease Diseases 0.000 abstract description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 83
- 108090000623 proteins and genes Proteins 0.000 description 47
- 102000004169 proteins and genes Human genes 0.000 description 39
- 235000018102 proteins Nutrition 0.000 description 36
- 102220350531 c.80A>G Human genes 0.000 description 27
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- 125000003275 alpha amino acid group Chemical group 0.000 description 21
- 239000003153 chemical reaction reagent Substances 0.000 description 18
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 16
- 102220638482 Protein PML_K68R_mutation Human genes 0.000 description 16
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 14
- 102220638483 Protein PML_K65R_mutation Human genes 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 14
- 230000006320 pegylation Effects 0.000 description 14
- 101001055320 Myxine glutinosa Insulin-like growth factor Proteins 0.000 description 13
- 210000004556 brain Anatomy 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 12
- 108010016616 cysteinylglycine Proteins 0.000 description 12
- 125000005647 linker group Chemical group 0.000 description 12
- 235000018977 lysine Nutrition 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 125000003277 amino group Chemical group 0.000 description 11
- 239000000872 buffer Substances 0.000 description 11
- 239000013598 vector Substances 0.000 description 11
- PQGCEDQWHSBAJP-TXICZTDVSA-N 5-O-phosphono-alpha-D-ribofuranosyl diphosphate Chemical compound O[C@H]1[C@@H](O)[C@@H](O[P@](O)(=O)OP(O)(O)=O)O[C@@H]1COP(O)(O)=O PQGCEDQWHSBAJP-TXICZTDVSA-N 0.000 description 10
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 10
- 101800000628 PDH precursor-related peptide Proteins 0.000 description 10
- OTZMRMHZCMZOJZ-SRVKXCTJSA-N Arg-Leu-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O OTZMRMHZCMZOJZ-SRVKXCTJSA-N 0.000 description 9
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 9
- BIYXEUAFGLTAEM-WUJLRWPWSA-N Thr-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(O)=O BIYXEUAFGLTAEM-WUJLRWPWSA-N 0.000 description 9
- 235000009697 arginine Nutrition 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 108010079547 glutamylmethionine Proteins 0.000 description 9
- 210000003000 inclusion body Anatomy 0.000 description 9
- 239000011541 reaction mixture Substances 0.000 description 9
- 108010007375 seryl-seryl-seryl-arginine Proteins 0.000 description 9
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 9
- 108010061238 threonyl-glycine Proteins 0.000 description 9
- 102000007469 Actins Human genes 0.000 description 8
- 108010085238 Actins Proteins 0.000 description 8
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 8
- QAMMIGULQSIRCD-IRXDYDNUSA-N Gly-Phe-Tyr Chemical compound C([C@H](NC(=O)C[NH3+])C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C([O-])=O)C1=CC=CC=C1 QAMMIGULQSIRCD-IRXDYDNUSA-N 0.000 description 8
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 8
- HXSUFWQYLPKEHF-IHRRRGAJSA-N Phe-Asn-Arg Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N HXSUFWQYLPKEHF-IHRRRGAJSA-N 0.000 description 8
- DXWNFNOPBYAFRM-IHRRRGAJSA-N Phe-Val-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N DXWNFNOPBYAFRM-IHRRRGAJSA-N 0.000 description 8
- 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 7
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 7
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 7
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- YJNDFEWPGLNLNH-IHRRRGAJSA-N Met-Tyr-Cys Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CS)C(O)=O)CC1=CC=C(O)C=C1 YJNDFEWPGLNLNH-IHRRRGAJSA-N 0.000 description 7
- 108091005804 Peptidases Proteins 0.000 description 7
- 239000004365 Protease Substances 0.000 description 7
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 7
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 210000002569 neuron Anatomy 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- BRPMXFSTKXXNHF-IUCAKERBSA-N (2s)-1-[2-[[(2s)-pyrrolidine-2-carbonyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H]1NCCC1 BRPMXFSTKXXNHF-IUCAKERBSA-N 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 6
- 108020004511 Recombinant DNA Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- DBKNLHKEVPZVQC-LPEHRKFASA-N Arg-Ala-Pro Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O DBKNLHKEVPZVQC-LPEHRKFASA-N 0.000 description 5
- CVLIHKBUPSFRQP-WHFBIAKZSA-N Cys-Gly-Ala Chemical compound [H]N[C@@H](CS)C(=O)NCC(=O)N[C@@H](C)C(O)=O CVLIHKBUPSFRQP-WHFBIAKZSA-N 0.000 description 5
- 206010012289 Dementia Diseases 0.000 description 5
- GJBUAAAIZSRCDC-GVXVVHGQSA-N Glu-Leu-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O GJBUAAAIZSRCDC-GVXVVHGQSA-N 0.000 description 5
- YQAQQKPWFOBSMU-WDCWCFNPSA-N Glu-Thr-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O YQAQQKPWFOBSMU-WDCWCFNPSA-N 0.000 description 5
- 206010018612 Gonorrhoea Diseases 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 5
- 108010005233 alanylglutamic acid Proteins 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000000502 dialysis Methods 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 208000001786 gonorrhea Diseases 0.000 description 5
- 108010028295 histidylhistidine Proteins 0.000 description 5
- 108010087846 prolyl-prolyl-glycine Proteins 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 229960002317 succinimide Drugs 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 4
- UDDITVWSXPEAIQ-IHRRRGAJSA-N Cys-Phe-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UDDITVWSXPEAIQ-IHRRRGAJSA-N 0.000 description 4
- MFVQGXGQRIXBPK-WDSKDSINSA-N Gly-Ala-Glu Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O MFVQGXGQRIXBPK-WDSKDSINSA-N 0.000 description 4
- JJGBXTYGTKWGAT-YUMQZZPRSA-N Gly-Pro-Glu Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O JJGBXTYGTKWGAT-YUMQZZPRSA-N 0.000 description 4
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- CGHXMODRYJISSK-NHCYSSNCSA-N Leu-Val-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(O)=O CGHXMODRYJISSK-NHCYSSNCSA-N 0.000 description 4
- BQVJARUIXRXDKN-DCAQKATOSA-N Met-Asn-His Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 BQVJARUIXRXDKN-DCAQKATOSA-N 0.000 description 4
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 4
- DCHQYSOGURGJST-FJXKBIBVSA-N Pro-Thr-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O DCHQYSOGURGJST-FJXKBIBVSA-N 0.000 description 4
- 108010009736 Protein Hydrolysates Proteins 0.000 description 4
- NLQUOHDCLSFABG-GUBZILKMSA-N Ser-Arg-Arg Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NLQUOHDCLSFABG-GUBZILKMSA-N 0.000 description 4
- KNCJWSPMTFFJII-ZLUOBGJFSA-N Ser-Cys-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(O)=O KNCJWSPMTFFJII-ZLUOBGJFSA-N 0.000 description 4
- ODXKUIGEPAGKKV-KATARQTJSA-N Thr-Leu-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)O)N)O ODXKUIGEPAGKKV-KATARQTJSA-N 0.000 description 4
- VLOYGOZDPGYWFO-LAEOZQHASA-N Val-Asp-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O VLOYGOZDPGYWFO-LAEOZQHASA-N 0.000 description 4
- 150000001768 cations Chemical class 0.000 description 4
- 210000003169 central nervous system Anatomy 0.000 description 4
- 108010079413 glycyl-prolyl-glutamic acid Proteins 0.000 description 4
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 230000000750 progressive effect Effects 0.000 description 4
- 238000001542 size-exclusion chromatography Methods 0.000 description 4
- 230000009261 transgenic effect Effects 0.000 description 4
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 3
- 208000037259 Amyloid Plaque Diseases 0.000 description 3
- PBVLJOIPOGUQQP-CIUDSAMLSA-N Asp-Ala-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O PBVLJOIPOGUQQP-CIUDSAMLSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- XABFFGOGKOORCG-CIUDSAMLSA-N Cys-Asp-Leu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O XABFFGOGKOORCG-CIUDSAMLSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- PXXGVUVQWQGGIG-YUMQZZPRSA-N Glu-Gly-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N PXXGVUVQWQGGIG-YUMQZZPRSA-N 0.000 description 3
- HKTRDWYCAUTRRL-YUMQZZPRSA-N Glu-His Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 HKTRDWYCAUTRRL-YUMQZZPRSA-N 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- SOYCWSKCUVDLMC-AVGNSLFASA-N His-Pro-Arg Chemical compound N[C@@H](Cc1cnc[nH]1)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CCCNC(=N)N)C(=O)O SOYCWSKCUVDLMC-AVGNSLFASA-N 0.000 description 3
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- HKXSZKJMDBHOTG-CIUDSAMLSA-N Lys-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CCCCN HKXSZKJMDBHOTG-CIUDSAMLSA-N 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- JEGFCFLCRSJCMA-IHRRRGAJSA-N Phe-Arg-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N JEGFCFLCRSJCMA-IHRRRGAJSA-N 0.000 description 3
- LEIKGVHQTKHOLM-IUCAKERBSA-N Pro-Pro-Gly Chemical compound OC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 LEIKGVHQTKHOLM-IUCAKERBSA-N 0.000 description 3
