HRP20010901A2 - Compositions of a-beta peptide and processes for producing same - Google Patents
Compositions of a-beta peptide and processes for producing same Download PDFInfo
- Publication number
- HRP20010901A2 HRP20010901A2 HR20010901A HRP20010901A HRP20010901A2 HR P20010901 A2 HRP20010901 A2 HR P20010901A2 HR 20010901 A HR20010901 A HR 20010901A HR P20010901 A HRP20010901 A HR P20010901A HR P20010901 A2 HRP20010901 A2 HR P20010901A2
- Authority
- HR
- Croatia
- Prior art keywords
- peptide
- preparation
- solution
- pharmaceutically acceptable
- suspension
- Prior art date
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 234
- 239000000203 mixture Substances 0.000 title claims description 116
- 238000000034 method Methods 0.000 title claims description 50
- 230000008569 process Effects 0.000 title claims description 16
- 239000000243 solution Substances 0.000 claims description 82
- 238000002360 preparation method Methods 0.000 claims description 80
- 108010064539 amyloid beta-protein (1-42) Proteins 0.000 claims description 75
- 239000000725 suspension Substances 0.000 claims description 55
- 239000000872 buffer Substances 0.000 claims description 44
- 150000003839 salts Chemical class 0.000 claims description 27
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 26
- 238000001914 filtration Methods 0.000 claims description 25
- 239000002671 adjuvant Substances 0.000 claims description 24
- 229940024606 amino acid Drugs 0.000 claims description 22
- 150000001413 amino acids Chemical class 0.000 claims description 21
- 239000007864 aqueous solution Substances 0.000 claims description 20
- 239000012528 membrane Substances 0.000 claims description 20
- 239000002253 acid Substances 0.000 claims description 18
- 239000004247 glycine and its sodium salt Substances 0.000 claims description 18
- 235000013905 glycine and its sodium salt Nutrition 0.000 claims description 18
- 229940029258 sodium glycinate Drugs 0.000 claims description 18
- WUWHFEHKUQVYLF-UHFFFAOYSA-M sodium;2-aminoacetate Chemical compound [Na+].NCC([O-])=O WUWHFEHKUQVYLF-UHFFFAOYSA-M 0.000 claims description 18
- 241000124008 Mammalia Species 0.000 claims description 16
- 239000004471 Glycine Substances 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 208000024827 Alzheimer disease Diseases 0.000 claims description 10
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical class [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 10
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 10
- 239000011148 porous material Substances 0.000 claims description 10
- 238000004108 freeze drying Methods 0.000 claims description 8
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 8
- 239000008181 tonicity modifier Substances 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 150000008044 alkali metal hydroxides Chemical class 0.000 claims description 6
- 239000002738 chelating agent Substances 0.000 claims description 6
- 230000009851 immunogenic response Effects 0.000 claims description 6
- 238000007911 parenteral administration Methods 0.000 claims description 6
- 230000003113 alkalizing effect Effects 0.000 claims description 5
- 230000005875 antibody response Effects 0.000 claims description 5
- 229940079593 drug Drugs 0.000 claims description 5
- 239000004094 surface-active agent Substances 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 4
- 230000002163 immunogen Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 150000007522 mineralic acids Chemical class 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- 150000007524 organic acids Chemical class 0.000 claims description 4
- 230000004044 response Effects 0.000 claims description 4
- 239000008174 sterile solution Substances 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 229960003121 arginine Drugs 0.000 claims description 2
- 230000008014 freezing Effects 0.000 claims description 2
- 238000007710 freezing Methods 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 229940037003 alum Drugs 0.000 claims 2
- 230000001939 inductive effect Effects 0.000 claims 2
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 claims 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims 1
- 230000000890 antigenic effect Effects 0.000 claims 1
- 229960003589 arginine hydrochloride Drugs 0.000 claims 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims 1
- 229920001477 hydrophilic polymer Polymers 0.000 claims 1
- 238000009472 formulation Methods 0.000 description 84
- 102000004196 processed proteins & peptides Human genes 0.000 description 25
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 22
- 229930006000 Sucrose Natural products 0.000 description 20
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 20
- 239000005720 sucrose Substances 0.000 description 20
- 229920000053 polysorbate 80 Polymers 0.000 description 18
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 16
- 229930195725 Mannitol Natural products 0.000 description 16
- 239000000594 mannitol Substances 0.000 description 16
- 235000010355 mannitol Nutrition 0.000 description 16
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 16
- 229940068968 polysorbate 80 Drugs 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 239000002245 particle Substances 0.000 description 15
- 238000004007 reversed phase HPLC Methods 0.000 description 15
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 12
- 239000002585 base Substances 0.000 description 11
- 238000012512 characterization method Methods 0.000 description 10
- -1 his Chemical compound 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 9
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 9
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 9
- 238000002955 isolation Methods 0.000 description 9
- 238000011146 sterile filtration Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 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 8
- 238000004458 analytical method Methods 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 235000000346 sugar Nutrition 0.000 description 8
- 239000007853 buffer solution Substances 0.000 description 7
- 238000002983 circular dichroism Methods 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 7
- 150000008163 sugars Chemical class 0.000 description 7
- 150000007513 acids Chemical class 0.000 description 6
- 125000003275 alpha amino acid group Chemical group 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 239000007979 citrate buffer Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 229920000136 polysorbate Polymers 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 108010064397 amyloid beta-protein (1-40) Proteins 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 5
- 238000004090 dissolution Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- 238000004448 titration Methods 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-M Aminoacetate Chemical compound NCC([O-])=O DHMQDGOQFOQNFH-UHFFFAOYSA-M 0.000 description 4
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 239000000370 acceptor Substances 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 238000001802 infusion Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 229950008882 polysorbate Drugs 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 238000011069 regeneration method Methods 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 208000037259 Amyloid Plaque Diseases 0.000 description 3
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 3
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000005755 formation reaction Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 239000012669 liquid formulation Substances 0.000 description 3
- 239000012931 lyophilized formulation Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000001471 micro-filtration Methods 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 235000011083 sodium citrates Nutrition 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- 150000005846 sugar alcohols Chemical class 0.000 description 3
- 238000000954 titration curve Methods 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 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 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 2
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- 101000823051 Homo sapiens Amyloid-beta precursor protein Proteins 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 239000000908 ammonium hydroxide Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 239000006172 buffering agent Substances 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001142 circular dichroism spectrum Methods 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 239000003002 pH adjusting agent Substances 0.000 description 2
- 238000010979 pH adjustment Methods 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- PBDAWQLIMPWEEF-JEDNCBNOSA-M sodium;(2s)-2,6-diaminohexanoate Chemical compound [Na+].NCCCC[C@H](N)C([O-])=O PBDAWQLIMPWEEF-JEDNCBNOSA-M 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000008215 water for injection Substances 0.000 description 2
- 239000000811 xylitol Substances 0.000 description 2
- 235000010447 xylitol Nutrition 0.000 description 2
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 2
- 229960002675 xylitol Drugs 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical group CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- BRMWTNUJHUMWMS-UHFFFAOYSA-N 3-Methylhistidine Natural products CN1C=NC(CC(N)C(O)=O)=C1 BRMWTNUJHUMWMS-UHFFFAOYSA-N 0.000 description 1
- 108010067409 AN-1792 Proteins 0.000 description 1
- ZSOICJZJSRWNHX-ACZMJKKPSA-N Ala-Ile Chemical compound CC[C@H](C)[C@@H](C([O-])=O)NC(=O)[C@H](C)[NH3+] ZSOICJZJSRWNHX-ACZMJKKPSA-N 0.000 description 1
- LNNSWWRRYJLGNI-NAKRPEOUSA-N Ala-Ile-Val Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O LNNSWWRRYJLGNI-NAKRPEOUSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 101800001718 Amyloid-beta protein Proteins 0.000 description 1
- 241000272478 Aquila Species 0.000 description 1
- KWTQSFXGGICVPE-WCCKRBBISA-N Arginine hydrochloride Chemical compound Cl.OC(=O)[C@@H](N)CCCN=C(N)N KWTQSFXGGICVPE-WCCKRBBISA-N 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000759568 Corixa Species 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 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