- 206010036790 Productive cough Diseases 0.000 description 3
- 108010034546 Serratia marcescens nuclease Proteins 0.000 description 3
- JQAWYCUUFIMTHE-WLTAIBSBSA-N Thr-Gly-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JQAWYCUUFIMTHE-WLTAIBSBSA-N 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 108010029539 arginyl-prolyl-proline Proteins 0.000 description 3
- 229910021538 borax Inorganic materials 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000002490 cerebral effect Effects 0.000 description 3
- 108010004073 cysteinylcysteine Proteins 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000006167 equilibration buffer Substances 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 125000003827 glycol group Chemical group 0.000 description 3
- 108010085325 histidylproline Proteins 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 102000028416 insulin-like growth factor binding Human genes 0.000 description 3
- 108091022911 insulin-like growth factor binding Proteins 0.000 description 3
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 229920001427 mPEG Polymers 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 108010014614 prolyl-glycyl-proline Proteins 0.000 description 3
- 108010077112 prolyl-proline Proteins 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 235000010339 sodium tetraborate Nutrition 0.000 description 3
- 208000024794 sputum Diseases 0.000 description 3
- 210000003802 sputum Anatomy 0.000 description 3
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 3
- VZXTWGWHSMCWGA-UHFFFAOYSA-N 1,3,5-triazine-2,4-diamine Chemical compound NC1=NC=NC(N)=N1 VZXTWGWHSMCWGA-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- SDMAQFGBPOJFOM-GUBZILKMSA-N Ala-Arg-Arg Chemical compound NC(=N)NCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SDMAQFGBPOJFOM-GUBZILKMSA-N 0.000 description 2
- TTXMOJWKNRJWQJ-FXQIFTODSA-N Ala-Arg-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N TTXMOJWKNRJWQJ-FXQIFTODSA-N 0.000 description 2
- NINQYGGNRIBFSC-CIUDSAMLSA-N Ala-Lys-Ser Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CO)C(O)=O NINQYGGNRIBFSC-CIUDSAMLSA-N 0.000 description 2
- MAZZQZWCCYJQGZ-GUBZILKMSA-N Ala-Pro-Arg Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MAZZQZWCCYJQGZ-GUBZILKMSA-N 0.000 description 2
- JGDGLDNAQJJGJI-AVGNSLFASA-N Arg-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)N JGDGLDNAQJJGJI-AVGNSLFASA-N 0.000 description 2
- YCYXHLZRUSJITQ-SRVKXCTJSA-N Arg-Pro-Pro Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 YCYXHLZRUSJITQ-SRVKXCTJSA-N 0.000 description 2
- AUIJUTGLPVHIRT-FXQIFTODSA-N Arg-Ser-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N AUIJUTGLPVHIRT-FXQIFTODSA-N 0.000 description 2
- RATOMFTUDRYMKX-ACZMJKKPSA-N Asp-Glu-Cys Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N RATOMFTUDRYMKX-ACZMJKKPSA-N 0.000 description 2
- SCQIQCWLOMOEFP-DCAQKATOSA-N Asp-Leu-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O SCQIQCWLOMOEFP-DCAQKATOSA-N 0.000 description 2
- PKNIZMPLMSKROD-BIIVOSGPSA-N Cys-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CS)N PKNIZMPLMSKROD-BIIVOSGPSA-N 0.000 description 2
- QJUDRFBUWAGUSG-SRVKXCTJSA-N Cys-Cys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)N QJUDRFBUWAGUSG-SRVKXCTJSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- MXPBQDFWIMBACQ-ACZMJKKPSA-N Glu-Cys-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O MXPBQDFWIMBACQ-ACZMJKKPSA-N 0.000 description 2
- 108010053070 Glutathione Disulfide Proteins 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- CSTNMMIHMYJGFR-IHRRRGAJSA-N His-His-Arg Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C1=CN=CN1 CSTNMMIHMYJGFR-IHRRRGAJSA-N 0.000 description 2
- STOOMQFEJUVAKR-KKUMJFAQSA-N His-His-His Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)C1=CNC=N1 STOOMQFEJUVAKR-KKUMJFAQSA-N 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 108010000521 Human Growth Hormone Proteins 0.000 description 2
- 239000000854 Human Growth Hormone Substances 0.000 description 2
- 108010031794 IGF Type 1 Receptor Proteins 0.000 description 2
- 102100039688 Insulin-like growth factor 1 receptor Human genes 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- IBMVEYRWAWIOTN-RWMBFGLXSA-N Leu-Arg-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(O)=O IBMVEYRWAWIOTN-RWMBFGLXSA-N 0.000 description 2
- RTIRBWJPYJYTLO-MELADBBJSA-N Leu-Lys-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N RTIRBWJPYJYTLO-MELADBBJSA-N 0.000 description 2
- BOJYMMBYBNOOGG-DCAQKATOSA-N Lys-Pro-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O BOJYMMBYBNOOGG-DCAQKATOSA-N 0.000 description 2
- RXWPLVRJQNWXRQ-IHRRRGAJSA-N Met-His-His Chemical compound C([C@H](NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)C1=CNC=N1 RXWPLVRJQNWXRQ-IHRRRGAJSA-N 0.000 description 2
- LHXFNWBNRBWMNV-DCAQKATOSA-N Met-Ser-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N LHXFNWBNRBWMNV-DCAQKATOSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 2
- PESQCPHRXOFIPX-UHFFFAOYSA-N N-L-methionyl-L-tyrosine Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- CGBYDGAJHSOGFQ-LPEHRKFASA-N Pro-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 CGBYDGAJHSOGFQ-LPEHRKFASA-N 0.000 description 2
- 241001415846 Procellariidae Species 0.000 description 2
- WBAXJMCUFIXCNI-WDSKDSINSA-N Ser-Pro Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O WBAXJMCUFIXCNI-WDSKDSINSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- OQCXTUQTKQFDCX-HTUGSXCWSA-N Thr-Glu-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N)O OQCXTUQTKQFDCX-HTUGSXCWSA-N 0.000 description 2
- 239000007984 Tris EDTA buffer Substances 0.000 description 2
- AZGZDDNKFFUDEH-QWRGUYRKSA-N Tyr-Gly-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 AZGZDDNKFFUDEH-QWRGUYRKSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 230000008499 blood brain barrier function Effects 0.000 description 2
- 210000001218 blood-brain barrier Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000001054 cortical effect Effects 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 125000006612 decyloxy group Chemical group 0.000 description 2
- 238000001739 density measurement Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011026 diafiltration Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 2
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 208000037906 ischaemic injury Diseases 0.000 description 2
- 150000002669 lysines Chemical class 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000003928 nasal cavity Anatomy 0.000 description 2
- 210000000933 neural crest Anatomy 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 108010012581 phenylalanylglutamate Proteins 0.000 description 2
- 210000002706 plastid Anatomy 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 108010070643 prolylglutamic acid Proteins 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001370 static light scattering Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- GYSCBCSGKXNZRH-UHFFFAOYSA-N 1-benzothiophene-2-carboxamide Chemical compound C1=CC=C2SC(C(=O)N)=CC2=C1 GYSCBCSGKXNZRH-UHFFFAOYSA-N 0.000 description 1
- JBGWMRAMUROVND-UHFFFAOYSA-N 1-sulfanylidenethiophene Chemical compound S=S1C=CC=C1 JBGWMRAMUROVND-UHFFFAOYSA-N 0.000 description 1
- IHWDSEPNZDYMNF-UHFFFAOYSA-N 1H-indol-2-amine Chemical compound C1=CC=C2NC(N)=CC2=C1 IHWDSEPNZDYMNF-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 238000010173 Alzheimer-disease mouse model Methods 0.000 description 1
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 1
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- QAXCZGMLVICQKS-SRVKXCTJSA-N Arg-Glu-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N QAXCZGMLVICQKS-SRVKXCTJSA-N 0.000 description 1
- BSYKSCBTTQKOJG-GUBZILKMSA-N Arg-Pro-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O BSYKSCBTTQKOJG-GUBZILKMSA-N 0.000 description 1
- KXOPYFNQLVUOAQ-FXQIFTODSA-N Arg-Ser-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O KXOPYFNQLVUOAQ-FXQIFTODSA-N 0.000 description 1
- ZTRJUKDEALVRMW-SRVKXCTJSA-N Asn-His-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CC(=O)N)N ZTRJUKDEALVRMW-SRVKXCTJSA-N 0.000 description 1
- GIQCDTKOIPUDSG-GARJFASQSA-N Asn-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)N)N)C(=O)O GIQCDTKOIPUDSG-GARJFASQSA-N 0.000 description 1
- ZUFPUBYQYWCMDB-NUMRIWBASA-N Asn-Thr-Glu Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZUFPUBYQYWCMDB-NUMRIWBASA-N 0.000 description 1
- AXXCUABIFZPKPM-BQBZGAKWSA-N Asp-Arg-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O AXXCUABIFZPKPM-BQBZGAKWSA-N 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000012617 Butyl Sepharose™ 4 Fast Flow Substances 0.000 description 1
- 102000003846 Carbonic anhydrases Human genes 0.000 description 1
- 108090000209 Carbonic anhydrases Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- GHVNFZFCNZKVNT-UHFFFAOYSA-N Decanoic acid Natural products CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 1
- DBAKFASWICGISY-DASCVMRKSA-N Dexchlorpheniramine maleate Chemical group OC(=O)\C=C/C(O)=O.C1([C@H](CCN(C)C)C=2N=CC=CC=2)=CC=C(Cl)C=C1 DBAKFASWICGISY-DASCVMRKSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-N Formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 1
- 201000011240 Frontotemporal dementia Diseases 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 208000003098 Ganglion Cysts Diseases 0.000 description 1
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 1
- XBGGUPMXALFZOT-VIFPVBQESA-N Gly-Tyr Chemical compound NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-VIFPVBQESA-N 0.000 description 1
- HQSKKSLNLSTONK-JTQLQIEISA-N Gly-Tyr-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 HQSKKSLNLSTONK-JTQLQIEISA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- YPLYIXGKCRQZGW-SRVKXCTJSA-N His-Arg-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O YPLYIXGKCRQZGW-SRVKXCTJSA-N 0.000 description 1
- QZAFGJNKLMNDEM-DCAQKATOSA-N His-Asn-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC1=CN=CN1 QZAFGJNKLMNDEM-DCAQKATOSA-N 0.000 description 1
- DQZCEKQPSOBNMJ-NKIYYHGXSA-N His-Thr-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DQZCEKQPSOBNMJ-NKIYYHGXSA-N 0.000 description 1
- 101000823051 Homo sapiens Amyloid-beta precursor protein Proteins 0.000 description 1
- 101000617546 Homo sapiens Presenilin-2 Proteins 0.000 description 1
- 208000023105 Huntington disease Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- 102000048143 Insulin-Like Growth Factor II Human genes 0.000 description 1
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- DEFJQIDDEAULHB-IMJSIDKUSA-N L-alanyl-L-alanine Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(O)=O DEFJQIDDEAULHB-IMJSIDKUSA-N 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- WSXTWLJHTLRFLW-SRVKXCTJSA-N Lys-Ala-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O WSXTWLJHTLRFLW-SRVKXCTJSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000003505 Myosin Human genes 0.000 description 1
- 108060008487 Myosin Proteins 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 1
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 1
- 108091006006 PEGylated Proteins Proteins 0.000 description 1
- FMMIYCMOVGXZIP-AVGNSLFASA-N Phe-Glu-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O FMMIYCMOVGXZIP-AVGNSLFASA-N 0.000 description 1
- FRMKIPSIZSFTTE-HJOGWXRNSA-N Phe-Tyr-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O FRMKIPSIZSFTTE-HJOGWXRNSA-N 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 208000000609 Pick Disease of the Brain Diseases 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- VXCHGLYSIOOZIS-GUBZILKMSA-N Pro-Ala-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 VXCHGLYSIOOZIS-GUBZILKMSA-N 0.000 description 1
- YXHYJEPDKSYPSQ-AVGNSLFASA-N Pro-Leu-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 YXHYJEPDKSYPSQ-AVGNSLFASA-N 0.000 description 1
- MRYUJHGPZQNOAD-IHRRRGAJSA-N Pro-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1 MRYUJHGPZQNOAD-IHRRRGAJSA-N 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- 241000239226 Scorpiones Species 0.000 description 1
- IOVBCLGAJJXOHK-SRVKXCTJSA-N Ser-His-His Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 IOVBCLGAJJXOHK-SRVKXCTJSA-N 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 208000005400 Synovial Cyst Diseases 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
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- FPCIBLUVDNXPJO-XPUUQOCRSA-N Val-Cys-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CS)C(=O)NCC(O)=O FPCIBLUVDNXPJO-XPUUQOCRSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000012382 advanced drug delivery Methods 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000012436 analytical size exclusion chromatography Methods 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000004671 cell-free system Anatomy 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 230000001713 cholinergic effect Effects 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 150000002009 diols Chemical group 0.000 description 1
- 210000005064 dopaminergic neuron Anatomy 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000010436 fluorite Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002518 glial effect Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 108010045126 glycyl-tyrosyl-glycine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 208000007386 hepatic encephalopathy Diseases 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 102000046783 human APP Human genes 0.000 description 1
- 102000044162 human IGF1 Human genes 0.000 description 1
- 102000055037 human PSEN2 Human genes 0.000 description 1
- 125000004356 hydroxy functional group Chemical group O* 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- LFKYBJLFJOOKAE-UHFFFAOYSA-N imidazol-2-ylidenemethanone Chemical compound O=C=C1N=CC=N1 LFKYBJLFJOOKAE-UHFFFAOYSA-N 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 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 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 108010053062 lysyl-arginyl-phenylalanyl-lysine Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 230000007171 neuropathology Effects 0.000 description 1
- 231100000189 neurotoxic Toxicity 0.000 description 1
- 230000002887 neurotoxic effect Effects 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 229910052762 osmium Inorganic materials 0.000 description 1
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- 150000002924 oxiranes Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- WXZMFSXDPGVJKK-UHFFFAOYSA-N pentaerythritol Chemical compound OCC(CO)(CO)CO WXZMFSXDPGVJKK-UHFFFAOYSA-N 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 210000000578 peripheral nerve Anatomy 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 108010025826 prolyl-leucyl-arginine Proteins 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000012070 reactive reagent Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000007151 ring opening polymerisation reaction Methods 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 208000009999 tuberous sclerosis Diseases 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
- 239000002023 wood Substances 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
- C07K14/575—Hormones
- C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Wood Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Diabetes (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Plant Pathology (AREA)
- Psychiatry (AREA)
- Hospice & Palliative Care (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
200819468 九、發明說明: 【發明所屬之技術領域】 本發明係關於用於製造類胰島素生長因子_1( 乙二醇(PEG)之社人物夕古、土人+ 〃、不 p 口物之方法,含有該等結合物之醫藥组 合物,及該等結合物之使用方法。 、、、 【先前技術】
阿茲海默氏病(AD)為日益流行之神經退化形式,其為年 齡65歲以上之人群中約5〇%_6〇%全部癡呆症病例之原因。 據估計其目前在世界範圍内影響15⑽萬人且由於人口中老 年人之相對增加,在以後20至3〇年中其流行率可能增加。 AD為進行性病症,在臨床症狀發作與死亡之間平均持續 時間為約8,5年。與較高心理功能相關之大腦區域中錐體 神經兀之死亡及神經元突觸之減少導致典型症狀,其特徵 在於總體及進行性認知功能障礙(Francis,ρ·τ•等人,L
Neurol. Neurosurg. Psychiatry 66 (1999) 137-147)。AD為 世界上老年癡呆症與初期老年癡呆症之最常見形式且在臨 床上硪別為持續進行性癡呆症,其表現為記憶、智力功能 哀退及語言障礙漸增(Merritt,A Textbook of Neurology, 弟 6版 ’ Lea & Febiger,Philadelphia (1979),第 484-489 頁)。在神經病理學上,AD之主要特點為存在兩種特徵性 病變··澱粉樣老年斑及神經原纖維纏結(NFT)。空斑係在 神經元外沈積,而纏結係在死後大腦中在神經元内觀察到 的。澱粉樣空斑核心之主要組份之一為病理性沈積之小澱 粉樣-β-肽(Αβ),其係由分泌酶自澱粉樣前驅蛋白質(APP) 123528.doc 200819468 歧解而得(Seik〇e,d.J·,Physiol· Rev· 81 (2001) 741-766 · rdy,J.及 Selkoe,D.J·,Science 297 (2002) 353-356 · ush,Α·Ι·及 Tanzi,R.E·,Pr〇c. Natl. Acad. Sci· USA 99 (2⑽2) 7317_7319)。Αβ是一種39-43個殘基之自聚集肽 (MW為約4 kDa),其係作為更大Αρρ(η〇·ΐ2〇 ki^)之部分 而合成。APP是一種具有大N末端細胞外域、單一跨膜域 及短細胞質尾之1型完整膜醣蛋白。Αβ區橫跨APP之部分 細胞外域及跨膜域。ΑΡρ參與AD中神經元細胞死亡之最常 見假說為殿粉樣蛋白假說。此假說假定空斑澱粉樣沈積或 部分聚集之可溶性Αβ引發神經毒性級聯,進而造成類似於 八〇病理之神經退化(Selkoe,D.J·,Physiol. Rev. 81 (2001) 741-766 ; Hardy,J.及 Selkoe,D.J·,Science 297 (2002) 353- 356) 〇 人類類胰島素生長因子I(IGF-I)為結構上與胰島素相關 之循環激素。傳統認為IGF-I為生長激素對周邊組織發揮 作用之主要介體。IGF-I由70個胺基酸組成且亦稱作體介 素C且係由SwissProt第P01343號定義。用途、活性及製備 描述於以下文獻中:例如le Bouc,Y.等人,FEBS Lett. 1 96 (1986) 108-1 12 ; de Pagter-Holthuizen,Ρ·等人,FEBS Lett. 195 (1986) 179_184 ; Sandberg Nordqvist,A.C·等人,
Brain Res. Mol. Brain Res. 12 (1992) 275-277 ; Steenbergh, Ρ·Η·等人,Biochem. Biophys· Res. Commun. 175 (1991) 507-5 14 ; Tanner,J.M·等人,Acta Endocrinol. (Copenh·) 84 (1977) 681-696 ; Uthne,K·等人,J. Clin· Endocrinol· 123528.doc 200819468