- 239000007836 KH2PO4 Substances 0.000 description 1
- 125000000998 L-alanino group Chemical group [H]N([*])[C@](C([H])([H])[H])([H])C(=O)O[H] 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-M L-lysinate Chemical compound NCCCC[C@H](N)C([O-])=O KDXKERNSBIXSRK-YFKPBYRVSA-M 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001092142 Molina Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- NTNWOCRCBQPEKQ-YFKPBYRVSA-N N(omega)-methyl-L-arginine Chemical compound CN=C(N)NCCC[C@H](N)C(O)=O NTNWOCRCBQPEKQ-YFKPBYRVSA-N 0.000 description 1
- JDHILDINMRGULE-LURJTMIESA-N N(pros)-methyl-L-histidine Chemical compound CN1C=NC=C1C[C@H](N)C(O)=O JDHILDINMRGULE-LURJTMIESA-N 0.000 description 1
- 125000003047 N-acetyl group Chemical group 0.000 description 1
- JJIHLJJYMXLCOY-BYPYZUCNSA-N N-acetyl-L-serine Chemical compound CC(=O)N[C@@H](CO)C(O)=O JJIHLJJYMXLCOY-BYPYZUCNSA-N 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 229910004835 Na2B4O7 Inorganic materials 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 108010050254 Presenilins Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 241001454523 Quillaja saponaria Species 0.000 description 1
- 235000009001 Quillaja saponaria Nutrition 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical class OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000012382 advanced drug delivery Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 239000002610 basifying agent Substances 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000005388 borosilicate glass Substances 0.000 description 1
- 238000011095 buffer preparation Methods 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- UHBYWPGGCSDKFX-UHFFFAOYSA-N carboxyglutamic acid Chemical compound OC(=O)C(N)CC(C(O)=O)C(O)=O UHBYWPGGCSDKFX-UHFFFAOYSA-N 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 150000001805 chlorine compounds Chemical class 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
- 238000000978 circular dichroism spectroscopy Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010960 commercial process Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000019262 disodium citrate Nutrition 0.000 description 1
- 239000002526 disodium citrate Substances 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- CEYULKASIQJZGP-UHFFFAOYSA-L disodium;2-(carboxymethyl)-2-hydroxybutanedioate Chemical compound [Na+].[Na+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O CEYULKASIQJZGP-UHFFFAOYSA-L 0.000 description 1
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- CETRZFQIITUQQL-UHFFFAOYSA-N dmso dimethylsulfoxide Chemical compound CS(C)=O.CS(C)=O CETRZFQIITUQQL-UHFFFAOYSA-N 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- VFRSADQPWYCXDG-LEUCUCNGSA-N ethyl (2s,5s)-5-methylpyrrolidine-2-carboxylate;2,2,2-trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.CCOC(=O)[C@@H]1CC[C@H](C)N1 VFRSADQPWYCXDG-LEUCUCNGSA-N 0.000 description 1
- 229940071106 ethylenediaminetetraacetate Drugs 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000005660 hydrophilic surface Effects 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229960000448 lactic acid Drugs 0.000 description 1
- GZQKNULLWNGMCW-PWQABINMSA-N lipid A (E. coli) Chemical compound O1[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)[C@@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@@H]1OC[C@@H]1[C@@H](O)[C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)O1 GZQKNULLWNGMCW-PWQABINMSA-N 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000012792 lyophilization process Methods 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 230000002887 neurotoxic effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000008288 physiological mechanism Effects 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920006393 polyether sulfone Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000013097 stability assessment Methods 0.000 description 1
- 238000012430 stability testing Methods 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000001665 trituration Methods 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0007—Nervous system antigens; Prions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Neurosurgery (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Neurology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Psychiatry (AREA)
- Hospice & Palliative Care (AREA)
- Dermatology (AREA)
- Marine Sciences & Fisheries (AREA)
- Organic Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
DOSADAŠNJE SPOZNAJE PREVIOUS KNOWLEDGE
Područje izuma Field of invention
Ovaj izum se općenito odnosi na farmaceutske pripravke koji sadrže proteine koji su korisni za povećanje odgovora antitijela u sisavcima. Specifičnije, ovaj izum se odnosi na farmaceutski prihvatljive pripravke koji sadrže količinu amiloidnog beta peptida učinkovitog da izazove imunogeni odgovor u sisavcu, te farmaceutski prihvatljiv razrjeđivač. Preferirano, razrjeđivač je sterilna parenetalno prihvatljiva vodena faza. This invention relates generally to pharmaceutical compositions containing proteins useful for enhancing antibody responses in mammals. More specifically, the present invention relates to pharmaceutically acceptable compositions comprising an amount of amyloid beta peptide effective to elicit an immunogenic response in a mammal, and a pharmaceutically acceptable diluent. Preferably, the diluent is a sterile parenterally acceptable aqueous phase.
Dosadašnja znanja Previous knowledge
Amiloidni beta peptid, također poznat kao A-beta ili Aβ peptid je produkt cijepanja proteina koji je amiloidni prekursor (APP). To je osnovna komponenta amiloidnog plaka u mozgu sisavaca stoje osnovna karakteristika Alzheimerove bolesti. Aβ peptid je 39-43 aminokiselinski lanac kojem se duljina promjenljiva zbog različitog postupka dobivanja iz APP, a od nekoliko proteaza. Amyloid beta peptide, also known as A-beta or Aβ peptide, is a cleavage product of the amyloid precursor protein (APP). It is the basic component of the amyloid plaque in the brain of mammals, which is the basic characteristic of Alzheimer's disease. Aβ peptide is a 39-43 amino acid chain, the length of which is variable due to the different process of obtaining it from APP, and from several proteases.
Nekoliko mutacija unutar APP proteina je korelirano s postojanjem Azheimerove bolesti. Vidi npr. Goate et al. Nature, 349, 704 (1991) (valin717 u izoeucin); Chartier Haran et al. Nature 353, 844 (1991) (valin717 u glicin); Murrell et al. Science 254, 97 (1991) (valin717 u fenilalanin); Mullan et al. Nature Genet. I, 345 (1992) (dvostruka mutacijamijenjanjalizin595-metionina596 u asparagin595-leucin596). Pokazano je da takve mutacije uzrokuju Alzheimerovu dolest, a povećanim ili promijenjenim procesuiranjem APP u Aβ peptid, posebice procesuiranjem APP u povećanu količinu Aβ42 i Aβ43. Mutacije u drugim genima, kao što su presenilin geni, PSI i PS2 se smatraju da neizravno djeluju na porcesuiranje APP i stvaranju povećane količine Aβ42 i Aβ43 (vidi Hardy, TINS 20, 154 (1997)). Opažanje pokazuje da je Aβ peptid, posebice Aβ42, uzročni element Alzheimerove bolesti. U mozgu dolazi do agregacije Aβ peptida i stvara se amilodna naslaga koja sadrži peptide organizirane u strukturu fibrila ili β-nabrane ploče. Several mutations within the APP protein have been correlated with the existence of Alzheimer's disease. See eg Goate et al. Nature, 349, 704 (1991) (valine717 to isoeucine); Chartier Haran et al. Nature 353, 844 (1991) (valine717 to glycine); Murrell et al. Science 254, 97 (1991) (valine717 to phenylalanine); Mullan et al. Nature Genet. I, 345 (1992) (double mutation changing lysine595-methionine596 to asparagine595-leucine596). It has been shown that such mutations cause Alzheimer's disease, and by increased or changed processing of APP into Aβ peptide, especially by processing APP into an increased amount of Aβ42 and Aβ43. Mutations in other genes, such as the presenilin genes, PSI and PS2, are thought to act indirectly to process APP and generate increased amounts of Aβ42 and Aβ43 (see Hardy, TINS 20, 154 (1997)). The observation shows that Aβ peptide, especially Aβ42, is the causative element of Alzheimer's disease. In the brain, aggregation of Aβ peptides occurs and amyloid deposits are formed, which contain peptides organized into the structure of fibrils or β-pleated plates.
Napor u nedavnog istraživanju terapeutskog tretmana ili prevencije Alzheimerove bolesti je usmjeren na zadržavanje ili usporavanje produkcije Aβ peptida u mozgu, a blokiranjem procesuiranja ili depozicije u amilodini plak nakon osobađanja. Jedan terapeutski prisup od posebne važnosti je primjena ovog izuma za upotrebu Aβ peptida za indukciju imuno odgovora tijela protiv toga. Vidi, primjerice PCT pubikaciju br. WO99/27944, koja je ovdje ugrađena citatom u potpunosti za sve svrhe. Efforts in recent research into the therapeutic treatment or prevention of Alzheimer's disease are focused on stopping or slowing down the production of Aβ peptides in the brain, by blocking processing or deposition into amyloidin plaques after personalization. One therapeutic approach of particular importance is the application of the present invention to the use of Aβ peptides to induce the body's immune response against it. See, for example, PCT publication no. WO99/27944, which is incorporated herein by reference in its entirety for all purposes.
Ovaj izum je upućen na nove neočekivane metode za upotrebu izuma opisane u PCT pubikaciji WO99/27944. Određenije, to obuhvaća davanje neke formulacije dugog oblika Aβ peptida pacijentu da se izazove imuni odgovor. Međutim, kao stoje naznačeno u struci, dulji oblici Aβ peptida su teško topljivi u konvencionalnim sustavima formulacija. This invention is directed to new unexpected methods for using the invention described in PCT publication WO99/27944. More particularly, it involves administering some formulation of the long form of Aβ peptide to a patient to induce an immune response. However, as indicated in the art, the longer forms of the Aβ peptide are poorly soluble in conventional formulation systems.