Metab. 39 (1974) 548-554 ; EP 0 123 228 ; EP 0 128 733 ; US 5,861,373 ; US 5,714,460 ; EP 0 597 033 ; W〇 02/32449 ; WO 93/02695。 IGF-I功能之調節相當複雜。在循環中,僅〇.2〇/()之IGF-I 係以游離形式存在,而大部分係與IGF結合蛋白(IGFBP)結 合,該等蛋白對IGF具有極高親和力且調節IGF-I功能。該 因子可藉由釋放IGF-I之機制(諸如IGFBP經蛋白酶蛋白質 水解)而局部釋放。 IGF-I在發育中及成熟之大腦中起旁分泌作用(Werther, G.A·等人,Mol. Endocrinol. 4 (1990) 773-778)。活體外研 究中指示IGF-I為CNS中數種類型神經元之強效非選擇性營 養劑(Knusel,B.等人,J. Neurosci· 10(1990) 558-570 ; Svrzic,D.及 Schubert,D·,Biochem. Biophys. Res. Commun. 172 (1990) 54·60),該等神經元包括多巴胺能神經元 (Knusel,Β·等人,J· Neurosci. 10(1990) 558-570)及寡突細 胞(McMords,F.A·及 Dubois-Dalcq, M.,J. Neurosci· Res. 21 (1988) 199-209 ; McMorris,F.A.等人,Proc. Natl. Acad, Sci· USA 83 (1986) 822-826 ; Mozell,R.L.及 McMorris, F.A·,J· Neurosci· Res. 30 (1991) 382-390))。US 5,093,3 1 7 提及膽鹼能神經元細胞之存活可藉由投與IGF_;[來增強。 此外’已知IGF-I刺激周邊神經再生(Kanje,M.等人,Brain Res· 486 (1989) 396-398)且增強鳥胺酸脫羧酶活性(us 5,〇93,3 17)。US 5,861,373及 WO 93/02695提及一種藉由增 加患者中樞神經系統中IGF-I及/或其類似物之活性濃度來 123528.doc 200819468 治療主要影.響神經膠質及/或非膽鹼能神經元細 ^ _員傷或疾病之方法。w〇咖洲係關於藉由向》 哺乳動物之鼻腔投與包含治療有效量之服七戈其生物活 性變異體之醫藥組合物來減少或預防哺乳動物中樞神經系 統之缺血性損傷之方法。服]或其變異體係以可有效減 少或預防與缺血性事件相關之缺血性損傷之量經由鼻腔吸 收且輸送至哺乳動物之中樞神經系統中。Ep 〇 874⑷主 張1仍-1或IGF-II用於製造治療或預防中柩神經系統中神經 兀損傷之藥物的用途,其中該神經元損傷係由以下疾病造 成:Ams相關性癡呆症、AD、中白金森氏病(恤⑹⑽,s Disease)、皮克氏病(Plck’s⑴咖叫、亨廷頓氏病 (HUntington’s Disease)、肝性腦病、皮質基底神經節症候 群、進行性癡呆症、家族性痕呆症伴癌擎、性靡疾、進行性 核上麻療、多發性硬化症、希爾德病(SeMider)之腦硬化症 或急性壞死出血性腦脊髓炎,其中藥物為可將有效量之該 IGF非經腸投與血腦屏障或血-脊髓屏障外部之形式。 大腦及血清游離IGF-I含量之降低與偶發性及家族性形 式之AD之發病機理相關。此外,IGIM保護神經元免受八^ 誘發之神經毒性影響(Niikura,T.等人,j. Neur〇sci. 21 (2_) 19〇2-191〇; Dore,S·等人,Pr〇c _ Α(^ % USA 94 (1997) 4772-4777 ; Dore,S.等人,Ann Νγ Acad Sci. 890 (1999) 356-364)。近來,已展示周邊投與 IGF_U& 夠降低大鼠及小鼠之大腦Αβ含量(Carr〇,E.等人,Nat. Med. 8 (2002) 1390-1397)。此外,研究證實在轉殖基因 123528.doc 200819468 AD小鼠模型中延長之IGF-Ι治療顯著降低大腦澱粉樣空斑 負荷。此等資料強烈支持IGF_I能夠藉由將Αβ自大腦清除 而減少大腦Αβ含量及與空斑相關之大腦癡呆症的觀點。 已證實用聚乙二醇(PEG)共價修飾蛋白質為延長蛋白質 在體内之循環半衰期的有效方法(Hershfield,M.S.等人, N· Engl. J· Med. 3 16 (1987) 589-596 ; Meyers,F.J.等人, Clin· Pharmacol· Ther. 49 (1991) 307-313 ; Delgado,C·等 人,Crit. Rev· Ther. Drug Carrier Syst· 9 (1992) 249-304 ;
Katre, Advanced Drug Delivery Reviews 10 (1993) 91- 114 ; EP-A 0 400 472 ; Monfardini,C·等人,Bi〇conjUgate Chem. 6 (1995) 62-69 ; Satake-Ishikawa,R·等人,Cell Struct· Fimct· 17 (1992) 157-160 ; Katre,N.V·等人,proc· Natl. Acad. Sci. USA 84 (1987) 1487-1491 ; Tsutsumi,Y_ 等 人 ’ Jpn. J. Cancer Res· 85 (1994) 9-12 ; Inoue,H.等人,J· Lab. Clin. Med· 124 (1994) 529-536 ; Chamow,S.M.等人, Bioconjugate Chem· 5 (1994) 133-140) 〇 PEG化之其他優點為溶解度增加及蛋白質免疫原性降低 (Katre,N.V·,J· Immunol. 144 (1990) 209-213)。蛋白質 PEG化之常用方法為使用經胺基反應性試劑(如…羥基琥轴 醯亞胺(NHS))活化之聚乙二醇。使用該等試劑,聚乙二醇 在游離第一胺基(諸如N末端α·胺基及離胺酸殘基之ε•胺基) 處與蛋白質連接。然而,此方法之主要侷限在於蛋白質通 常含有相當大量之離胺酸殘基且因此聚乙二醇基團係以非 特異性方式在所有游離心胺基處與蛋白質連接,從而產生 123528.doc -10 - 200819468
Ik機PEG化蛋白之異質產物混合物。因此,由於較低比活 性,許多NHS-PEG化蛋白不適於商業用途。生物活性所需 之一或多個離胺酸殘基或义末端胺基殘基之共價修飾或聚 乙二醇殘基在接近或在蛋白質活性位點處之共價連接可產 生滅活作用。舉例而言,已發現使用NHS-PEG化試劑修飾 人類生長激素使蛋白質之生物活性降低1〇倍以上(Clark,R. 等人,J. Biol· Chem. 271 (1996) 21969-21977)。除 N-末端 胺基酸之外,人類生長激素含有9個離胺酸。某些此等離 胺酸係位於已知對於受體結合關鍵之蛋白質區域中 (Cunningham,B.C·等人,Science 254 (1991) 821-825)。另 外,藉由使用胺基反應性聚乙二醇試劑修飾紅血球生成素 亦導致生物活性幾乎完全喪失(Wojchowski,D.M.等人,
Biochim· Biophys. Acta 910 (1987) 224-232)。使用胺基反 應性PEG化试劑共價修飾干擾素_α2導致生物活性喪失 40%-75%(美國專利第5,382,657號)。G-CSF之類似修飾導 致大於60%之活性喪失(Tanaka,η·等人,Cancer Res· 51 (1991) 3710-37 14)且介白素_2之類似修飾導致大於90%之 生物活性喪失(Goodson,R. J.及Katre,N. V·,BioTechnology 8 (1990) 343-346) 〇 W〇94/12219及WO 95/32003主張包含PEG及IGF或經半 胱胺酸突變之IGF之聚乙二醇結合物,該PEG係在突變蛋 白之N末端區域中之游離半胱胺酸處與該突變蛋白連接。 WO 2004/603 00描述 N末端 PEG化之 IGF-I。
IgA蛋白酶之識別位點描述為Ya^pro.LXaa-Pro。Yaa代 123528.doc 11 200819468 表Pro(或極少地代表Pro與Ala、Gly或Thr之組合:Pro-Ala、Pro-Gly 或 Pr〇-Thr)。Xaa代表丁hr、Ser 或 Ala(Pohlner, J·等人,Bio/Technology 10 (1992) 799-804 ; Pohlner,J·等 人,Nature 325 (1987) 458-462 ;及 US 5,427,927)。Wood, S.G·及 Burton J·,Infect· Immun· 59 (1991) 18 18-1 822 已鑑 別天然裂解位點。來自淋病雙球菌(2型)之免疫球蛋白A1蛋 白酶之合成肽底物為自我蛋白水解位點Lys-pr〇-Ala胃 Pro.I.Ser^Pro > VaKAla-Pro-Pro.! .Ser-Pro > Pro-Arg-Pro-Pro.!.Ala-Pro > Pro-Arg-Pro-Pro.! .Ser-Pro > Pro-Arg-Pro-Pro.LThr-Pro 及 IgAl 裂解位點 pro-Pr〇-Thr_Pr〇 ! Ser_Pr〇& Ser-Thr-Pro-Pro.!.Thr-Pro。 WO 2006/066891揭示由類胰島素生長因子變異 體及或兩個I乙一醇基團組成之結合物,其特徵在於該 IGF-I變異體具有在野生型igF-I胺基酸序列之多達三個胺 基酸位置27、37、65、68處之胺基酸改變以使得該等胺基 酸中之一或兩者為離胺酸且胺基酸27為非離胺酸之極性胺 基酸,其係經由該(等)離胺酸之第一胺基結合且據揭示該 (荨)聚乙一醇基團具有20 kDa至1〇〇 kDa之總分子量。該等 結合物適用於治療神經退化性病症,如阿茲海默氏病。 WO 2006/074390 提及 IGF-I 融合多肽。 【發明内容】 本發明包含一種用於製造離胺酸_PECHtIGF_I或離胺酸· PEG化IGF-I變異體之方法,該變異體包含一或兩個選自由 獨立經另一極性胺基酸取代之離胺酸27、65及/或Μ組成 123528.doc 12 200819468 之群的胺基酸,其特徵在於: a) 培養包含含有編碼融合蛋白之核酸之表現載體的原核 宿主細胞,其中該融合蛋白包含N末端與原肽之C末端 連接之該IGF-I或IGF-I變異體, b) 藉此該原肽在C末端由胺基酸-Y-Pro終止,其中Y係選 i*Pro、Pro-Ala、Pro-Gly、Pro-Thr、Ala-Pro、Gly-Pro、Thr-Pro、Arg-Pro 或 Pro-Arg-Pro 組成之群, c) 回收及PEG化該融合蛋白, d) 用IgA蛋白酶裂解該PEG化融合蛋白,及 e) 回收該PEG化IGF-I或IGF-I變異體。 在一實施例中,該方法之特徵在於離胺酸-PEG化IGF-I 變異體為RRK且該變異體係在離胺酸殘基68處經單PEG化 或離胺酸-PEG化IGF-I變異體為RKR且該變異體係在離胺 酸殘基65處經單PEG化。 在另一實施例中,該方法之特徵在於離胺酸-PEG化IGF-f 1變異體為RKK且該變異體係在離胺酸殘基65及68處經單 PEG4 匕或匕。 在另一實施例中,該方法之特徵在於離胺酸-PEG化IGF-I變異體為RRK、RKR或RKK之混合物且該變異體係在離 胺酸殘基65及68處經單PEG化或二PEG化。 本發明之另一實施例為一種包含N末端與原肽之C末端 連接之該IGF-I或IGF-I變異體的融合蛋白,其特徵在於該 原肽在C末端由胺基酸-Y-Pro終止,其中Y係選自由Pro、 Pro-Ala、Pro-Gly、Pro-Thr、Ala-Pro、Gly-Pro、Thr- 123528.doc -13 - 200819468
Pro、Arg-Pro 或 Pro-Arg-Pro 組成之群。由於 _γ_ρΓ〇 序列, 原肽可藉由IgA蛋白酶處理而自該或IGF」變異體分 根據本發明之融合蛋白之特徵較佳在於式Met_Xl_HiSn-X2-Y-Pro-[IGF-I或 IGF-I變異體],其中 • IGF-I表示人類IGF-i且IGF-I變異體表示其中一或兩 •個選自由離胺酸27、65及/或68組成之群之胺基酸獨 立經另一極性胺基酸取代之IGF-I, (' • Met表示甲硫胺酸, • X1為一鍵、絲胺酸或天冬醯胺酸, • His為組胺酸, • η為〇至1〇 ’較佳〇至6之數, • Χ2為連接肽’其係選自由肽SEQ ID NO:6-10組成之 群, • Pro為脯胺酸,且 ^ / Y 係适自由 Pro、Pro-Ala、Pro-Gly、Pro-Thr、Ala-
Pro Gly-Pro、Thr-Pro、Arg-Pro 或 Pro-Arg-Pro 組成 之群。
• 根據本發明之融合蛋白較佳係選自由融合蛋白SEQ ID . N〇:2-5 及 SEQ ID N〇:22-25 組成之群。 原狀k佳係由式Met-XpHiSn-XrY-Pro-展示,其中 • 表示甲硫胺酸, • X〗為一鍵、絲胺酸或天冬醯胺酸, • His為組胺酸, 123528.doc -14- 200819468 •η為0至1〇,較佳〇至6之數, • Χ2為連接肽’其係選自由肽SEQ ID NO:6-10組成之 群, • Pro為脯胺酸,且
Pr〇、Gly"Pro、Thr-Pr〇、Arg-Pro 或 pro-Arg-Pro 組成 之群。 原肽在C末端與igf-ι或igf-ι變異體之N末端(甘胺酸)連 接。原肽較佳不包括離胺酸殘基。原肽較佳具有至多30個 胺基酸之長度。\較佳為一鍵。n較佳為〇或6。&較佳為 肽 SEQ ID NO:7。Y較佳為 Pr…Arg-Pr〇。 本發明之另一實施例為一種離胺酸邛£〇化IGF-I,其特 徵在於不包含N末端曱硫胺酸。 本發明之另一實施例為一種離胺酸_peg化iGIM變異 體,其中一或兩個選自由離胺酸27、65及/或68組成之群 之胺基酸獨立經另一極性胺基酸取代,其特徵在於該變異 體不包含N末端甲硫胺酸。 该(等)極性胺基酸較佳獨立為精胺酸、麩酸胺酸或天冬 醯胺酸,尤其為精胺酸。 較佳之IGF-I變異體及融合蛋白中之變異體為:rkk、 RKR、RRK。IGF-I變異體如下命名:K65意謂胺基酸μ為 離胺酸’ R27意謂胺基酸27為精胺酸等。帶有胺基酸 R27、Κ6 5、Κ68之IGF-I變異體命名為RKK,且該RKK變 異體之其餘胺基酸與SEQ ID Ν〇·1之IGF-I野生型中相同。 123528.doc 200819468 因此,RKK為IGF-Ι野生型之變異體,其藉由在位置27處 將K替換為R而突變。rkR為IGF-I野生型之變異體,其藉 由在位置27及68處將K替換為R而突變。rrk為IGF-I野生 型之變異體,其藉由在位置27及65處將K替換為R而突 變。其中無胺基酸改變之IGF-I野生型命名為KKK。
離胺酸-PEG化IGF-I較佳係在離胺酸殘基27、65及68隨 機PEG化,較佳為單PEG化或二pEG化(每個沁以分子一或 兩個PEG)。離胺酸-PEGKiGFj變異體較佳係在離胺酸殘 基65及68單PEG化或二PEG化,較佳係在K65或K68單PEG 化。水(乙一醇)基團係經由離胺酸之一級胺基與該或 IGF-I變異體結合。 根據本發明之方法包含離胺酸_PEG化IGF-I或IGF-I變異 體之製備’其中每個IGF-I或IGF-1變異體之該(等)聚(乙二 醉)基團具有較佳至少20 kDa,更佳約20 kDa至1〇〇 kDa, 尤其較佳20 kDa至80 kDa之總分子量,係藉由根據本發明 之IGF-I融合蛋白與活化之聚乙二醇在使該聚乙二醇經由 IGF-I或IGF-I變異體之離胺酸一級胺基與該IGIM中間物化 學結合之條件下反應。離胺酸-PEG化IGF-I或IGF-I變異 體較佳係單PEG化。變異體較佳係選自由分別關於胺基酸 位置27、65及68之RKK、RKR、RRK組成之群。PEG較佳 具有30 kDa至45 kDa,尤其30 kDa或40 kDa之平均總分子 量。 本發明進一步包含含有根據本發明之離胺酸^^〇化1(31^ I或IGF I、欠異體,較佳連同醫藥學上可接受之載劑的醫藥 123528.doc -16- 200819468 組合物。 本發明進一步包含用於製造含有根據本發明之離胺酸_ PEG化IGF-Ι或IGF-Ι變異體之醫藥組合物的方法。 本發明進一步包含根據本發明之離胺酸—PEG化IGF_J或 IGF-I變異體用於製備治療AD之藥物的用途。 本發明進一步包含用於治療AD之方法,其特徵在於將 醫藥學上有效里之Jk基反應性PEG化IGF-I或IGF-I變異體 投與需要該治療之患者’較佳每週施用一至兩次。 【實施方式】 意外地發現IgA蛋白酶(較佳為來自淋病雙球菌之IgA蛋 白酶)能夠裂解胺基酸序列Y-Pro.!,Gly-Pro。Y係選自由
Pro、Pro-Ala、Pro-Gly、pro-Thr、Ala-Pro、Gly-Pro、
Thr-Pro、Arg-Pro或Pro-Arg-Pro組成之群。較佳適用作裂 解位點的為 Pro-Pro.!.Gly-Pro(SEQ ID NO:15) *Pro-Arg-
Pro-Pro.!.Gly-Pro(SEQ ID N0:11)(·!·:裂解位置)。根據本 發明之方法所用之IgA蛋白酶裂解位點具有胺基酸一致序 列Y-Pro.!,Gly-Pro,藉此Gly-Pro為IGF-I之前兩個胺基 酸。Y較佳表示由胺基酸Pro、Pro-Ala、Arg-Pro或Pro-Arg-Pro終止之胺基酸序列。該等Y胺基酸序列(尤其是Pr〇-Arg-Pro)可由另一 Ala或Pro-Ala基團延長,如例如在Ala-Pro-Arg-Pro(SEQ ID NO:12)或 Pro-Ala-Pro-Arg-Pro(SEQ ID NO:13)中。尤其較佳的為裂解胺基酸序列Pr〇-Ala_ Pro.!.Gly-Pro(SEQ ID N0:14)、Pro-Pro_!.Gly-Pro(SEQ ID N〇:15)、Pro-Arg-Pro-Pro·!· Gly-Pro(SEQ ID NO:16)、Ala- 123528.doc 200819468
Pro-Arg-Pro-Pro.!.Gly-Pro(SEQ ID NO:17)或 Pro-Ala-Pro-Arg-Pro-Pro.!.Gly-Pro(SEQ ID NO:18)。 根據本發明,術語nIgA蛋白酶n包括特異性裂解IgA之蛋 白酶且其已例如由Kornfeld,S.J.及Plaut,A.G·在Rev· 11^61〈1:.0丨8.3 (198 1) 52 1-5 34中以(例如)來自淋病雙球菌(2 型)之IgAl蛋白酶來描述。諸如DE-A 36 22 221 ; Koomey,
J.M·等人,Proc. Natl. Acad. Sci. USA 79 (1982) 7881· 7885 ; Bricker,J.等人,proc. Natl· Acad· Sci. USA 80 (1983) 268 1· 2685 ; Pohlner,J.等人,Nature 325 (1987) 458 462,及 Halter,R·專人,EMBO J. 3 (1984) 1 595.1601 中所述之重組IgA蛋白酶亦恰為適合的。該IgA蛋白酶較佳 為來自淋病雙球菌(較佳2型)之IgA蛋白酶。 較佳將編碼融合蛋白之基因置於適合(較佳可誘導)表現 h唬之控制下以使得可根據需要製造融合蛋白。可使用適 合原核或真核(植物以及動物)細胞作為用於製造蛋白融合 物之宿主細胞;然而,不含細胞之系統亦為可能的。 根據本發明之方法之一較佳實施例的特徵在於將宿主細 月已用重組DNA或重組載體轉化,其中DNA或載體含有至少 一個編碼根據本發明之融合蛋白之基因的複本,且將經轉 化細胞於適合培養基中培養,使編碼融合蛋白之基因在經 I中表現,將融合蛋白PEG化且隨後用IgA蛋白酶 W解且分離PEG化IGF_UiUGF_It異體。 與不含離胺 不含離胺酸 根據本發明之融合蛋白之表現可(例如)藉由 -夂之β半乳糖苦酶基因之片段融合(亦即Y含有 123528.doc -18 - 200819468 之β-半乳糖苷酶蛋白之部分)而在DNA水平上改良。專家 瞭解用於增加融合蛋白表現之其他替代方法。可藉由與其 他多肽,詳言之與多荷電多肽或蛋白質(例如聚(Lys, 或可以高親和力與特定物質結合之多肽或蛋白質(例如抗 生蛋白鏈菌素)融合來促進表現產物之純化及分離(例如參 見EP-A 0 089 626、EP_A 〇 3〇6 61〇)。尤其較佳之連接狀 為肽SEQ ID NO:6-1〇,其N末端之前較佳
(SEQ ID NO:19)、NHHHHHH(SEQ ID NO:2G)或 HHHHHH (SEQ ID NO:21) 〇 本發明亦提供一種(重組)核酸,其編碼根據本發明之融 合蛋白且其中在原肽與IGF_;[或IGiM變異體之間的接合區 域中併入IgA蛋白酶裂解位點。 根據本發明之重組DNA可以熟習分子生物學領域之技術 者已知的方式獲得。對此,通常將含有編碼ZGPq或 變異體之胺基酸序列之DNA序列的載體用限制性核酸内切 酶在此基因之5’端區域中裂解且與含有所需序列之寡核苷 酸再接合。 另外’本發明亦提供一種重組載體,其含有至少一個根 據本發明之重組DNA之複本。專家瞭解適用作原核生物體 中蛋白質表現之基礎的載體。此載體較佳為使得根據本發 明之重組DNA可高度表現之載體。載體上之重組dNA較佳 係在可誘導表現信號(例如,X、tax、iac或鄉啟動子)之控 制下。 根據本發明之載體可存在於染色體外(例如質體)以及整 123528.doc -19- 200819468 合於佰主生物體之基因組中(例如噬菌體λ)。根據本發明之 為質體。_子生物學領域之技術者已知在特 疋佰主生物體中在各情況下適用於基因表現之載體。其可 二、/、/載體但車乂佳為原核載體。適用於在原核生物中表 現根據本發明之DNA之載體的實例為⑼如)市售puc及 pUR載體。 本發明亦提供-種經根據本發明之重組dna或/及經根 據本發明之重組載體轉化之細胞,較佳為原核細胞,尤其 較佳為大腸桿菌(Ε· c〇H)細胞。 八 田融合蛋白在原核生物中表現時,形成無活性之微溶聚 集體(折射體、包涵體)。因此,必須將融合蛋白轉化為其 活性形式。使用熟習此項技術者熟知之程序(例如參見£?_ A 0 219 874、EP A 0 1 14 506、WO 84/0371 1),首先藉由 添加變性劑進行溶解,隨後複性且必要時進行其他純化步 驟。在融合蛋白PEG化之後,用igA蛋白酶來處理融合蛋 白0 用IgA蛋白酶裂解PEG化IGF-I或IGF-1變異體之處理所需 之條件並非關鍵的。然而,在此方法中,較佳地peg化 IGF_I或IGF-I變異體與IgA蛋白酶之重量比為1:1至5〇〇:;[, 較佳為100:1。較佳在pH值為6.5至8.5之緩衝水溶液中進行 反應。緩衝液濃度較佳在介於50 mmol/l與500 mmol/1之 間之範圍内’必要時可添加〇 -1 〇〇 mm〇l/l氣化鈉。裂解較 佳在室溫下進行至少60分鐘至長達5天,較佳介於24小時 至72小時之間。 123528.doc -20- 200819468 在溶解、複性、PEG化及用IgA蛋白酶裂解之後,較佳 將以此方式獲得之PEG化裂解產物藉助於離子交換層析、 疏水性相互作用層析及/或根據尺寸分餾來純化。以此方 式製得之PEG化lGF-i或IGF-I變異體在位置1處不含甲硫胺 酸且較佳不含其他蛋白質(如非PEG化IGIM或iGF]變異 體)’且較佳不含N末端PEG化原肽,5%(w/w)或更低。 如本文中所用之”PEG化IGF-I或IGF-I變異體,,或”胺基反 應性PEG化”意謂IGIM或IGIM變異體係經由胺基反應性偶 合與聚乙二醇基團共價鍵結。PEG基團係在離胺酸側鏈之 第一 ε-胺基之IGF_b^IGF·;[變異體分子之位點處連接。此 外’ PEG化亦有可能另外發生在原肽之n末端心胺基上。 知因於所用之合成方法及融合蛋白,PEG化igf-I或igf-I 變異體可由混合物組成,藉此PEG化位點在不同分子中可 不同或關於每個分子聚乙二醇侧鏈之量及/或分子中pEG化 位點之量可大體上均勻。IGIM或IGF-;[變異體較佳係經單 PEG4b&/或二lPEG 化。 如本文中所用之胺基反應性PEG化指示一種藉由使用反 應性(經活化)聚乙二醇,較佳藉由使用(較佳)甲氧基聚乙 二醇之N-羥基琥珀醯亞胺基酯使聚乙二醇鏈與πρ]或 IGF-I變異體之第一離胺酸胺基隨機連接之方法。偶合反 應使聚乙二醇與離胺酸殘基之反應性第一 ε _胺基及視情況 與融合蛋白之Ν-末端胺基酸之α-胺基連接。此項技術中熟 知PEG與蛋白質之此胺基結合。舉例而言,Vej^eSe F.M·,Biomaterials 22 (2001) 405-417提供了 此等方法之概 123528.doc -21 - 200819468 述。根據Veronese,PEG與蛋白質之第一胺基之結合可藉 由使用實施該等第一胺基之烷基化之經活化pEG來進行。 對於此反應而言,可使用經活化烷基化PEG,例如阳G 醛、PEG-三氟乙磺醯氯或PEG環氧化物。其他適用試劑為 u 醯化PEG,諸如羧酸化PEG或其中末端羥基經氣甲酸酯或 羰基咪唑活化之PEG的羥基琥珀醯亞胺基酯。其他適用之 PEG試劑為具有胺基酸臂之PEG。該等試劑可含有所喟分 ( ' 枝PEG,藉此至少兩個相同或不同之pEG分子經肽間隔基 (較佳為離胺酸)連接在一起且(例如)以離胺酸間隔基之經 活化羧酸自旨之形式與IGF-I或IGF-I變異體結合。 如本文中所用之”PEG或聚乙二醇”意謂市售或可根據此 項技術中熟知之方法(Kodera,γ·等人,Pr〇gress in
Polymer Science 23 (1998) 1233-1271 ; Francis,G,E•等 人,IrU. J. Hematol· 68 (199δ) h8)藉由乙二醇之開環聚 合來製備之水溶性聚合物。術語”PEG,,廣泛用於涵蓋任何 ί / 聚乙二醇分子,其中乙二醇單元之數目為至少460,較佳 為460至2300且尤其較佳為460至1 840(230 PEG單元係指約 10 kDa之分子量)。PEG單元之上限數目僅由離胺酸邛eg - 化1GF·1或WF·1變異體之溶解度限制。通常不使用大於含 有2300個單元之PEG的PEG。本發明中所用之PEG較佳在 一端由羥基或曱氧基終止(曱氧基PEG,mPEG)且在另一端 經由醚氧鍵與連接基團部分共價連接。pEG聚合物為線性 或分枝的。PEG聚合物較佳為分枝的。分枝pEG描述於(例 如)Veronese,F.M·等人,Journal 〇fBi〇active — c〇mpatiMe 123528.doc -22- 200819468
Polymers 12 (1997) 196_2〇7 中。適用之 pEc}試劑可(例如) 自 Nektar Therapeutics (www.nektar.com)獲得。分枝 PEG 可 (例如)藉由將聚氧化乙烯添加至各種多元醇(包括甘油、異 戊四醇及山梨糖醇)中來製備。舉例而言,四臂分枝可 • 由異戊四醇及環氧乙烷來製備。分枝PEG通常具有2至8條 臂且描述於(例如)EP-A 〇 473 〇84及美國專利第5,932,462 唬中。具有經由離胺酸之第一胺基連接之兩個pE(}側鏈的 ( PEG(縮寫為PEG2)尤其較佳(Monfardini,C等人,
Bioconjugate Chem· 6 (1995) 62-69)。 按貫際需要,可使用(例如)約2〇千道爾頓⑴…至丨⑽kDa 之任何分子質量的PEG(n為460至2300)。PEG中之重複單 元數目,n’f係關於以道爾頓計之分子質量進行估計。舉例 而言’若兩個PEG分子與連接基團連接,在各pEG分子具 有10 kDa之相同分子質量(各η為約23 〇)之情況下,則連接 基團上PEG之總分子質量為約2〇 kDa。與連接基團連接之 (J PEG之分子質量亦可不同,例如在連接基團上之兩個分子 中’一PEG分子可為5 kDa且另一 PEG分子可為15 kDa。分 子質量通常意謂平均分子質量。 • 在以下實例中,描述用於製造胺基反應性jGF-zstgfj .變異體之一些較佳試劑。應瞭解可在程序中(例如)根據
Veronese,F.M·, Biomaterials 22 (2001) 405-417所述之方 法進行修飾,只要該方法產生根據本發明之離胺酸_PEG化 IGF-I或IGF-I變異體即可。 本發明提供用於製造具有改良性質之1(^]或IGiM變異 123528.doc -23 - 200819468 體之PEG化形式之方法。該等離胺酸-PEG化IGF-Ι或IGF-I 變異體含有與其隨機連接之線性或分枝PEG,藉此離胺酸-PEG化IGF-I或IGF-I變異體中所有PEG基團之總分子量較 佳為約20 kDa至80 kDa。熟習此項技術者清楚瞭解此分子 量範圍之較小偏差係可能的,只要PEG化IGF-I或IGF-I變 異體展示出降低大腦中Αβ肽含量之活性即可。具有大於80 kDa之分子量之PEG亦可產生較高生物可用性。然而,預 期隨著分子量增加,由於降低之IGF-I受體活化及血腦屏 障輸送,此活性降低。因此,應瞭解對於PEG之分子量20 kDa至100 kDa之範圍為適用於有效治療患有AD之患者之 離胺酸-PEG化IGF-I或IGF-I變異體的最佳範圍。 較佳製造選自由RKK、RKR及RRK組成之群之單PEG化 IGF-I變異體,其中分枝PEG基團具有30-45,較佳40-45 kDa之分子量(約920個PEG單元)。舉例而言,根據PEG之 44 kDa之平均分子量及IGF-I之7.6 kDa之分子量,此單 ?£0-10卩-1之經計算平均分子量為約51.6 1^0&。尤其較佳使 用分子量為40 kDa之經N-羥基琥珀醯亞胺基活化之分枝 PEG 酉旨(mPEG2-NHS)(Monfardini,C.等人,Bioconjugate Chem· 6 (1995) 62-69 ; Veronese,F.M.等人,J. Bioactive Compatible Polymers 12 (1997) 197-207 ;美國專利第 5,932,462號)。亦較佳製造其中PEG具有30 kDa或40 kDa之 平均分子量之單PEG化IGF-I。 如本文中所用,π分子量”意謂PEG之平均分子量。 以下IGF-1或IGF-I變異體之PEG化形式為較佳產物且可 123528.doc -24- 200819468 藉由根據本發明之方法來獲得: -K 68單PEG化IGF-Ι變異體,較佳為RRK或RKK變異體, 更佳為RRK變異體,其中PEG基團具有20 kDa至80 kDa 之分子量(460至1840個PEG單元); -K 65單PEG化IGF-Ι變異體,較佳為RKR或RKK變異體, 更佳為RKR變異體,其中PEG基團具有20 kDa至80 kDa 之分子量(460至1840個PEG單元); - 二PEG化IGF-Ι變異體,較佳為RKK變異體,其中PEG基 團各具有約10-50 kDa之分子量(230至1150個PEG單 元);及其混合物; -K 68單PEG化IGF-Ι變異體,較佳為RRK或RKK變異體, 更佳為RRK變異體,其包含分子量為40 kDa之PEG2基 團; -K 65單PEG化IGF-Ι變異體,較佳為RKR或RKK變異體, 更佳為RKR變異體,其包含分子量為40 kDa之PEG2基 團; - 單PEG化IGF-Ι,其中PEG基團具有20 kDa至80 kDa之分 子量(460至1840個PEG單元); - 二PEG化IGF-Ι,其中PEG基團各具有約10-50 kDa之分 子量(230至11 50個PEG單元); - 單PEG化IGF-Ι,其包含分子量為40 kDa之PEG2基團。 PEG化IGF-Ι或IGF-Ι變異體大體上為均勻的。製劑可含 有少量未反應(亦即無PEG基團)之蛋白質。如由肽定位所 確定,變異體之純度為至少90%(w/w)。該等製劑之進一步 123528.doc -25- 200819468 純化(包括分離單PEG化及/或二叩〇化IGIM或Ι<3ρΜ變異 體)可藉由常用純化方法,較佳藉由尺寸排阻層析、疏水 性相互作用層析及/或離子交換層析,尤其藉由陽離子交 換層析來進行。 如本文中所用之”單PEG化’’意謂IGF-I或IGF-I變異體在 每個IGF-ί或— 〗變異體分子中僅在一個離胺酸處經ρΕ〇 化。 在此項技術之目前發展水平熟知”經活化pEG或經活化 PEG試劑”。較佳使用經親電子活化之pEG,諸如聚乙二醇 之燒氧基丁酸琥珀醯亞胺基酯(”低碳烷氧基_PEg-Sba”)或 聚乙二醇之烷氧基丙酸琥珀醯亞胺基酯(,,低碳烷氧基_ PEG-SPA”)或經N-羥基琥珀醯亞胺活化之pEG。可利用使 經活化酯與胺反應以形成醯胺之任何習知方法。在經活化 PEG與IGF-I之反應中,例示性琥珀醯亞胺基酯為促使醯胺 形成之離去基。琥珀醯亞胺基酯用於製造與蛋白質之結合 物之用途揭示於美國專利第5,672,662號中。 所用反應條件對於不同PEG化之IGIM4IGF_pt異體之 相對$具有影響。藉由操控反應條件(例如,試劑比、 pH、派度、蛋白質濃度、反應時間等)可改變不同pEG化 種類之相對ϊ。反應較佳於視情況含有多達3〇%(Wv)乙醇 之緩衝水溶液(pH 8-1〇)中進行。蛋白質:pEG莫耳比較佳 為1 · 1至1 ·6,較佳為丨:2至丨:5。反應溫度及反應時間可根 據热習此項技術者之知識改變,高溫及長反應時間可使得 PEG化增加。若製備單PEG化蛋白,則較佳在4。〇與22它之 123528.doc -26- 200819468 間操作且歷時長達3〇分鐘或長達60分鐘。當PEG化試劑與 IGF-I或IGF-I變異體於較佳由50 mM硼酸鈉及25%乙醇組成 之反應緩衝液(pH約9·0-9·5)中,蛋白質:PEG比約1:3至 1:4,及反應溫度4°C組合時,產生單PEG化種類、二PEG 化種類、及痕量三PEG化種類之混合物,視蛋白質中存在 之Lys殘基而定。 根據本發明之離胺酸_PEG化IGF-I或IGF-I變異體可藉由 使IGF-I或IGF_I變異體之離胺酸一級胺基與一種雙官能試 劑共價反應以形成具有一個醯胺鍵之中間物及使該含有醯 胺鍵之中間物與一種活化之聚(乙二醇)衍生物共價反應以 形成離胺酸-PEG化IGF-I或IGF_I變異體來製備。在前述方 法中,雙官能試劑較佳為N-琥珀醯亞胺基-S-乙醯基硫代 丙酉文S曰或N -破》轴亞胺基-S _乙酿基硫代乙酸5旨,活化之 聚(乙二醇)衍生物較佳係選自由碘-乙醯基-曱氧基-PEG、 甲氧基- PEG-乙細基石風、及曱氧基-p E G -順丁稀二酿亞胺組 成之群。 活化之PEG衍生物在此項技術中已知,例如pEG_乙烯基 颯描述於 Morpurgo,M·等人,J. Bioconjugate Chem. 7 (1996) 363-368中。直鏈及分支鏈PEG種類均適於製備式工 化合物。反應性PEG試劑之實例為碘-乙醯基·曱氧基_pEG 及甲氧基-PEG-乙烯基砜。此等碘活化物質之用途在此項 技術中已知,例如已由Hermanson,G.T·在Bioconjugate Techniques,Academic Press,San Diego (1996),第 147_!48 頁中描述。 123528.doc -27- 200819468 如本文中所用之”極性胺基酸”係指選自由半胱胺酸(c)、 天冬胺酸(D)、麵胺酸(E)、組胺酸(H)、天冬醯胺酸(n)、 麩醯胺酸(Q)、精胺酸(R)、絲胺酸(8)及蘇胺酸(τ)組成之 群之胺基酸。離胺酸亦為極性胺基酸,但將其排除在外, 因為離胺酸根據本發明經取代。精胺酸較佳用作極性胺基 酸。 醫藥調配物 根據本發明製造之PEG化IGF_I4IGF-It異體提供改良 之循環穩定性’從而使得可在較低施用間隔下遍及全身持 續接觸IGF-I受體。 本發明之化合物可根據用於製備醫藥組合物之方法進行 調配’熟習此項技術者已知該等方法。為製備該等組合 物,較佳藉由對含有醫藥組合物之所需成份之水溶液透析 或透濾將根據本發明之PEG化IGIM或IGF小變異體與醫藥 學上可接受之載劑以混合物形式組合。該等可接受之載劑 描述於(例如)由〇sl〇等人編之Remingt〇nfs pharmaceutical 心咖,第 18 版,Mack Publishing c〇mpany(例 士第143 5-1712頁中)中。典型組合物含有有效量之根據 本t明之物質(例如約01 mg/mls 1〇〇 ,以及適合 置之載劑。組合物可非經腸投與。根據本發明之PEG化 IGF I或IGF-Ι變異體較佳係經由腹膜内、皮下、靜脈内或 鼻内施用而投與。 <根據本發明之醫藥調配物可根據此項技術中已知之方法 來衣備通$,將PEG化IGF-I或IGF-I變異體之溶液對意 123528.doc -28- 200819468 欲用於醫藥組合物中之緩衝液透析或透濾且藉由濃縮或稀 釋來調節所需最終蛋白質濃度。 提供以下實例及圖式用以幫助理解本發明,本發明之真 實範疇係在所附申請專利範圍中提出。應瞭解可在不悖離 本發明之精神之情況下對所提出之程序作出修改。胺基酸 之名稱係使用一個字母編碼(例如R)或三個字母編碼(例如 Arg)來縮寫。 序列清單 SEQ ID ΝΟ:1 人類10?-1之胺基酸序列(來自8\¥丨33?1*〇1 P01343 之胺基酸 49-118)。 SEQ ID NO:2 融合蛋白 px3036—IAG_R K27R K65R K68之胺基酸序列 SEQ ID NO:3 融合蛋白 px3036_IAEE_Fl K27R K65R K68之胺基酸序列 SEQ ID NO:4 融合蛋白 px3036—IAFX—FI K27R K65R L) SEQ ID NO:5 K68之胺基酸序列 融合蛋白 px3036_IAFX—F2 K27R K65R K68之胺基酸序列 SEQ ID NO:6-10 連接子 SEQ ID ΝΟ:11-18 裂解序列 SEQ ID NO:19-21 其他 SEQ ID NO:22 融合蛋白 px3036—IAG_R K27R K65 K68R之胺基酸序列 SEQ ID NO:23 融合蛋白 px3036—IAEE_F1 K27R K65 123528.doc -29- 200819468 K68R之胺基酸序列 SEQ ID NO:24 融合蛋白 px3036—IAFX_F 1 K27R K65 K68R之胺基酸序列 SEQ ID NO:25 融合蛋白 px3036—IAFX_F2 K27R K65 K68R之胺基酸序列 實例 實例1 製得以下(式I)之具有原肽之突變體(包含IGF-I-變異體 RRK):
突變體 Xi X2 n Y Px3036JAG K27R K65R K68 無 KAKRFKKH 6 PRPP Px3036JAG_R K27R K65R K68 無 RARRFRRH 6 PRPP Px3036 IAEE FI K27RK65RK68 s NTEHNREH 6 PRPP Px3036 IAFX FI K27RK65RK68 N IEGRH 6 PRPP Px3036 IAFX F2 K27R K65R K68 N TEFENIEH 6 PRPP K68_單-PEG化IGF-I-變異體(RRK變異體)之製備 所用表現載體及大腸桿菌菌株描述於EP 0 972 838中。 自生長於選擇性瓊脂培養盤上表現融合蛋白px3036_IAG_R K27R K65R K68、px3036_IAEE_F 1 K27R K65R K68、 px3036__IAFX__Fl K27R K65R K68 或 px3036—IAFXJF2 K27R K65R K68之大腸桿菌純系,將一個接種環轉移至 (100 ml)選擇性培養基中且在37°C下培養13小時至光學密 度(578 nm)為2-4。其後6小時將此培養物儲存於冰上,隨 後在37°C下進行主要培養物之自動接種。藉由添加1.0 mM IPTG在50之光學密度(578 nm)下起始IGF-I突變體之表 現。整個醱酵持續長達16小時。藉由在SDS-PAGE凝膠上 123528.doc -30- 200819468 比較產物蛋白質帶與IGF標準物帶之體積強度來以密度量 測法測定蛋白質之量。藉由離心收集培養物肉湯。 為獲得經純化包涵體(IB)物質,使用以下程序來處理自 標準醱酵收集之生物質:將生物質用TrisMgS〇4緩衝液 7)再懸浮且補充〇·3 g/i〇〇 g生物乾重。將溶菌酶及5 u/i呂 生物乾重Benz〇nase培育20分鐘且均質化。添加3〇 un §生 • 物乾重Benzonase且在37t下培育60分鐘。添加每公升〇·5 (, [Brij緩衝液且在室溫下培育3〇分鐘。在離心後,將離心 塊再懸浮於300 ml Tris-EDTA緩衝液/100 g生物濕重(經純 化IB濕重)中’在室溫下培育3 〇分鐘且離心。在室溫下將 每公升1 g IB溶解於6.8 Μ胍-HC卜0.1 M TrisHC卜0.1 Μ DTT(PH 8,5)中隔夜。在4°C下將混濁溶液對6·8 μ胍_HC1、 〇·1 M TrisHCl(PH 8·0)透析。透析後,藉由離心移除不溶 性組份。藉由在室溫下將prodGF]溶液5〇倍稀釋於〇 8 Μ 精胺酸、(Μ M TrisHCM、〇·1 Μ胍-HCM、1 mM GSH、1 mM GSSG(PH 8·5)中來進行摺疊。兩小時後,將溶液補充 2 Μ氯化鈉,過濾且以1〇 mi/min之流動速率施用於HIC管 柱(Butyl Sepharose 4 Fast Flow ; GE5 Amersham • Biosciences)上,該管柱已在室溫下用含有2 M NaC卜0.8 • M精胺酸、0.1 M TrisHCl、〇·ΐ Μ胍-HC1之緩衝液(ΡΗ 8·5) 平衡。將管柱用平衡緩衝液洗滌直至達到基線且隨後用1〇 倍管柱體積之以平衡緩衝液起始且以含有〇, 1 M TrisHC1、 5%乙二醇之緩衝液(pH 8·5)終止之線性梯度溶離。藉由逆 相高效層析(rpHPLC)來分析經溶離之溶離份。彙集含有具 123528.doc -31- 200819468 有正確形成之SS橋之蛋白質的溶離份。在4。〇下將池對5〇 mM硼酸鈉(pH 9.〇)透析。將經NHS活化之40 kDa分枝 PEG(mPEG MW 2〇’〇〇〇之N_羥基琥珀醯亞胺(NHs)醋 (mPEG2NHS ^ US 5?932?462,Nektar Shearwater P〇lymers? H_sville,AL))溶解於冰冷之2瘦HQ中且立即添加至經 透析蛋白質溶液(PEG-試劑/蛋白質莫耳比為2:1)中。在冰 上培育1小時及2小時後,將相同量之酸性㈤即⑺屮則溶 液添加至蛋白質/PEG反應混合物中。在第三次添加相同量 之酸性mPEG2-NHS溶液後(總共6倍莫耳過量之pEG試 劑),將反應物在冰上再培fl小時。藉由添加固體氯化銨 且再培育45分鐘來終止反應且隨後調節至pH 8〇(參見圖 1) 。將蛋白質/PEG反應混合物補充來自淋病雙球菌(2型)之 IgAl蛋白酶(w/w比為1:5〇)且在室溫下培育隔夜(參見圖 2) 。將反應混合物用50 mM乙酸(pH 4.5)1:2稀釋且隨後施 用於陽離子 IEC 管柱(MacroCap SP support ; GE,Amersham
Bioscnences,Uppsala, Sweden)上,該管柱已用 5〇 mM 乙酸 平衡。將管柱洗滌直至達到基線且隨後用2〇倍管柱體積之 以50 mM乙酸起始且以補充! “氣化鈉之5〇 酸終止 之線性.梯度溶離。藉由SDS-PAGE來分析經溶離之溶離 份。將含有具有約60 kDa之估算相對分子尺寸之單一帶的 溶離份彙集作為K68-單PEG化IGF-I(參見圖3)。藉由分析 尺寸排阻層析(SEC)以靜態光散射偵測、騰蛋白酶水解物 之MS分析、Asp-N水解物之MS分析及分析陽離子iec來證 實鑑別K68-單PEG化IGF-I。除K68-單PEG化IGF_I之外, 123528.doc -32- 200819468 未發現其他PEG化異變異體(isovariant)。 實例2 製得以下(式I)之具有原肽之突變艟(包含IGF-I·變異體 RKR):