Hilbich et al. J. Mol. Biol., 218 (1), str. 149-64 (1991) su pokazali da Aβ-43 peptid topljiv do neko stupnja u čistoj vodi, dodatak ionskih komponenata kao što su puferi ili soli, ili orgnaska otapala uzrokuju da se peptid taloži u otopini u obliku amorgfnog agregata. Primjerice, Hibich je našao da fiziološka otopina fosfatnog pufera ("PBS" koji ovdje sadrži 137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4 · 2H2O i 2 mM KH2PO4 pri pH 7.5) čini 90-94% peptida netopljivim u pripravku. PBS je uobičajeni nosač parenteralnih pripravaka, približne toničnost i pH razine živom sustavu. Pet (5) mM NaCl uzrokuje da se 42-50% peptida taloži, (ibid, str. 153, Tablica 2). Otopina peptida u čistoj vodi bi bila hipotonična, pH 5.5 takve otopine je određen od Hibich (idem.). Tipično, pH krvi u čovjeka je 7.4. Dyrks et al. su također pokazali da je Aβ42 netopljiv u fiziološkim uvjetima. Dyrks, T., Weidemann, A., Multhaup, G., et al. EMBO J.. 7, str. 949-57 (1988). Hilbich et al. J. Mol. Biol., 218 (1), p. 149-64 (1991) have shown that the Aβ-43 peptide is soluble to some degree in pure water, the addition of ionic components such as buffers or salts, or organic solvents cause the peptide to precipitate in solution in the form of amorphous aggregates. For example, Hibich found that physiological phosphate buffer solution ("PBS" here containing 137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4 · 2H2O and 2 mM KH2PO4 at pH 7.5) made 90-94% of peptides insoluble in the preparation. PBS is a common carrier of parenteral preparations, approximating tonicity and pH levels to the living system. Five (5) mM NaCl causes 42-50% of the peptide to precipitate, (ibid, p. 153, Table 2). A peptide solution in pure water would be hypotonic, pH 5.5 of such a solution was determined by Hibich (idem.). Typically, the pH of human blood is 7.4. Dyrks et al. have also shown that Aβ42 is insoluble under physiological conditions. Dyrks, T., Weidemann, A., Multhaup, G., et al. EMBO J.. 7, p. 949-57 (1988).
Konformacija Aβ peptida u otopim se može mjeriti upotrebom spektroskopije cirkularnog dikroizma (C.D.). Konformacijska istraživanja Aβ peptida i fragmenata upotrebom C. D. su prikazani od Hilbich et al. idem. Vidi također monografiju od M. Manning pod naslovom: Protein Structure and Stability Assesment by Circular Dicroism Spectroscopy, od Bioctalyst design for Stability and Specificity; Himmel M. E. i Georiou, G., editori, ACS Symposium Series 516 (1993) na str. 36. Ta referenca se nakon ovog odnosi na Manningovu referencu, koja je ovdje ugrađena citatom u cijelosti za sve svrhe. The conformation of the Aβ peptide in solution can be measured using circular dichroism (C.D.) spectroscopy. Conformational studies of Aβ peptides and fragments using C. D. are presented by Hilbich et al. I am going to. See also the monograph by M. Manning entitled: Protein Structure and Stability Assessment by Circular Dichroism Spectroscopy, by Bioctalyst design for Stability and Specificity; Himmel M. E. and Georiou, G., editors, ACS Symposium Series 516 (1993) at p. 36. That reference hereinafter refers to the Manning reference, which is incorporated herein by quotation in its entirety for all purposes.
Kline et al., U. S. Patent br. 5,851,996 ('996 patent) i 5,753,624 ('624 patent) opisuju davanje vrlo male količline (10-2 mg ili manje) Aβ peptida ili njegovog fragmenta danog sublinvalno u tekućini ili čvrstom nosaču, kao stoje fenilirana fiziološka otopina. '996 patent tvrdi da amiloidni beta protein "postoji u različitim sturkturnim oblicima" (kolona 2, rad 31) što se može usporediti za tretman Alzheimerove bolesti. Na drugom mjestu nije definirano što se mislilo s različitim strukturnim oblicima, niti je bio koji karakteriziran osim fragmenta od 28 aminokiselina korištenih u primjerima. U patentu '996 se tvrdi da su doze Aβ peptida od 10-10 do 10-12 mg (kolona 8, redovi 442-32). Kline et al., U.S. Patent No. 5,851,996 ('996 patent) and 5,753,624 ('624 patent) describe the administration of a very small amount (10-2 mg or less) of Aβ peptide or fragment thereof given sublinally in a liquid or solid carrier, such as phenylated saline. The '996 patent claims that amyloid beta protein "exists in various structural forms" (column 2, paper 31) which can be compared for the treatment of Alzheimer's disease. Elsewhere, what was meant by the various structural forms was not defined, nor was any characterized other than the 28 amino acid fragment used in the examples. The '996 patent claims doses of Aβ peptide from 10-10 to 10-12 mg (column 8, lines 442-32).
Sa gornjeg stanovišta, prethodne spoznaje su pokazale poteškoću u otapanju Aβ peptida održavanju ih otopljenim. Nadalje, netopljivost dugih oblika Aβ peptida predstavlja poteškoće sa stanovišta sterilizacije i standardizacije. Većina standardnih metoda sterilizacije su nekompatibilni s formulacijama peptida, uključujući i radijaciju, sterilizaciju autoklavnim ili kemijskim tehnikama, kao što je plin etienoksid ili glutaraldehid, od kojih svi uzrokuju degradaciju peptida. Stoga, filtracija peptida bi bila metoda od izbora za sterilizaciju formulacije Aβ peptida. Nažalost, netopljivost Aβ peptida uzrokuje začepljenje filtracijskih membrana i sprječava izolaciju dovoljne količine Aβ peptida u postupku na komercijanom mjerilu. From the above point of view, previous findings have shown the difficulty in dissolving Aβ peptides and keeping them dissolved. Furthermore, the insolubility of long forms of Aβ peptides presents difficulties from the point of view of sterilization and standardization. Most standard sterilization methods are incompatible with peptide formulations, including radiation, sterilization by autoclave or chemical techniques, such as ethylene oxide gas or glutaraldehyde, all of which cause peptide degradation. Therefore, peptide filtration would be the method of choice for sterilization of Aβ peptide formulation. Unfortunately, the insolubility of Aβ peptides causes clogging of filtration membranes and prevents the isolation of a sufficient amount of Aβ peptides in a commercial-scale process.
Sažetak izuma Summary of the invention
Ovaj izum je upućen naieočekiano otkriće da vodene otopine koje sadrže visoku koncentraciju Aβ peptida se mogu pripraviti podešavanjem kiselosti/bazičnosti takvih vodenih otopina na pH učinkovit da otopi Aβ peptid. Preferirano pH je podešen na raspon pH od oko 8.5 do oko 12, preferiranije od oko 9 do oko 10. This invention is directed to the anticipated discovery that aqueous solutions containing a high concentration of Aβ peptide can be prepared by adjusting the acidity/basicity of such aqueous solutions to a pH effective to dissolve the Aβ peptide. Preferably the pH is adjusted to a pH range of from about 8.5 to about 12, more preferably from about 9 to about 10.
Ovaj izum je nadalje upućen na otkriće da se otopljeni Aβ peptid u otopini može sterilno filtrirati preko pogodnih filtera s mikroporama, a uz izolaciju najmanje 50% Aβ peptida nakon sterilne filtracije. Preferirano se izolira 70% Aβ peptida nakon sterilne filtracije, a još preferiranije se izolira 90% Aβ peptida nakon sterilne filtracije. Takve sterilne otopine se mogu formulirati kao farmaceutski pripravci koji sadrže dovoljnu količinu A p peptida koja izaziva imunogeni odgovor kada je dan sisavcu. Preferirano, takvo davanje je parenteralno davanje, u obliku suspenzije pripravka. This invention is further directed to the discovery that dissolved Aβ peptide in solution can be sterile filtered through suitable micropore filters, with isolation of at least 50% of the Aβ peptide after sterile filtration. Preferably, 70% of the Aβ peptide is isolated after sterile filtration, and even more preferably, 90% of the Aβ peptide is isolated after sterile filtration. Such sterile solutions can be formulated as pharmaceutical compositions containing a sufficient amount of A p peptide to elicit an immunogenic response when administered to a mammal. Preferably, such administration is parenteral administration, in the form of a suspension of the preparation.