突變體 Xi X2 n Y Px3036JAG K27R K65 K68R 無 KAKRFKKH 6 PRPP Px3036_IAG R_K27R K65 K68R 無 RARRFRRH 6 PRPP Px3036JAEE_Fl K27RK65 K68R S NTEHNREH 6 PRPP Px3036 IAFX FI K27RK65 K68R N IEGRH 6 PRPP Px3036—IAFX—F2 K27R K65 K68R N TEFENIEH 6 PRPP K65_單_PEG化IGF-I·變異體(RKR變異體)之製備 所用表現載體及大腸桿菌菌株描述於EP 0 972 838中。 自生長於選擇性瓊脂培養盤上表現融合蛋白px3036_IAG_R K27R K65 K68R、px3036_IAEE__F 1 K27R K65 K68R、 px3036_IAFX^Fl K27R K65 K68R 或 px3036_IAFX_F2 K27R K65 K68R之大腸桿菌純系,將一個接種環轉移至 (100 ml)選擇性培養基中且在37°C下培養13小時至光學密 (J 度(578 nm)為2-4。其後6小時將此培養物儲存於冰上,隨 後在37°C下進行主要培養物之自動接種。藉由添加1·〇 ηιΜ IPTG在50之光學密度(578 nm)下起始IGF-I突變體之表 - 現。整個醱酵持續長達16小時。藉由在SDS-PAGE凝膠上 比較產物蛋白質帶與IGF標準物帶之體積強度來以密度量 測法測定蛋白質之量。藉由離心收集培養物肉湯。 為獲得經純化包涵體(IB)物質,使用以下程序來處理自 標準醱酵收集之生物質:將生物質用TrisMgS04緩衝液(pH 7)再懸浮,且補充0.3 g/100 g生物乾重。將溶菌酶及5 U/1 123528.doc -33- 200819468 g生物乾重Benzonase培育20分鐘且均質化。添加3〇 u/;l g 生物乾重Benzonase且在37t;下培育6〇分鐘。添加每公升 〇·5 L Brij緩衝液且在室溫下培育3〇分鐘。在離心後,將離 心塊再懸浮於300 ml Tris-EDTA緩衝液/loo g生物濕重(經 純化IB濕重)中,在室溫下培育3〇分鐘且離心。在室溫下 將每公升1 g IB溶解於6·8 Μ胍-HC卜0.1 M TrisHCl、0.1 M DTT(pH 8.5)中隔夜。在4°c下將混濁溶液對6 8 乂胍_ HC1、〇·1 M TrisHCl(pH 8·0)透析。透析後,藉由離心移除 不溶性組份。藉由在室溫下將pr〇_IGF-I溶液5〇倍稀釋於 0.8 Μ 精胺酸、0.1 M TrisHCl、〇·1 Μ 胍-HC1、1 mM GSH、1 mM GSSG(pH 8.5)中來進行摺疊。2小時至48小時 後’較佳2小時至24小時後,將溶液補充2 μ氣化鈉,過濾 且以10 ml/min之流動速率施用於HIC管柱(Butyl Sephar〇se
4 Fast Flow; GE,Amersham Biosciences)上,該管柱已在 室溫下用含有2 M NaC卜〇·8 M精胺酸、0.1 M TrisHCl、 〇,l M胍-HC1之緩衝液(pH 8.5)平衡。將管柱用平衡緩衝液 洗條直至達到基線且隨後用1 〇倍管柱體積之以平衡緩衝液 起始且以含有0·1 M TrisHCl、5%乙二醇之緩衝液(PH 8.5) 終止之線性梯度 >谷離。藉由逆相高效層析(rpHpLc)來分析 經’谷_之 >谷離份。茱集含有具有正確形成之$ ^橋之蛋白質 的溶離份。在4°C下將池對50 mM硼酸鈉(pH 9.0)透析。將 經NHS活化之40 kDa分枝PEG(mPEG MW 20,000之N-羥基 琥轴醯亞胺(NHS)酯(mPEG2NHS,US 5,932,462,Nektar Shearwater Polymers,Huntsville,AL))溶解於冰冷之 2 mM 123528.doc -34- 200819468 HC1中且立即添加至經透析蛋白質溶液(peg_試劑/蛋白質 莫耳比為2:1)中。在冰上培育丨小時及2小時後,將相同量 之酸性mPEG2_NHS溶液添加至蛋白質/pEG反應混合物 中。在第三次添加相同量之酸性mPEG2_NHS溶液後(總共6 仏莫耳過$之pEG試劑),將反應物在冰上再培育1小時。 2由添加固體氯化銨且再培育45分鐘來終止反應且隨後調 節至pH 8.0(參見圖υ。將蛋白質/PEG反應混合物補充來自 淋病雙球菌(2型)之igAl蛋白酶(w/w比為1:5〇)且在室溫下 坧月隔夜(麥見圖2)。將反應混合物用5〇 mM乙酸(pH 4·5)1:2稀釋且隨後施用於陽離子IEC管柱㈧狀⑺以口 ^pport ; GE? Amersham Bi〇sciences? Uppsala, Sweden) 上"亥官柱已用5 0 mM乙酸平衡。將管柱洗滌直至達到基 線且卩返後用2〇倍管柱體積之以5〇 mM乙酸起始且以補充^ Μ氯化鈉之5〇 mM乙酸終止之線性梯度溶離。藉由SDS_ PAGE來分析經溶離之溶離份。將含有具有約之估 算相對分子尺寸之單一帶的溶離份彙集作為&65_單pEG化 WF-I。藉由分析尺寸排阻層析(SEC)以靜態光散射偵測、 胰蛋白酶水解物之MS分析、Asp-N水解物之MS分析及分 析陽離子IEC來證實鑑別K65-單PEG化IGF_I。 實例3 在活體内K68單PEG化IGF-I-變異體RRK使大腦可溶性Ap 減少 為#估K68單PEG化IGF-I變異體rrk(40 kD, PEG2)(PEG-RRK)關於降低可溶性Αβ含量之功效,將具有 123528.doc -35- 200819468 嚴重殿粉樣空斑負荷之9_ i Q個月大之B6. i 52H小鼠(表現人 類APP及PS2突變體之雙轉瘦基因小鼠)藉由每週兩次皮下 庄射5 pg/kg pEG_RRK重複治療。在μ天後偵測皮質 APP、Αβ及肌動蛋白含量。pEG_RRK未顯著改變App/肌動 蛋白比率(圖4A),從而表明在14天内pEG_RRK對轉殖基因 表現無影響。相比之下,PEG_RRK顯著降低Αβ/肌動蛋白 (圖4Β)及Αβ/ΑΡΡ(圖4〇比率。此指示pEG_RRi^Ap清除
C/ 之與其由轉殖基因製造無關之積極影響。 【圖式簡單說明】 圖1 :單PEG化融合蛋白之肽分析。 PEG化之前及之後摺疊融合蛋白iSDS_pAGE分析。 色帶1,標準蛋白質之混合物(牛肺抗蛋白酶,6.0 kDa ;雞蛋白溶菌酶,14·4 kDa ;黃豆胰蛋白酶抑制 劑’ 2 1 · 5 kDa ;牛紅血球碳酸酐酶,3 1 · 0 kDa ;豬肌 肉乳酸脫氫酶,36·5 kDa;牛肝麩胺酸脫氫酶,55.4 kDa ;牛血清白蛋白,66.3 kDa ;兔肌肉石粦酸化酶 b,97.4 kDa ;大腸桿菌β-半乳糖苷酶,97.4 kDa ; 兔肌肉肌凝蛋白,200 kDa);色帶2,PEG化前之 pro-IGF-I ;色帶 3,PEG化後之 pro-IGF-I。 圖2 :單PEG化IGF-I變異體之肽分析。
IgA裂解之前及之後PEG化融合蛋白之SDS-PAGE分 析。色帶1,標準蛋白質之混合物(與圖1中相同); 色帶2,IgA蛋白酶裂解前之反應混合物;色帶3, I g A蛋白酶裂解後之反應混合物。 123528.doc -36 * 200819468 圖 3 : SDS PAGE。 PEG化之前及之後摺疊融合蛋白之SDS-PAGE分析。 色帶1,標準蛋白質之混合物(與圖1中相同);色帶 2,IEC前之反應混合物;色帶3,IEC之流過;色帶 4-19,自IEC之單一溶離溶離份。 圖4 : B6.152H小鼠中PEG-RRK之活體内大腦AP降低。 將9-10個月齡之雙轉殖基因B6.152H小鼠用媒劑 (NaCl)或PEG-RRK(5 pg/kg,皮下,每週兩次)處理 1 4天。製備可溶性大腦提取物且如所述評估APP、 Αβ及肌動蛋白含量。評估APP/肌動蛋白、Αβ/肌動 蛋白及Αβ/ΑΡΡ比率,以對照物之%表示。A,ΑΡΡ/ 肌動蛋白;Β,Αβ/肌動蛋白;C,Αβ/ΑΡΡ。上部圖 表展示單一動物資料點,下部圖表展示包括統計學 差異之條形圖表示(平均士 SEM)(*,對未經處理之對 照物ρ<0·05 ; **,對未經處理之對照物ρ<0·01, η=10) 〇 123528.doc -37- 200819468 序列表 <110>瑞士商赫孚孟拉羅股份有限公司
<120>製造類胰島素生長因子I及聚乙二醇之結合物之方法 <130> 23907 FT <140> 096132101 <141> 2007-08-29 <150> EP06018170 <151> 2006-08-31 <160> 25 <170> Patentln version 3.2 T嫫o R.i 1 7 p人 >>>> 0 12 3 t—I I—ί I—< T—I 2 2 2 2 <220> <221〉 misc_feature <223> " 人類IGF-I之胺基酸序列(來自SwissProt P01343之胺基酸49-118) <400> 1
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gin Phe 1 5 10 15
Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 20 25 30
Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly lie Val Asp Glu Cys Cys 35 40 45
Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 50 55 60
Lys Pro Ala Lys Ser Ala 65 70 <21〇> 2 <211> 89 <212> PRT <213〉 人造 <220> <223〉融合蛋白px3〇36jAG_R K27R K65R K68之胺基酸序列 <400> 2 123528.doc 200819468
Met His His His His His His Arg Ala Arg Arg Phe Arg Arg His Pro 1 5 10 15
Arg Pro Pro Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala 20 25 30
Leu Gin Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Arg Pro Thr 35 40 45
Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly He Val Asp 5〇 55 60
Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys 65 70 75 80
Ala Pro Leu Arg Pro Ala Lys Ser Ala 85 0 12 3 f—I i—I ϊi *—f 2 2 2 2 < v < <
<220> <223> 融合蛋白 Px3036JAEE一F1 <400> 3
Met Ser His His His His His His Asn His Asn Arg Glu His Pro Arg 10 15
Pro Pro Gly pr〇 Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu 20 25 30
Gin Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Arg Pro Thr Gly 35 40 45
Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly He Val Asp Glu 50 55 60
Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala 65 70 75 80
Pro Leu Arg pr〇 Ala Lys Ser Ala 85 123528.doc 2- 200819468 <210> 4 <211〉 87 <212> PRT <213〉 人造 <220> <223> 融合蛋白px3036JAFX_Fl K27R K65R K68之胺基酸序列 <400> 4
Met Asn His His His His His His lie Glu Gly Arg His Pro Arg Pro 1 5 10 15
Pro Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gin 20 25 30
Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Arg Pro Thr Gly Tyr 35 40 45
Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly lie Val Asp Glu Cys 50 55 60
Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro 65 70 75 80
Leu Arg Pro Ala Lys Ser Ala 85 <210〉 5 <211> 90 <212> PRT <213〉人造 <22〇> <223〉融合蛋白px3036_IAFX_F2 K27R K65R K68之胺基酸序列 <400> 5
Met Asn His His His His His His Thr Glu Phe Glu Asn lie Glu His 1 5 10 15
Pro Arg Pro Pro Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp 20 25 30
Ala Leu Gin Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Arg Pro 35 40 45 123528.doc 200819468
Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly lie Val 50 55 60
Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr 65 70 75 80
Cys Ala Pro Leu Arg Pro Ala Lys Ser Ala 85 90 <21〇> 6 <211> 8 <212> PRT <213〉 人造 <220> <223>連接子 <400> β
Lys Ala Lys Arg Phe Lys Lys His 1 5 <210> 7 <211> 8 <212> PRT <213〉 人造 <220> <223> 連接子 <4〇〇> 7
Arg Ala Arg Arg Phe Arg Arg His 1 5
<210〉 8 <211> 8 <212> PRT <213〉 人造 <22〇> <223〉連接子 <4〇0> 8
Asn Thr Glu His Asn Arg Glu His 1 5 <210> 9 <211> 5 <212> PRT <213〉人造 4- 123528.doc 200819468 <22〇> <223>連接子 <400> 9 lie Glu Gly Arg 1
His 5 Γ: <210> 10 <211> 8 <212> PRT <213〉 人造 <220> <223>連接子 <400> 10 Thr Glu Phe Glu 1
Asn lie Glu His 5 <210> 11 <211> 6 <212> PRT <213〉 人造 <220> <223〉裂解序列 <400> 11 Pro Arg Pro Pro 1
Gly Pro 5
<210> 12 <211> 4 <212> PRT <213〉人造 <22〇> <223>裂解序列 <400> 12 Ala Pro Arg Pro 1 <210> 13 <211> 5 <212> PRT <213〉 人造 <22〇> <223>裂解序列 123528.doc 200819468 <4〇0> 13
Pro Ala Pro Arg Pro 1 5 <21〇> <211> 14 5 <212> <213> PRT 人造 <220> <223> 裂解序列 <400> 14
Pro Ala Pro Gly Pro 1 5 <21〇> <211> 15 4 <212> <213> PRT 人造 <22〇> <223〉裂解序列 <4〇〇> 15
Pro Pro Gly Pro <21〇> <211> 16 6 <212> <213> PRT 人造 <22〇> <223> 裂解序列 <40〇> 16
Pro Arg Pro Pro Gly Pro 1 5 <210> <211> 17 7 <212> <213> PRT 人造 <22〇> <223> 裂解序列 <4〇0> 17 123528.doc -6 200819468
Ala Pro Arg Pro Pro Gly Pro 1 5 <210〉 18 <211> 8 <212> PRT <213〉 人造 <22〇> <223〉 裂解序列 <400> 18 Pro Ala Pro Arg 1
Pro Pro Gly Pro 5
<210〉 19 <211> 7 <212> PRT <213> 人造 <220> <223〉 其他 <40〇> 19 Ser His His His 1
His His His 5 <210〉 20 <211> 7 <212> PRT <213〉人造 <22 0〉 <223〉其他 <4〇〇> 20 Asn His His His 1
His His His 5 <210> 21 <211> 6 <212> PRT <213> 人造 <220> <223> 其他 <400> 21
His His His His His His 1 5 123528.doc 200819468 <210> 22 <211> 89 <212> PRt <213〉 人造 <220> <22 3〉融合蛋*px3〇36_lAG_R K27R K65 K68R之胺基酸序列 <400> 22
Met His His His His His His Arg Ala Arg Arg Phe Arg Arg His Pro 1 5 10 15
Arg Pro Pro Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala 20 25 30
Leu Gin Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Arg Pro Thr
Gly Tyr Qly ser Ser Ser Arg Arg Ala Pro Gin Thr Gly He Val Asp 50 55 60
Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys 65 70 7 5 80
Ala Pro Leu Lys Pro Ala Arg Ser Ala 85 <210> 23 <211> 88 <212〉 pr丁 <213〉人造 <220> <223>融合蛋白px3036JAEE_Fl K27R K65 K68R之胺基酸序列 <400> 23
Met Ser His His His His His His Asn His Asn Arg Glu His Pro Arg 1 5 10 15
Pro Pr〇 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu 20 25 30
Gin Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Arg Pro Thr Gly 35 4〇 45 123528.doc 200819468
Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly lie Val Asp 5 0 55 60
Glu
Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys 65 70 75
Ala 80
Pro Leu Lys Pro Ala Arg Ser Ala 85 <210> 24 <211> 87 <212> PRT <213〉 人造 <220> <223> 融合蛋白px3036_IAFX_Fl K27R K65 K68R之胺基酸序列f : <400> 24 Met Asn His His His His His His lie Glu Gly Arg His Pro Arg 1 5 . 10 15
Pro
Pro Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu 20 25 30
Gin
Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Arg Pro Thr Gly 35 40 45
Tyr
Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly lie Val Asp Glu 50 55 60
Cys L)
Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala 65 7〇 75
Pro 80
Leu Lys Pro Ala Arg Ser Ala 85 <21〇> 25 <211> 90 <212〉 PRT <213〉 人造 <220> <223〉融合蛋白px3036_IAFX_F2 K27R K65 K68R之胺基酸序列 <400> 25 123528.doc 200819468
Met Asn His His His His His His Thr Glu Phe Glu Asn lie Glu His 15 10 15
Pro Arg Pro Pro Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp 20 25 30
Ala Leu Gin Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Arg Pro 35 40 45
Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gin Thr Gly lie Val 50 55 60
Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr 65 70 75 80
Cys Ala Pro Leu Lys Pro Ala Arg Ser Ala 85 90 -10- 123528.doc
Claims (1)
- 200819468 十、申請專利範圍: 1. 一種用於製造離胺酸-PEG化IGF-Ι或IGF-Ι變異體之方 法,該變異體包含一或兩個選自由離胺酸27、65及/或68 組成之群的胺基酸獨立經另一個極性胺基酸取代,其特 徵在於 a) 培養包含一種表現載體含有編碼一種融合蛋白之核酸 之原核宿主細胞,其中該融合蛋白包含該IGF-Ι或 IGF-Ι變異體N端與一種原肽(pro peptide)之c端連接, b) 該原肽在C端以胺基酸-Y-pro終止,其中γ係選自由 Pro、Pro-Ala、pr〇-Gly、Pro-Thr、Ala-Pro、Gly-Pro、Thr-Pro、Arg-Pro 或 Pro-Arg-Pro 組成之群, c) 回收及PEG化該融合蛋白, d) 用IgA蛋白麵裂解該peg化融合蛋白,及 e) 回收該PEG化IGF-Ι或IGF-Ι變異體。 2. 如請求項1之方法,其特徵在於該原肽係由下式顯示 Met-X 丨- Hisn-X2-Y-Pr〇*» 其中 • Met表示曱硫胺酸, • χι為一鍵、絲胺酸或天冬醯胺酸, • His為組胺酸, • η為一個〇至1〇之數, • X2為一個連接肽,其係選自由肽此卩ID ν〇:6-1(κ& 成之群, • Pro為脯胺酸,及 123528.doc 200819468 • Υ係選自由?1'〇、?1*〇-八比、?1'〇-〇以、?1*〇-丁111'、八^ Pro、Gly-Pro、Thr-Pro、Arg-Pro 或 Pro-Arg-pro 組 成之群。 3. 如請求項1或2之方法,其特徵在於該原肽不包含離胺酸 殘基。 4. 如請求項1或2之方法,其特徵在於該(等)極性胺基酸獨 立地為精胺酸、麩醯胺酸或天冬醯胺酸。 5. 如凊求項1或2之方法,其特徵在於該離胺酸_peg化IGF-Ϊ變異體係在離胺酸殘基65及68單PEG化或二PEG化。 6·如睛求項5之方法’其特徵在於該離胺酸—peg化IGF-Ι變 異體為RRK且該變異體係在離胺酸殘基68單peg化,或 該離胺酸-PEG化IGF_I變異體為RKR且該變異體係在離 胺酸殘基65單PEG化。 7·如凊求項5之方法,其特徵在於該離胺酸—peg化IGF-I變 異體為RKK且該變異體係在離胺酸殘基65及68單PEG化 或 ^一 P E G 4 匕。 8. 如晴求項5之方法,其特徵在於該離胺酸_peg化IGF-I變 異體為RRK、RKR或RKK之混合物且該變異體係在離胺 酸殘基65及68單PEG化或二PEG化。 9. 如睛求項1或2之方法,其特徵在於該離胺酸_pEC^IGF_ Ϊ係隨機單PEG化或二PEG化。 10·如請求項1或2之方法,其特徵在於該pEG具有20 kDa至 1 00 kDa之總分子量。 1 1 ·如请求項1或2之方法,其特徵在於該(等)聚(乙二醇)基 123528.doc 200819468 團為分枝聚(乙二醇)基團。 12· —種融合蛋白,其包含離胺酸_peg化IGF_;^ IGIM變異 體,该變異體包含一或兩個選自由離胺酸27、65及/或68 組成之群的胺基酸獨立經另一個極性胺基酸取代,N端 與種原肽之c端連接,其特徵在於該原肽在c端以胺基 酸-Y-Pro終止’其中 γ係選自由 Pr〇、pr〇_Ala、pr〇_Gly、 Pro-Thr、Ala-Pro、Gly-Pro、Thr-Pro、Arg-Pro 或 Pro- Arg-Pro組成之群。 13.如請求項12之融合蛋白,其特徵在於該原肽具有多至3〇 個胺基酸之長度。 14·如請求項12或13之融合蛋白,其特徵在於式 Met-XrHiSn-XrY-Pro-RGF-I或 IGF-I變異體], 其中 • IGF-I表示人類igF-I,IGF-I變異體表示igF-I中一 或兩個選自由離胺酸27、65及/或68組成之群之胺 基酸獨立經另一個極性胺基酸取代, • Met表示甲硫胺酸, • X1為一鍵、絲胺酸或天冬醯胺酸, • His為組胺酸, • η為一個〇至1〇之數, • χ2為一個連接肽,其係選自由肽SEQ ID N0:6-l 0組 成之群, • Pro為脯胺酸,及 • Y係選自由 Pro、Pro-Ala、Pro-Gly、pr〇-Thr、Ala- 123528.doc . ^ 200819468 Pro、Gly-Pro、Thr-Pro、Arg-Pro 或 Pro-Arg-Pro 組 成之群。 1 5 · —種離胺酸-PEG化IGF-I,其特徵在於不包含一個N端曱 硫胺酸。 16. —種離胺酸-PEG化IGF_I變異體,其中一或兩個選自由 離胺酸27、65及/或68組成之群之胺基酸獨立經另一個極 性fee基取代’其特徵在於該變異體不包含一個n端甲 硫胺酸。1 7. —種醫藥組合物,其包含如請求項丨5之離胺酸化 IGF-I或如請求項16之離胺酸·ρ]Ε〇化異體。 阿茲 18· 一種如請求項15之離胺酸-PEG化IGF-I或如請求項16之 離胺酸-PEG化IGF-I變異體之用途,其係用於製備治療 默氏病(Alzheimer’s disease)(AD)之藥物。 123528.doc 200819468 七、指定代表圖: (一) 本案指定代表圖為:第(1)圖。 (二) 本代表圖之元件符號簡單說明: (無元件符號說明) 八、本案若有化學式時,請揭示最能顯示發明特徵的化學式: (無) 123528.doc
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP06018170 | 2006-08-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
TW200819468A true TW200819468A (en) | 2008-05-01 |
Family
ID=37667695
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
TW096132101A TW200819468A (en) | 2006-08-31 | 2007-08-29 | Method for the production of conjugates of insulin-like growth factor I and poly(ethylene glycol) |
Country Status (21)
Country | Link |
---|---|
US (4) | US7625996B2 (zh) |
EP (1) | EP2059261B1 (zh) |
JP (1) | JP5184532B2 (zh) |
KR (1) | KR101106931B1 (zh) |
CN (1) | CN101511390B (zh) |
AR (1) | AR062575A1 (zh) |
AU (1) | AU2007291502B2 (zh) |
BR (1) | BRPI0715943A2 (zh) |
CA (1) | CA2662062C (zh) |
CL (1) | CL2007002502A1 (zh) |
CO (1) | CO6251278A2 (zh) |
CR (1) | CR10591A (zh) |
ES (1) | ES2397660T3 (zh) |
IL (1) | IL196736A (zh) |
MA (1) | MA30660B1 (zh) |
MX (1) | MX2009002011A (zh) |
NO (1) | NO20090451L (zh) |
PE (1) | PE20080912A1 (zh) |
RU (1) | RU2009106118A (zh) |
TW (1) | TW200819468A (zh) |
WO (1) | WO2008025528A1 (zh) |
Families Citing this family (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1674113A1 (en) | 2004-12-22 | 2006-06-28 | F. Hoffmann-La Roche Ag | Conjugates of insulin-like growth factor-1 (IGF-1) and poly(ethylene glycol) |
US8168181B2 (en) | 2006-02-13 | 2012-05-01 | Alethia Biotherapeutics, Inc. | Methods of impairing osteoclast differentiation using antibodies that bind siglec-15 |
CA2638823A1 (en) | 2006-02-13 | 2007-08-23 | Alethia Biotherapeutics Inc. | Polynucleotides and polypeptide sequences involved in the process of bone remodeling |
CL2007002502A1 (es) | 2006-08-31 | 2008-05-30 | Hoffmann La Roche | Variantes del factor de crecimiento similar a insulina-1 humano (igf-1) pegilados en lisina; metodo de produccion; proteina de fusion que la comprende; y su uso para tratar la enfermedad de alzheimer. |
WO2008025527A1 (en) | 2006-08-31 | 2008-03-06 | F. Hoffmann-La Roche Ag | Method for the production of insulin-like growth factor-i |
EP2249853A4 (en) | 2008-01-30 | 2012-12-26 | Univ Indiana Res & Tech Corp | PEPTIDE PRODRUGS BASED ON ESTERS |
AU2009231394B2 (en) * | 2008-04-03 | 2013-09-05 | F. Hoffmann-La Roche Ag | Use of PEGylated IGF-I variants for the treatment of neuromuscular disorders |
EP2376520B1 (en) | 2008-12-19 | 2014-02-12 | Indiana University Research&Technology Corporation | Insulin analogs |
WO2010080607A1 (en) * | 2008-12-19 | 2010-07-15 | Indiana University Research And Technology Corporation | Yl-based insulin-like growth factors exhibiting high activity at the insulin receptor |
CN102245624B (zh) | 2008-12-19 | 2016-08-10 | 印第安纳大学研究及科技有限公司 | 基于酰胺的胰岛素前药 |
US20110152188A1 (en) | 2009-12-23 | 2011-06-23 | Hanns-Christian Mahler | Pharmaceutical compositions of igf/i proteins |
KR101485646B1 (ko) * | 2010-02-11 | 2015-01-22 | 에프. 호프만-라 로슈 아게 | Igf―ⅰ 폴리(에틸렌 글리콜) 컨쥬게이트 |
JP5969469B2 (ja) | 2010-06-16 | 2016-08-17 | インディアナ ユニバーシティー リサーチ アンド テクノロジー コーポレーションIndiana University Research And Technology Corporation | インスリン受容体に対して高い活性を示す単鎖インスリンアゴニスト |
JP5912112B2 (ja) | 2010-06-24 | 2016-04-27 | インディアナ ユニバーシティー リサーチ アンド テクノロジー コーポレーションIndiana University Research And Technology Corporation | アミド系インスリンプロドラッグ |
GB201012784D0 (en) | 2010-07-29 | 2010-09-15 | Ucb Pharma Sa | Method |
WO2013096386A1 (en) | 2011-12-20 | 2013-06-27 | Indiana University Research And Technology Corporation | Ctp-based insulin analogs for treatment of diabetes |
CA2928851A1 (en) | 2012-07-19 | 2014-01-23 | Alethia Biotherapeutics Inc. | Anti-siglec-15 antibodies |
CN108383902A (zh) | 2012-09-26 | 2018-08-10 | 印第安纳大学研究及科技有限公司 | 胰岛素类似物二聚体 |
RU2678134C2 (ru) | 2013-03-14 | 2019-01-23 | Индиана Юниверсити Рисерч Энд Текнолоджи Корпорейшн | Конъюгаты инсулин-инкретин |
WO2014205617A1 (en) * | 2013-06-24 | 2014-12-31 | Shandong University | Lanthanide labeled peptide and use thereof |
BR112016005716A2 (pt) | 2013-10-02 | 2017-09-12 | Nat Institutes Of Health | miméticos de fator de crescimento semelhante à insulina para uso em terapia |
UY35874A (es) | 2013-12-12 | 2015-07-31 | Novartis Ag | Un proceso para la preparación de una composición de proteínas pegiladas |
EP3197912B1 (en) | 2014-09-24 | 2023-01-04 | Indiana University Research & Technology Corporation | Lipidated amide-based insulin prodrugs |
US10232020B2 (en) | 2014-09-24 | 2019-03-19 | Indiana University Research And Technology Corporation | Incretin-insulin conjugates |
EP3420087B1 (en) * | 2016-02-23 | 2021-09-01 | The Regents of The University of Colorado, A Body Corporate | Peptide-based methods for treating neurological injury |
EP3911668A1 (en) | 2019-01-18 | 2021-11-24 | The Regents of the University of Colorado, a body corporate | Amphipathic alpha-helical antimicrobial peptides treat infections by gram-negative pathogens |
Family Cites Families (46)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE220101T1 (de) | 1983-04-25 | 2002-07-15 | Chiron Corp | Hybrid-dns-synthesis von reifen insulinähnlichen wachstumsfaktoren |
IL71991A (en) | 1983-06-06 | 1994-05-30 | Genentech Inc | Preparation of human FGI and FGE in their processed form through recombinant AND tranology in prokaryotes |
US4904584A (en) | 1987-12-23 | 1990-02-27 | Genetics Institute, Inc. | Site-specific homogeneous modification of polypeptides |
US6235488B1 (en) | 1988-09-29 | 2001-05-22 | Agilent Technologies, Inc. | Surface preparation for chemical-specific binding |
EP0400472B1 (en) | 1989-05-27 | 1996-04-03 | Sumitomo Pharmaceuticals Company, Limited | Process for preparing polyethylene glycol derivatives and modified protein. |
US5093317A (en) * | 1989-06-05 | 1992-03-03 | Cephalon, Inc. | Treating disorders by application of insulin-like growth factor |
US6723699B1 (en) * | 1989-06-05 | 2004-04-20 | Cephalon, Inc. | Treating disorders by application of insulin-like growth factors and analogs |
DE3924705A1 (de) | 1989-07-26 | 1991-01-31 | Boehringer Mannheim Gmbh | Heterobifunktionelle verbindungen |