Stoga, u jednom aspektu pripravka iz izuma, nakon sterilne filtracije se pH pripravka podesi na fiziološki prihvatljiv pH da se stvori suspenzija peptida koja sadrži barem 0.1 mg/mL Aβ peptida. Pripravak je koristan za parenteralno davanje. pH suspenzije pripravka je između pH 5 do oko pH 7, preferirano ozmeđu pH 5.5 i 6.5. Najpreferiranije, pripravak sadrži dovoljnu količinu QS-21 skupa s Aβ peptidom da se stvori vizualno bistra, sterilna suspenzija. Therefore, in one aspect of the composition of the invention, after sterile filtration, the pH of the composition is adjusted to a physiologically acceptable pH to create a peptide suspension containing at least 0.1 mg/mL of Aβ peptide. The preparation is useful for parenteral administration. The pH of the preparation suspension is between pH 5 and about pH 7, preferably between pH 5.5 and 6.5. Most preferably, the composition contains a sufficient amount of QS-21 together with the Aβ peptide to form a visually clear, sterile suspension.
Ovaj izum je ju nadalje upućen na otkriće da je otopljene i sterilne otopine Aβ peptida moguće liofilizirati da se dobiju liofilizirane formulacije koje sadrže Aβ peptid. Ovi pripravci se mogu prirediti u odgovarajuće vrijeme da se dobije vodeni pripravak koji sadrži A peptid. This invention is further directed to the discovery that thawed and sterile solutions of Aβ peptide can be lyophilized to obtain lyophilized formulations containing Aβ peptide. These preparations can be prepared at appropriate times to obtain an aqueous preparation containing the A peptide.
U još jednom od aspekata pripravka, izum je upućen na vodene otopine koje sadrže najmanje 0.01 mg/mL Aβ peptida pri čemu je rečena vodena otopina održavana pri pH dovoljnom da se rečeni Aβ peptid otopi. Preferirano je otopina održavana pri takvom pogodnom pH upotrebom učinkovite količine farmaceutski prihvatljivog pufera. In another aspect of the preparation, the invention is directed to aqueous solutions containing at least 0.01 mg/mL of Aβ peptide, wherein said aqueous solution is maintained at a pH sufficient to dissolve said Aβ peptide. Preferably, the solution is maintained at such a suitable pH using an effective amount of a pharmaceutically acceptable buffer.
U još jednom od aspekata pripravka, izum je upućen na sterilne vodene otopine koje sadrže barem 0.1 mg/mL Aβ peptida pri čemu je rečena vodena otopina održavana pri pH dovoljnom da rečeni Aβ peptid otopi. Preferirano je otopina održavana pri takvom pogodnom pH upotrebom učinkovite količine farmaceutski prihvatljivog pufera. In another aspect of the preparation, the invention is directed to sterile aqueous solutions containing at least 0.1 mg/mL of Aβ peptide, wherein said aqueous solution is maintained at a pH sufficient to dissolve said Aβ peptide. Preferably, the solution is maintained at such a suitable pH using an effective amount of a pharmaceutically acceptable buffer.
U još jednom od aspekata pripravka, izum je upućen na liofilizirane pripravke koji sadrže liofilizirani pripravak s Aβ peptidom, a pripravak je priređen postupkom: In another aspect of the preparation, the invention is directed to lyophilized preparations containing a lyophilized preparation with Aβ peptide, and the preparation is prepared by the following procedure:
a) zamrzavanja sterilne vodene otopine koja sadrži barem 0.01 mg/mL Aβ peptida, pri čemu je rečena vodena otopina održavana pri pH dovoljnom da otopi Aβ peptid, te a) freezing a sterile aqueous solution containing at least 0.01 mg/mL Aβ peptide, wherein said aqueous solution is maintained at a pH sufficient to dissolve the Aβ peptide, and
b) liofilizacije zamrznutog pripravka pripravljenom gore u a). b) lyophilization of the frozen preparation prepared above in a).
Preferirano, pripravak iz izuma sadrži dugi oblik (dolje definiran) Aβ peptida. Preferiranije, pripravak sadrži farmaceutski prihvatljiv pufer koji je preferirano odabran iz skupine koji čine: aminokiseline, soli ili njihovi derivati; farmaceutski prihvatljiva sredstva za zaluživanje, hidroksidi alkalijskim metala i amonijevi hidroksidi, organske i anorganske kiseline i njihove soli; te njihove smjese. Preferably, the composition of the invention contains the long form (defined below) of the Aβ peptide. More preferably, the preparation contains a pharmaceutically acceptable buffer which is preferably selected from the group consisting of: amino acids, salts or derivatives thereof; pharmaceutically acceptable alkalizing agents, alkali metal hydroxides and ammonium hydroxides, organic and inorganic acids and their salts; and their mixtures.
U još jednom od aspekata pripravka, izum je upućen na pripravak koji sadrži vodenu otopinu koja sadrži najmanje 0.01 mg/mL Aβ peptida pri čemu je rečena vodena otopina održavana pri pH dovoljnim da se rečeni Aβ peptid otopi, te nadalje pri čemu je Aβ peptid uglavnom u konformaciji slučajnog klupka. In another aspect of the preparation, the invention is directed to a preparation containing an aqueous solution containing at least 0.01 mg/mL of Aβ peptide, wherein said aqueous solution is maintained at a pH sufficient to dissolve said Aβ peptide, and further wherein Aβ peptide is mainly in a random coil conformation.
Pripravci iz izuma se mogu formulirati u farmaceutske pripravke pogodne za unošenje u sisavca koji ima Alzheimerovu bolest ili rizik od razvitka Alzheiomerove bolesti. Aspekti pripravka iz izuma su upućeni na farmaceutske pripravke koji su u konformaciji slučajnog klupka stabilnog Aβ peptida, i u stabilnoj vodenoj suspenziji najmanje 0.1 mg/mL Aβ peptida suspendiranog u rečenom pripravku, ili liofilizirani pripravak, a bilo koji od njih može biti sterilan i dan parenteralno. The compositions of the invention can be formulated into pharmaceutical compositions suitable for administration to a mammal having Alzheimer's disease or at risk of developing Alzheimer's disease. Aspects of the preparation of the invention are directed to pharmaceutical preparations that are in the conformation of a random ball of stable Aβ peptide, and in a stable aqueous suspension of at least 0.1 mg/mL of Aβ peptide suspended in said preparation, or a lyophilized preparation, and any of them can be sterile and given parenterally .
U jednom od aspekata postupka, ovaj izum je upućen na postupak priprave sterilnih pripravaka Aβ peptida dugog oblika koji sadrži: In one of the aspects of the process, this invention is directed to the process of preparing sterile preparations of long-form Aβ peptides containing:
podešavanje pH vodene otopine tako da se Aβ peptid u njoj otopi, otapanje u otopini takve količine Aβ peptida koja je dovoljna da se postigne imunogena koncetracija za sisavce, adjusting the pH of the aqueous solution so that the Aβ peptide dissolves in it, dissolving in the solution such an amount of Aβ peptide as is sufficient to achieve an immunogenic concentration for mammals,
filtriranje nastale otopine preko membrane jednake veličine pora, a veličina rečenih pora je u rasponu koji omogućuje isključivanje bakterija, a prolazak gotovo cijele količine Aβ peptida kroz membranu, te filtering the resulting solution through a membrane of the same pore size, and the size of said pores is in the range that allows the exclusion of bacteria, and the passage of almost the entire amount of Aβ peptide through the membrane, and
za otopine koje sadrže 0.1 mg/mL ili više Aβ peptida, moguće podešavanje pH nastale otopine između od oko pH 5 do oko pH 7, a da se dobije suspenzija peptida. for solutions containing 0.1 mg/mL or more of Aβ peptide, it is possible to adjust the pH of the resulting solution between about pH 5 and about pH 7, to obtain a peptide suspension.
U još jednom aspektu postupka, izum je upućen na postupak prevencije ili tretmana Azheimerove bolesti kod sisavca, a metoda sadrži davanje rečenom sisavcu dovoljnu količinu sterilnog vodenog pripravka koji sadrži najmanje 0.05 mg/mL Aβ peptida, a da se inducira imunogeni odgovor rečenog sisavca. In another aspect of the method, the invention is directed to a method of preventing or treating Azheimer's disease in a mammal, and the method comprises administering to said mammal a sufficient amount of a sterile aqueous preparation containing at least 0.05 mg/mL Aβ peptide, to induce an immunogenic response of said mammal.
Najpreferiranije, postupak filtriranje iz ovog izuma koristi Aβ peptid koji je uglavnom u konformaciji slučajnog klupka. Most preferably, the filtration process of the present invention utilizes an Aβ peptide that is predominantly in a random coil conformation.