US5158875A (en) * | 1989-08-25 | 1992-10-27 | Amgen Inc. | Production of biologically active insulin-like growth factor i from high expression host cell systems |
NZ236819A (en) | 1990-02-03 | 1993-07-27 | Max Planck Gesellschaft | Enzymatic cleavage of fusion proteins; fusion proteins; recombinant dna and pharmaceutical compositions |
US5681814A (en) | 1990-06-07 | 1997-10-28 | Genentech, Inc. | Formulated IGF-I Composition |
JP3051145B2 (ja) | 1990-08-28 | 2000-06-12 | 住友製薬株式会社 | 新規なポリエチレングリコール誘導体修飾ペプチド |
US5861373A (en) | 1991-08-01 | 1999-01-19 | Genentech, Inc | IGF-1 to improve the neural condition |
DK0597033T3 (da) | 1991-08-01 | 1997-06-02 | Genentech Inc | IGF-1 til forbedring af den neurale tilstand |
AU6048294A (en) | 1992-11-25 | 1994-06-22 | Amgen Boulder Inc. | Modified insulin-like growth factors |
WO1995032003A1 (en) | 1994-05-24 | 1995-11-30 | Amgen Boulder Inc. | Modified insulin-like growth factors |
US5824784A (en) | 1994-10-12 | 1998-10-20 | Amgen Inc. | N-terminally chemically modified protein compositions and methods |
US5932462A (en) | 1995-01-10 | 1999-08-03 | Shearwater Polymers, Inc. | Multiarmed, monofunctional, polymer for coupling to molecules and surfaces |
US5741776A (en) * | 1995-05-22 | 1998-04-21 | Genentech, Inc. | Method of administration of IGF-I |
US5672662A (en) | 1995-07-07 | 1997-09-30 | Shearwater Polymers, Inc. | Poly(ethylene glycol) and related polymers monosubstituted with propionic or butanoic acids and functional derivatives thereof for biotechnical applications |
EP1017794A1 (en) | 1997-02-06 | 2000-07-12 | Novo Nordisk A/S | Polypeptide-polymer conjugates having added and/or removed attachment groups |
US20030069177A1 (en) * | 2000-05-16 | 2003-04-10 | Genentech, Inc. | Method for treating cartilage disorders |
US20030204864A1 (en) * | 2001-02-28 | 2003-10-30 | Henry Daniell | Pharmaceutical proteins, human therapeutics, human serum albumin, insulin, native cholera toxic b submitted on transgenic plastids |
US6767892B1 (en) | 1997-11-07 | 2004-07-27 | Chrion Corporation | Compositions providing for increased IGF-I solubility |
US7067485B2 (en) | 1997-11-07 | 2006-06-27 | Chiron Corporation | IGF-I composition and its use |
US6248546B1 (en) | 1998-03-09 | 2001-06-19 | Diagnostic Systems Laboratories, Inc. | Assay of IGFBP complex |
WO1999055362A1 (en) | 1998-04-29 | 1999-11-04 | Genentech, Inc. | Spray dried formulations of igf-i |
US6436897B2 (en) | 1998-06-01 | 2002-08-20 | Celtrix Pharmaceuticals, Inc. | Pharmaceutical formulations for IGF/IGFBP |
EP0972838B1 (en) | 1998-07-15 | 2004-09-15 | Roche Diagnostics GmbH | Escherichia coli host/vector system based on antibiotic-free selection by complementation of an auxotrophy |
IL143834A0 (en) | 1999-01-06 | 2002-04-21 | Genentech Inc | Insulin-like growth factor (igf) i mutant variants |
EP1141014B1 (en) | 1999-01-06 | 2004-12-08 | Genentech, Inc. | Insulin-like growth factor (igf) i mutant variant |
PT1165119E (pt) | 1999-04-08 | 2004-02-27 | Genentech Inc | Composicao baseada em polipeptidos com cargas opostas |
US6596849B1 (en) | 1999-05-28 | 2003-07-22 | Academia Sinica | Monoclonal-antibody for analysis and clearance of polyethylene glycol and polyethylene glycol-modified molecules |
US7431921B2 (en) | 2000-04-14 | 2008-10-07 | Maxygen Aps | Interferon beta-like molecules |
WO2001088149A1 (fr) * | 2000-05-16 | 2001-11-22 | Center For Advanced Science And Technology Incubation, Ltd. | Polypeptides de clivage de repetitions telomeriques |
US20040014652A1 (en) | 2000-06-01 | 2004-01-22 | Andre Trouet | Tumor activated prodrug compounds and methods of making and using the same |
WO2001091798A2 (en) | 2000-06-01 | 2001-12-06 | Universite Catholique De Louvain | Tumor activated prodrug compounds |
EP1370282A2 (en) | 2000-10-13 | 2003-12-17 | Chiron Corporation | Method for treating ischemic events affecting the central nervous system |
CA2716959A1 (en) | 2000-12-07 | 2002-06-13 | Eli Lilly And Company | Glp-1 fusion proteins |
WO2002094853A2 (en) | 2001-05-21 | 2002-11-28 | Nektar Therapeutics Al, Corporation | Antibodies specific for poly(ethylene glycol) |
KR101162908B1 (ko) * | 2002-12-26 | 2012-07-06 | 마운틴 뷰 파마슈티컬즈, 인크. | 수용체-결합 활성이 보존된, 사이토카인, 케모카인,성장인자, 폴리펩티드 호르몬 및 이들의 길항제의 중합체접합체 |
EP1674113A1 (en) | 2004-12-22 | 2006-06-28 | F. Hoffmann-La Roche Ag | Conjugates of insulin-like growth factor-1 (IGF-1) and poly(ethylene glycol) |
JP4988599B2 (ja) | 2005-01-07 | 2012-08-01 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | Igf−1融合ポリペプチドおよびその治療的使用 |
US7612178B2 (en) * | 2006-03-28 | 2009-11-03 | Biogen Idec Ma Inc | Anti-IGF-1R antibodies and uses thereof |
CL2007002502A1 (es) | 2006-08-31 | 2008-05-30 | Hoffmann La Roche | Variantes del factor de crecimiento similar a insulina-1 humano (igf-1) pegilados en lisina; metodo de produccion; proteina de fusion que la comprende; y su uso para tratar la enfermedad de alzheimer. |
AU2009231394B2 (en) | 2008-04-03 | 2013-09-05 | F. Hoffmann-La Roche Ag | Use of PEGylated IGF-I variants for the treatment of neuromuscular disorders |
-
2007
- 2007-08-28 CL CL200702502A patent/CL2007002502A1/es unknown
- 2007-08-29 AU AU2007291502A patent/AU2007291502B2/en not_active Ceased
- 2007-08-29 PE PE2007001177A patent/PE20080912A1/es not_active Application Discontinuation
- 2007-08-29 ES ES07801961T patent/ES2397660T3/es active Active
- 2007-08-29 AR ARP070103823A patent/AR062575A1/es not_active Application Discontinuation
- 2007-08-29 CN CN2007800319871A patent/CN101511390B/zh not_active Expired - Fee Related
- 2007-08-29 US US11/846,857 patent/US7625996B2/en not_active Expired - Fee Related
- 2007-08-29 EP EP07801961A patent/EP2059261B1/en active Active
- 2007-08-29 JP JP2009525971A patent/JP5184532B2/ja not_active Expired - Fee Related
- 2007-08-29 WO PCT/EP2007/007540 patent/WO2008025528A1/en active Application Filing
- 2007-08-29 RU RU2009106118/10A patent/RU2009106118A/ru not_active Application Discontinuation
- 2007-08-29 MX MX2009002011A patent/MX2009002011A/es active IP Right Grant
- 2007-08-29 BR BRPI0715943-9A patent/BRPI0715943A2/pt not_active IP Right Cessation
- 2007-08-29 KR KR1020097003975A patent/KR101106931B1/ko not_active IP Right Cessation
- 2007-08-29 TW TW096132101A patent/TW200819468A/zh unknown
- 2007-08-29 CA CA2662062A patent/CA2662062C/en not_active Expired - Fee Related
-
2009
- 2009-01-26 IL IL196736A patent/IL196736A/en active IP Right Grant
- 2009-01-29 CR CR10591A patent/CR10591A/es not_active Application Discontinuation
- 2009-01-29 NO NO20090451A patent/NO20090451L/no not_active Application Discontinuation
- 2009-01-30 CO CO09008773A patent/CO6251278A2/es not_active Application Discontinuation
- 2009-02-25 MA MA31660A patent/MA30660B1/fr unknown
- 2009-10-09 US US12/576,266 patent/US20100035817A1/en not_active Abandoned
-
2010
- 2010-04-27 US US12/767,829 patent/US20100210547A1/en not_active Abandoned
-
2012
- 2012-08-21 US US13/590,549 patent/US8476232B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
US8476232B2 (en) | 2013-07-02 |
CN101511390A (zh) | 2009-08-19 |
AU2007291502A1 (en) | 2008-03-06 |
EP2059261A1 (en) | 2009-05-20 |
MA30660B1 (fr) | 2009-08-03 |
US20130065831A1 (en) | 2013-03-14 |
CN101511390B (zh) | 2013-04-24 |
PE20080912A1 (es) | 2008-07-19 |
CR10591A (es) | 2009-04-03 |
EP2059261B1 (en) | 2012-10-24 |
CO6251278A2 (es) | 2011-02-21 |
CL2007002502A1 (es) | 2008-05-30 |
WO2008025528A1 (en) | 2008-03-06 |
IL196736A (en) | 2013-10-31 |
AU2007291502B2 (en) | 2012-05-31 |
US20100035817A1 (en) | 2010-02-11 |
KR20090046875A (ko) | 2009-05-11 |
AR062575A1 (es) | 2008-11-19 |
RU2009106118A (ru) | 2010-10-10 |
CA2662062C (en) | 2015-06-23 |
US20080119409A1 (en) | 2008-05-22 |
JP5184532B2 (ja) | 2013-04-17 |
MX2009002011A (es) | 2009-03-05 |
KR101106931B1 (ko) | 2012-01-25 |
US7625996B2 (en) | 2009-12-01 |
US20100210547A1 (en) | 2010-08-19 |
CA2662062A1 (en) | 2008-03-06 |
ES2397660T3 (es) | 2013-03-08 |
BRPI0715943A2 (pt) | 2013-07-30 |
NO20090451L (no) | 2009-02-26 |
JP2010501192A (ja) | 2010-01-21 |
IL196736A0 (en) | 2009-11-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
TW200819468A (en) | Method for the production of conjugates of insulin-like growth factor I and poly(ethylene glycol) | |
EP2729160B1 (en) | Engineered polypeptides having enhanced duration of action and reduced immunogenicity | |
CA2658736C (en) | Method for the production of insulin-like growth factor-i | |
KR20130009761A (ko) | 메티오닌-감마-라이아제 효소를 이용하여 유전자조작된 효소 및 이의 약리학적 제제 | |
US20160060317A1 (en) | Novel neurturin conjugates for pharmaceutical use | |
JP2017205120A (ja) | コリンエステラーゼ部分とポリマーとのコンジュゲート | |
JP2022118184A (ja) | コンジュゲートされたc1エステラーゼインヒビター及びその使用 | |
KR20200006568A (ko) | 고호모시스테인혈증 및 호모시스틴뇨증 환자를 치료하기 위한 인간-효소 매개된 호모시스테인의 고갈 | |
JP2019520392A (ja) | ヒト酵素媒介性シスチン枯渇 | |
TW201838647A (zh) | 對胰島素受體有降低親和性之胰島素類似物之接合物及其用途 | |
KR20160095133A (ko) | Peg화 단백질의 조성물을 제조하는 방법 | |
JP2024527613A (ja) | 代謝性障害のための融合ポリペプチド | |
CN117586423A (zh) | 用于代谢病症的融合多肽 | |
JP2022505689A (ja) | 治療的使用のための、遺伝子操作された霊長類シスチン/システイン分解酵素 |