Kratki opis slika Short description of the pictures
Slika 1 je C. D. spekar koji pokazuje srednju vrijednost ostatak eliptičnog mjerenja prikazanog kao funkcija valne duljine dvaju različitih otopina Aβ42. Iscrtkana linija pokazuje da je apsorbancija pri pH 6 pripisana β-nabranoj ploči iz molekule. Puna linija je krivulja apsorbancije otopine Aβ42 pri pH 9 i pokazuje da je peptid u konformaciji slučajnog klupka. Figure 1 is C. D. spekar showing the mean value of the residual ellipticity measurement plotted as a function of wavelength for two different Aβ42 solutions. The dashed line shows that the absorbance at pH 6 is attributed to a β-pleated sheet from the molecule. The solid line is the absorbance curve of the Aβ42 solution at pH 9 and shows that the peptide is in a random coil conformation.
Slika 2 je krivulja Aβ42 u otopini prema izračunatoj površini signala za količinu otopljenog peptida, a određeno visokotlačnom tekućinskom kromatorafijom reverzne faze, pokazujući topljivost Aβ42. Figure 2 is a curve of Aβ42 in solution versus the calculated signal area for the amount of dissolved peptide, as determined by reverse phase high pressure liquid chromatography, showing the solubility of Aβ42.
Detaljni opis izuma Detailed description of the invention
Ovaj izum upućen je na pripravke i metode koje koriste vodene pripravke koji sadrže terapijski učinkovitu koncentraciju Aβ peptida. Međutim, prije rasprave izuma u daljnje detalje, definirat će se sljedeći termini. This invention is directed to compositions and methods using aqueous compositions containing a therapeutically effective concentration of Aβ peptide. However, before discussing the invention in further detail, the following terms will be defined.
Definicije Definitions
Termin "dovoljna identičnost" označuje da dvije peptidne sekvencije, a kada su optimalno uspoređene, kao stoje programima Aβ ili BEDFIT korištenjem zadanog jaza masa, dijele barem 65 posto identičnosti sekvencije, preferirano barem 80-90 posto identičnosti sekvnecije, preferiranije najmanje 95 posto identičnosti sekvencije ili više (npr. 99 posto identičnosti sekvencije ili više). Preferirano se položaji ostatak koji nisu identični razlikuju konzervativnom aminokiselinskom supstitucijom. The term "sufficient identity" means that two peptide sequences, when optimally compared, such as by the Aβ or BEDFIT programs using a given mass gap, share at least 65 percent sequence identity, preferably at least 80-90 percent sequence identity, more preferably at least 95 percent sequence identity or higher (eg, 99 percent sequence identity or higher). Preferably, non-identical residue positions differ by conservative amino acid substitution.
Za usporedbu sekvencija, tipično jedna sekvnecija djeluje kao referentna sekvencija, i s njom je uspoređena testirana sekvnecija, specifično, dolje prikazana 42 aminokiselinska sekvnecija. Drugi pogodni oblici bi bili skraćeni obići, kao što je Aβ39 ili prošireni oblik, Aβ43 (s dodatnom treoninskom skupinom na C-terminalnom kraju). Kada se koristi algoritam usporedbe sekvencija, testirana i referentna sekvencija su ulazni podaci u kompjutor, naznače se koordinate podsekvencija ako je potrebno, te se naznače parametri programa za algoritam sekvencije. Program za algoritam usporedbe sekvencija se zatim računa postotak identičnosti sekvnecije za testiranu sekvenciju (sekvncije), relativno prema referentnoj sekvneciji, a zasnovano na naznačenim parametrima programa. For sequence comparison, typically one sequence acts as a reference sequence, and the tested sequence, specifically, the 42 amino acid sequence shown below, is compared to it. Other suitable forms would be truncated forms, such as Aβ39 or the extended form, Aβ43 (with an additional threonine group at the C-terminal end). When the sequence comparison algorithm is used, the tested and reference sequences are input data to the computer, the coordinates of the subsequences are indicated if necessary, and the program parameters for the sequence algorithm are indicated. The sequence comparison algorithm program then calculates the sequence identity percentage for the tested sequence(s), relative to the reference sequence, based on the specified program parameters.
Optimalno poravnavanje sekvnecija za usporedbu se može provesti npr. algoritmom lokalne homologije od Smith & Waterman, Adv. Appl. Math. 2:482 (1981), algoritmom homolognim poravnavanjem od Needeman & Wunsch, J. Mol. Biol. 48:443 (1970), metodom ispitivanja sličnosti od Pearson & Lipman, Proc. Nat'll. Acad. Sci. USA 85:2444 (1988), kompjuteriziranom implemntacijom ovih algoritama (GAP, BESTF1T, FASTA, te TFASTA u Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) ili vizualnom provjerom (općenito vidi Ausubel et al supra). Jedan primjer algoritma koji je pridodan za određivanje postotka sekvencijske identičnosti je algoritam BLAST koji opisuje Altschul et al. J. Mol. Biol. 215:403-410 (1990). Software za izvođenje BLAST analize je dostupan preko Natinal Center for Botechnology Information (http://www.ncbi.nm.nih.gov/). Zadani parametri programa se tipično mogu koristiti za izvođenje usporedbe sekvencije, mada se također može koristiti podešavanje parametara. Za sekvnciju aminokiselina BLASTP program kao zadani parametar koristi duljinu riječi (wordength, W) od 3, očekivanje (expectation, E) od 10, a BOSUM62 matricu (vidi Henikoff&Henikoff, Proc. Natl, Acad. Sci. USA 89, 10915(1989)). Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needeman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity test method of Pearson & Lipman, Proc. Nat'll. Acad. Sci. USA 85:2444 (1988), by computerized implementation of these algorithms (GAP, BESTF1T, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by visual inspection (see generally Ausubel et al supra ). One example of an algorithm that has been added to determine percent sequence identity is the BLAST algorithm described by Altschul et al. J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is available through the National Center for Biotechnology Information (http://www.ncbi.nm.nih.gov/). Default program parameters can typically be used to perform a sequence comparison, although parameter tuning can also be used. For the amino acid sequence, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10, and a BOSUM62 matrix as default parameters (see Henikoff&Henikoff, Proc. Natl, Acad. Sci. USA 89, 10915(1989) ).
Za svrhe klasificiranja aminokiselinske supstitucije kao konzervativnu ili nekonzervativnu, aminokiseline su grupirane su sljedeće skupine: Grupa I (hidrofobni bočni lanci): norleucin, met, ala, val, leu, ile; Grupa I (neutralni hidrofilni bočni lanci): cys, ser, thr; Grupa III (kiseli bočni lanci): asp, glu; Grupa IV (bazni bočni lanci): asn, gln, his, lys, arg; Grupa V (ostaci koji djeluju na orijentaciju lanca): ly, pro; te Grupa VI (aromatski bočni lanci): trp, tyr, phe. Konzervativna supstitucija obuhvaća aminokiseline iz iste skupine. Nekonzervativana supstitucija čini zamjenu člana jedne skupine s članom druge skupine. For purposes of classifying amino acid substitution as conservative or non-conservative, amino acids are grouped into the following groups: Group I (hydrophobic side chains): norleucine, met, ala, val, leu, ile; Group I (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues affecting chain orientation): ly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitution includes amino acids from the same group. A non-conserved substitution replaces a member of one group with a member of another group.
APP695, APP751, te APP770, odnosi se na aminokiselinske ostatke 695, 751 i 770 dugih polipeptida kodiranih od humanih APP gena. Vidi Kan et al., Nature 325, 773 (1987); Ponte et al., Nature 331, 525 (1988); te Kitaguchi et al., Nature 331, 530 (1988). Aminokiselinama unutar humanog amiloidnog prekursora proteina (APP) su pridruženi brojevi prema sekvenciji APP770 izoforma. APP695, APP751, and APP770 refer to amino acid residues 695, 751, and 770 of long polypeptides encoded by human APP genes. See Kan et al., Nature 325, 773 (1987); Ponte et al., Nature 331, 525 (1988); and Kitaguchi et al., Nature 331, 530 (1988). Amino acids within the human amyloid precursor protein (APP) are assigned numbers according to the sequence of the APP770 isoform.
U ovom izumu i u literaturi, masa Aβ peptida predstavlja oko 70% do oko 85% Aβ peptida i oko 15% do oko 30% soli i vode. To je određeno analizom aminokiselina i/ili analizom elementarnog dušika. Primjerice, kada je 0.1 mg Aβ42 peptida korigirano za sadržaj peptida, to predstavlja 0.075 mg Aβ42 peptida i 0.25 mg vode i soli; 0.6 mg Aβ40 peptida predstavlja 0.45 mg Aβ40 peptida i 0.15 mg vode i soli; 2.0 mg Aβ42 peptida predstavja 1.5 mg Aβ42 peptida i 0.5 mg vode i soli. In this invention and in the literature, the mass of Aβ peptide represents about 70% to about 85% Aβ peptide and about 15% to about 30% salt and water. This is determined by amino acid analysis and/or elemental nitrogen analysis. For example, when 0.1 mg of Aβ42 peptide is corrected for peptide content, it represents 0.075 mg of Aβ42 peptide and 0.25 mg of water and salt; 0.6 mg of Aβ40 peptide represents 0.45 mg of Aβ40 peptide and 0.15 mg of water and salt; 2.0 mg of Aβ42 peptide represents 1.5 mg of Aβ42 peptide and 0.5 mg of water and salt.
Aβ peptid, kao što se koristi u ovog izumu, odnosi se na segment Aβ peptida koji može tvoriti konformaciju β-nabrane ploče i povećati imunogeni odgovor kada je dan sisavcu sam ili zajedno s adjuvansom.. Određivanje konformacije β-nabrane ploče npr. mjerenjima cirkularnog dikroizma je unutar umijeća struke. Imunogenost se može odrediti kao što je opisano u dijelu Biološka aktivnost u donjim Primjerima. Aβ peptide, as used in the present invention, refers to a segment of the Aβ peptide that can form a β-pleated sheet conformation and enhance an immunogenic response when administered to a mammal alone or together with an adjuvant. Determination of β-pleated sheet conformation by, for example, measurements of circular of dichroism is within the skill of the profession. Immunogenicity can be determined as described in the Biological Activity section of the Examples below.
Termin "dugi oblici Aβ" peptida uključuju prirodne oblike Aβ38, Aβ39, Aβ40, Aβ41, Aβ42 i Aβ43 i peptidne skevencije koje su dovoljno identične ovima, te da su preferirano u humanom obliku. Aβ38, Aβ39, Aβ40, Aβ41, Aβ42 i Aβ43 se odnosi na peptid koji sadrži aminokiselinske ostatke 1-39, 1-40, 1-41, 1-42 i 1-43, aminokiselinse skraćene na C-terminalnom kraju peptida. Stoga, Aβ41, Aβ40, i Aβ39 se razlikuju od Aβ42 izostavjanjem Ala, Ala-Ile i Ala-Ile-Val is C-terminalnog kraja, kao što se može vidjeti iz niže navedene skevencije Aβ peptida. Aβ43 se razlikuje po prisutnosti treoninskog ostatka na C-terminalnom kraju. Sekvnecije tih peptida i njihov odnos prema APP je ilustriran na Slici 1 od Hardy et al., TINS 20, 155 (1997). The term "long forms of Aβ" peptides include naturally occurring forms of Aβ38, Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43 and peptide sequences sufficiently identical to these, and preferably in human form. Aβ38, Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43 refers to a peptide containing amino acid residues 1-39, 1-40, 1-41, 1-42 and 1-43, amino acids truncated at the C-terminal end of the peptide. Therefore, Aβ41, Aβ40, and Aβ39 differ from Aβ42 by omitting Ala, Ala-Ile, and Ala-Ile-Val from the C-terminal end, as can be seen from the Aβ peptide sequence below. Aβ43 is distinguished by the presence of a threonine residue at the C-terminal end. The sequences of these peptides and their relationship to APP is illustrated in Figure 1 of Hardy et al., TINS 20, 155 (1997).
Aβ42 imasekvenciju:H2N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-ln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-ly-Leu-Met-Val-Gy-Gly-Val-Val-Ile-Ala-OH. Aβ42 has the sequence: H2N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-ln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val -Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-ly-Leu-Met-Val-Gy-Gly-Val-Val-Ile-Ala-OH.
Termin "dugi oblici Aβ" peptida uključuju njihove analoge. Analozi uključuju alergične specije i induciran varijante. Analozi se tipično razlikuju od prirodnih peptida na jednom ili nekoliko položaja, često zbog konzervativne supstitucije. Analozi tipično imaju najmanje 89% identičnosti sekvencije s prirodnim peptidima. Neki analozi također uključuju sintetske aminokiseline ili modifikacije N ili C terminalnih aminokiselina. Primjeri sintetskih aminokiselina su α,α-disupstituirane aminokiseline, N-alkil-aminokiseline, mliječna kiselina, 4-hidroksiprolin, γ-karboksiglutamat, ε-N,N,N-trimetilizin, ε-N-acetiizin, O-fosfoserin, N-acetilserin, N-formilmetilnin,3-metil-histidin, 5-hidroksiizin, ω-N-metilarginin. The term "long forms of Aβ" peptides includes their analogs. Analogues include allergic species and induced variants. Analogues typically differ from natural peptides at one or several positions, often due to conservative substitution. Analogues typically have at least 89% sequence identity to native peptides. Some analogs also include synthetic amino acids or modifications of the N or C terminal amino acids. Examples of synthetic amino acids are α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetiizine, O-phosphoserine, N- acetylserine, N-formylmethylnine, 3-methyl-histidine, 5-hydroxyisine, ω-N-methylarginine.
Aβ se može koristiti u pripravcima i postupcima iz izuma od oko 0.05 mg/mL do gornje granice topljivosti od oko 2.0 mg/mL (odnosi se na Sliku 2). Preferirani raspon peptida je od oko 0.1 do oko 0.8 mg/mL, a najpreferiraniji je u rasponu od oko 0.3 do oko 0.6 mg/mL. Aβ can be used in the compositions and methods of the invention from about 0.05 mg/mL to an upper solubility limit of about 2.0 mg/mL (refer to Figure 2). The preferred range of the peptide is from about 0.1 to about 0.8 mg/mL, and the most preferred range is from about 0.3 to about 0.6 mg/mL.
Pokazano je da je netopljivi oblik Aβ peptida nađen u amiloidnom plaku u konformaciji P-nabrane ploče, Soto, C., et al. NeuroscienceLetters, 186 (2-3), pp. 115-118 (1995). Također su Simmons, L. K. et al. Molec, PharmacoL, 45 (3) str. 373-379 (1994) pokazali da je konformacija β-nabrane ploče povezana s neurotoksičnim efektima peptida, dok je oblik slučajnog klupka samo slabo toksičan ili neaktivan. Kao stoje gore naznačeno, metode i pripravci iz izuma mogu koristiti konformaciju slučajnog klupka Aβ peptida. Aplikanti ovdje pokazuju da je konformacija slučajnog klupka sposobna povećati imunogeni odgovor u testiranim sisavcima. Konformacija slučajnog klupka je najpreferiranija u postupku mikrofiltracije. It has been shown that the insoluble form of the Aβ peptide is found in the amyloid plaque in a P-pleated sheet conformation, Soto, C., et al. Neuroscience Letters, 186 (2-3), pp. 115-118 (1995). Also Simmons, L.K. et al. Molec, PharmacoL, 45 (3) p. 373-379 (1994) showed that the β-pleated sheet conformation is associated with the neurotoxic effects of the peptide, while the random coil form is only weakly toxic or inactive. As indicated above, the methods and compositions of the invention can utilize the random coil conformation of the Aβ peptide. Applicants herein demonstrate that the random coil conformation is capable of enhancing the immunogenic response in tested mammals. The random coil conformation is the most preferred in the microfiltration process.
Termin "slučajno klupko" odnosi se na otvorenu lančastu konformaciju Aβ peptida. Slučajno klupko je sekundarna konformacija peptidnog skeleta i nije uređena u regularnu konformaciju kao što su oblici α-uzvojnice ili β-nabrane ploče. Slučajnom klupku je poremećena regularnost strukture zbog hidrofobnog strukturiranja i interkacija preko vodikove veze koju karakteriziraju ostali ragularnije uređeni oblici. Slučajno klupko još uvijek ima neke oblike peptidnog skeleta ili djelomičnog uređenja, međutim, takve karakteristike su slučajne i dinamičke, pa stoga nisu tipične za sve populacije slučajnog klupka. U konformaciji slučajnog klupka peptidi imaju dobru topljivost i mogućnost fitriranja. α-Uzvojnica i β-nabrana ploča su dobro poznate konformacije peptida. One su opisane, primjerice u Lehniner, Biochemistry (2. izdanje, Worth Izdavači, 1975) str. 128-9, za α-uzvojnicui β-nabranu ploču, na str 133-4, stoje ovdje ugrađeno citatom. The term "random coil" refers to the open chain conformation of the Aβ peptide. A random coil is a secondary conformation of the peptide backbone and is not ordered into a regular conformation such as the α-coil or β-pleated sheet forms. The regularity of the random coil structure is disturbed due to hydrophobic structuring and hydrogen bond interactions, which are characterized by other more irregularly ordered forms. A random coil still has some form of peptide skeleton or partial ordering, however, such characteristics are random and dynamic, and thus not typical of all random coil populations. In the random coil conformation, peptides have good solubility and filterability. α-Helix and β-pleated sheet are well-known peptide conformations. They are described, for example, in Lehniner, Biochemistry (2nd edition, Worth Publishers, 1975) p. 128-9, for α-coil and β-corrugated plate, on pp. 133-4, are incorporated here by quotation.
Konformacija slučajnog klupka Aβ peptida je karakterizirana odgovarajućim cirkularnim dikroizmom ("CD") i lako ju je razlikovati od konformacije β-nabrane ploče. Kako je predstavljeno CD spektrom, za slučajno klupko je tipična jaka negativna vrpca između 190 i 200 nm, i mali CD signal opažen pri valnoj duljini većoj od 215 nm. To je pokazano na Slici l u Crtežima. Nasuprot tome, konformacija β-nabrane ploče pokazuje jaki pozitivni signal centriran oko 200 nm. Za revijalni pregled sekundarne strukture određene CD spektrima, valja se pozvati na Manninovu referencu citiranu supra. The random coil conformation of the Aβ peptide is characterized by the corresponding circular dichroism ("CD") and is easily distinguished from the β-pleated sheet conformation. As represented by the CD spectrum, a random coil is characterized by a strong negative band between 190 and 200 nm, and a small CD signal observed at wavelengths greater than 215 nm. This is shown in Figure 1 in the Drawings. In contrast, the β-pleated sheet conformation shows a strong positive signal centered around 200 nm. For a review of the secondary structure determined by CD spectra, reference should be made to Mannin's reference cited above.
Aβ peptid je dovoljno u konformaciji slučajnog klupka kada je više od 50% Aβ peptida u konformaciji slučajnog klupka. Preferirano je više od 70% ili 80%, a najpreferiranije više od 85% ili 90% Aβ peptida u konformaciji slučajnog klupka. The Aβ peptide is sufficiently in the random coil conformation when more than 50% of the Aβ peptide is in the random coil conformation. More than 70% or 80%, and most preferably more than 85% or 90% of the Aβ peptide is in a random coil conformation is preferred.
"Parenteralne pripravci" su oni pripravci koji su sterilizirani i pogodni za davanje izravno u tijelo, primjerice injekcijom ili infuzijom, tj. putovima kojima odmah dolaze u kontakt s krvi, a bez barijere ili zaštite imunim sustavom, a davanjem oblika koji ulaze u tijelo preko kože, sluzi, preko probanog ili respiratornog sustava. Zbog tih razloga, potrebna je sterilnost parenteralnog pripravka. "Parenteral preparations" are those preparations that are sterilized and suitable for administration directly into the body, for example by injection or infusion, i.e. by the routes by which they immediately come into contact with the blood, without barrier or protection by the immune system, and by giving forms that enter the body through skin, mucus, through the gut or respiratory system. For these reasons, sterility of the parenteral preparation is required.
"Infuzija" označuje davanje lijeka, a obuhvaća dovoljno kontinuirani polagani protok otopine lijeka i krvotok u relativno dugom vremenskom periodu. "Injekcija", nasuprot tome, je brzo davanje jedinice doze otopine ili suspenzije. "Infusion" refers to the administration of a drug, and includes a sufficiently continuous slow flow of the drug solution and blood flow over a relatively long period of time. An "injection," in contrast, is the rapid administration of a unit dose of a solution or suspension.
Intravaskularna (ili intravenozna IV), intrmuskularna (IM), intraperitonalna (IP), subkutana (SC) i intrasternana se odnose na načine davanja parenteranih pripravaka. Oni opisuju anatomskim terminima dijelove tijela u koje valja uvesti injekciji ili infuziju iz izuma. Mišljeno je da se pripravci iz ovog izuma koji imaju fiziološki prihvatljiv pH mogu davati na bilo koji od gornjih načina, ovisno o pojedinom pacijentu i prosudbi liječnika. Otopine s višim pH (pH>8) je najpogodnije davati polaganom IV infuzijom ili IV ukapavanjem. Intravascular (or intravenous IV), intramuscular (IM), intraperitoneal (IP), subcutaneous (SC), and intrasternal refer to routes of administration of parenteral preparations. They describe in anatomical terms the parts of the body where the injection or infusion from the invention should be introduced. It is contemplated that compositions of the present invention having a physiologically acceptable pH may be administered by any of the above routes, depending on the individual patient and the judgment of the physician. Solutions with a higher pH (pH>8) are best administered by slow IV infusion or IV drip.
Podrazumijeva se da se uz ovdje korištene termine "pufer" i "sredstvo za puferiranje" treba prisjetiti da titracijska krivulja kiseline ili baze ima relativno ravnu zonu koja se proteže oko 1.0 jedinica pH s bio koje strane titracijske točke infleksije. U točki infleksije, prisutna je ekvivalentna količina proton-donorskih i proton-akceptorskih čestica kiseline ili baze. U toj zoni, pH sustava se relativno malo mijenja kada se doda malo H* ili OH". To je zona u kojoj konjugirani par kiseline i baze djeuju kao pufer, sustav koji se odupire promjeni pH kada se doda malo H+ ili OH-. Pri razini pH izvan te zone, kapacitet pufera da se odupire promjeni pH je manji. Snaga pufera je maksimalna kad je pH točno jednak pH u točki infleksije titracijske krivulje, tj. kada je koncentracija proton akceptora i proton donora i pH jednaka pK+ (konstanta disocijacije kiseline). Pripravci pufera su opisani detaljno u Data for Biochemical Research, Rex, M. C., Oxford Scinece, Publications, 1995. It is understood that with the terms "buffer" and "buffering agent" used herein, it should be remembered that the titration curve of an acid or base has a relatively flat zone extending about 1.0 pH units on either side of the titration inflection point. At the inflection point, an equivalent amount of proton-donor and proton-acceptor particles of the acid or base is present. In this zone, the pH of the system changes relatively little when a little H* or OH is added". This is the zone where the conjugate acid-base pair acts as a buffer, a system that resists a change in pH when a little H+ or OH- is added. At at a pH level outside this zone, the capacity of the buffer to resist the change in pH is lower. The power of the buffer is maximal when the pH is exactly equal to the pH at the inflection point of the titration curve, i.e. when the concentration of proton acceptor and proton donor and pH is equal to pK+ (acid dissociation constant ).Buffer preparations are described in detail in Data for Biochemical Research, Rex, M.C., Oxford Science, Publications, 1995.
Mnogi fiziološki mehanizmi djeluju unutar tijela da bi održava i pH krvi unutar uske granice od 7.35 do 7.45. Dok su neki glavni puferski mehanizmi zasnovani na ravnoteži karboksilne kiseline ili fosforne kiseline, mnogi drugi mehanizmi obuhvaćaju aminokiseline i proteine. Primjerice, pH suza je održavan pri 7.4 proteinskim puferom. Pojedine aminokiseline su također korisni puferi, mada pokazuju kompleksnije tietracijske krivulje zbog toga što imaju atome koji su proton donori i proton akceptori unutar iste molekule. Molekule to tipa se nazivaju dipolarni ionu, tj. postoje u obliku koji ima pozitivni o negativni naboj unutar iste molekule. Aminokiseline su sposobne biti puferi dodatkom H+ i OH- iona, kao stoje dolje prikazano. Many physiological mechanisms work within the body to maintain blood pH within a narrow range of 7.35 to 7.45. While some major buffering mechanisms are based on carboxylic acid or phosphoric acid balance, many other mechanisms involve amino acids and proteins. For example, the pH of tears is maintained at 7.4 with a protein buffer. Certain amino acids are also useful buffers, although they show more complex titration curves due to the fact that they have atoms that are proton donors and proton acceptors within the same molecule. Molecules of this type are called dipolar ions, i.e. they exist in a form that has a positive or negative charge within the same molecule. Amino acids are capable of being buffered by the addition of H+ and OH- ions, as shown below.
[image] [image]
Za svrhe ovog izuma "farmaceutski prihvatljiv pufer" je široko definiran i uključuje konjugirani par kiselina - baza kao i kisele ili bazne spojeve koji imaju sposobnost podešavanja ili održavanja H otopine Aβ peptida pri željenoj razini. Postupci otapanja/filtracije iz ovog izuma izvode se pri pH 8.5 ili višem ili u drugom aspektu ispod pH 4. For the purposes of this invention, a "pharmaceutically acceptable buffer" is broadly defined and includes conjugated acid-base pairs as well as acidic or basic compounds that have the ability to adjust or maintain the H solution of the Aβ peptide at a desired level. The dissolution/filtration processes of this invention are carried out at pH 8.5 or higher or in another aspect below pH 4.
Preferirano su takvi puderi odabrani iz skupine koju čine: aminokiseline, soli ili njihovi derivati, farmaceutski prihvatljive tvari za zaluživanje, hidroksidi alkalijskim metala, organske i anorganske soli i njihove soli, i njihove smjese. Puferi se koriste u koncentracijama dovoljnim da se dostigne i održava željeni pH i stoga koncentracije ovise o kiselosti/bazičnosti pojedinog pufera ili o kombinaciji odabranih. Odabir učinkovite koncentracije je unutar umijeća struke, a koriste se primjerice pH metar i tehnike titracije. Such powders are preferably selected from the group consisting of: amino acids, salts or their derivatives, pharmaceutically acceptable alkalizing substances, alkali metal hydroxides, organic and inorganic salts and their salts, and their mixtures. Buffers are used in concentrations sufficient to reach and maintain the desired pH and therefore the concentrations depend on the acidity/basicity of the individual buffer or on the combination of the selected ones. The selection of an effective concentration is within the skill of the profession, and for example a pH meter and titration techniques are used.
Primjeri klasa spojeva korisnih u ovom izumu su hidroksidi, uključujući hidrokside alkalijskih metala, sredstva za zaluživanje poznata u farmaceutskoj struci uključujući ali bez ograničenja na njih, tris, borat (Na2B4O7) i dinatrijev citrat, aminokiseline, soli ili esteri ili amidi aminokiselina i njihovi jednostavni derivati, primjerice N-acetil derivati aminokiselina. Posebno su preferirani puferi su glicin (npr. natrijev glicinat), arginin i lizin, natrijev hidroksid i amonijev hidroksid. Exemplary classes of compounds useful in this invention are hydroxides, including alkali metal hydroxides, basifying agents known in the pharmaceutical art including, but not limited to, tris, borate (Na2B4O7) and disodium citrate, amino acids, amino acid salts or esters or amides, and their simple derivatives, for example N-acetyl derivatives of amino acids. Particularly preferred buffers are glycine (eg sodium glycinate), arginine and lysine, sodium hydroxide and ammonium hydroxide.
Primjeri farmaceutski prihvatljivih soli za prakticiranje metoda iz ovog izuma su, bez ograničenja, klorovodična kiselina, fosforna kiselina, limunska kiselina, jabučna kiselina i jantarna kiselina i slično. Uz to, te kiseline se mogu koristiti za titraciju bazne otopine do nižu farmaceutski prihvatljiviju razinu pH, čime nastaje suspenzija pripravka. Examples of pharmaceutically acceptable salts for practicing the methods of this invention are, without limitation, hydrochloric acid, phosphoric acid, citric acid, malic acid and succinic acid and the like. In addition, these acids can be used to titrate the base solution to a lower, more pharmaceutically acceptable pH level, resulting in a suspension of the preparation.
Nasuprot tome, bazni spojevi kao što su oni nabrojani gore se mogu koristiti za titraciju filtrirane otopine niske vrijednosti pH, a da se dobije farmaceutski prihvatljiviji pH, čime također nastaje suspenzija peptida. In contrast, base compounds such as those enumerated above can be used to titrate a low pH filtered solution to a more pharmaceutically acceptable pH, thereby also forming a peptide suspension.
Razmatrane su također kombinacije sredstava za podešavanje pH. Parovi konjugiranih baza i kiselina su primjeri soli kao što je amonijev acetat, također su unutar obujma sredstava za puferiranje u ovom izumu. Takav konjugirani par se može tvoriti, primjerice, triracijom bazne otopine amonijevog hidroksida s octenom kiseline do skoro neutralne otopine amonijevog acetata. Combinations of pH adjusting agents were also considered. Conjugate base and acid pairs exemplified by salts such as ammonium acetate are also within the scope of the buffering agents of this invention. Such a conjugated pair can be formed, for example, by trituration of a basic solution of ammonium hydroxide with acetic acid to a nearly neutral solution of ammonium acetate.
Kako se ovdje koristi "modifikator toničnosti" uključuje sredstva koja doprinose osmotskom tlaku otopine. Primjeri modifikatora toničnosti pogodnih u ovom izumu uključuju, ali nisu na njih ograničeni, saharide (šećeri) kao što je manitol, saharoza i glukoza i soli kao što je natrijev klorid, kalijev klorid i slično. Preferirano, sredstva za modifikaciju toničnosti će se koristiti u tolikoj količini da se dobije konačna osmotska vrijednost manja od oko 350 mOs,/kg, preferiranije između od oko 250 do oko 350 mOs/kg. Valja naznačiti da nabijeni spojevi koji služe kao puferi formulacije mogu također djelovati na toničnost. Stoga, toničnost puferskih otopina Aβ peptida je prvo određena prije daljnjeg podešavanja sredstva za modifikaciju toničmosti. As used herein "tonicity modifier" includes agents that contribute to the osmotic pressure of a solution. Examples of tonicity modifiers useful in the present invention include, but are not limited to, saccharides (sugars) such as mannitol, sucrose, and glucose, and salts such as sodium chloride, potassium chloride, and the like. Preferably, the tonicity modifier will be used in such an amount as to give a final osmotic value of less than about 350 mOs/kg, more preferably between about 250 to about 350 mOs/kg. It should be noted that charged compounds that serve as formulation buffers can also affect tonicity. Therefore, the tonicity of the Aβ peptide buffer solutions was first determined before further adjustment of the tonicity modifying agent.
Može se koristiti sredstvo za stvaranje kelata u postupcima i pripravcima iz izuma. Primjeri preferiranih sredstava za tvorbu kelata uključuju etilendiaminotetraoctenu kiselinu (EDTA) i njene soli (kao što je natrijeva) koji se normalno koriste pri koncentracijama od 0.05 do 50 mM, preferiranije pri koncentracijama od 0.05 do 10 mM ili od oko 0.1 do 5 mM, stoje najpreferiranije. Mogu se koristiti ostala poznata sredstva za stvaranje kelata, kao stoje polivinilni alkohol. A chelating agent can be used in the methods and compositions of the invention. Examples of preferred chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts (such as sodium) which are normally used at concentrations of 0.05 to 50 mM, more preferably at concentrations of 0.05 to 10 mM or from about 0.1 to 5 mM, as most preferred. Other known chelating agents can be used, such as polyvinyl alcohol.
Pripravci mogu sadržavati površinski aktivna sredstva ili deterente kao što je polisorbat (npr. Tween®) ili 4-(1,1,4,4-tetrametibutil)feniloksipolietoksietanoli (Triton®) ili polimeri polietilenpropilpropilen likola (Pluronics®). Površinski aktivne tvari se kreću od oko 0.005 do 1%, a preferirano je od oko 0.02 do 0.75% sredstva. Preferirani polisorbat je PS-80 koji je komercijalno pristupačan kao Tween® 80. The preparations may contain surfactants or detergents such as polysorbate (eg Tween®) or 4-(1,1,4,4-tetramethylbutyl)phenyloxypolyethoxyethanol (Triton®) or polyethylenepropylpropylene glycol polymers (Pluronics®). Surfactants range from about 0.005 to 1%, and about 0.02 to 0.75% of the agent is preferred. A preferred polysorbate is PS-80 which is commercially available as Tween® 80.
Klizna sredstva su također razmatrana za ekcipijente korisne u izumu. Polietilenglikoli, npr. PE3350 su korisni za modifikaciju asocijacije Aβ čestica i stoga je topljivost povezana s polimernom površinom i orijentacijom hidrofilnih ostataka prema vodenoj fazi. Klizna sredstva mogu biti prisutna od 0.5 do 5% (w/v). Gliding agents are also contemplated as excipients useful in the invention. Polyethylene glycols, eg PE3350, are useful for modifying the association of Aβ particles and therefore solubility is related to the polymer surface and the orientation of hydrophilic residues towards the aqueous phase. Lubricants can be present from 0.5 to 5% (w/v).
Farmacetuski prihvatljivi šećeri (primjerice saharoza, dekstroza, maltoza ili laktoza) ili farmaceutski prihvatljivi šećerni alkoholi (primjerice manitol, ksilitol ili sorbitol) nemaju utjecaj na medicinski učinak aktivne tvari. U jednom aspektu se mogu koristiti šećeri ili šećerni alkoholi koji imaju molekulsku masu manju od 500, i sposobni su lako se disperirati i otapati u vodi. Primjeri šećera i šećernih alkohola koji se mogu koristiti u ovom izumu su: ksilitol, manitol, sorbitol, arabinoza, riboza, ksiloza, glukoza, manoza, galaktoza, saharoza, laktoza i slično. Mogu se koristiti sami ili kao smjesa dvaju ili više spojeva. Najpreferiraniji šećer je manitol, posebno u liofiliziranom pripravku, a također je preferirana saharoza u otopini pripravka. Pharmaceutically acceptable sugars (for example sucrose, dextrose, maltose or lactose) or pharmaceutically acceptable sugar alcohols (for example mannitol, xylitol or sorbitol) have no influence on the medicinal effect of the active substance. In one aspect, sugars or sugar alcohols that have a molecular weight of less than 500 and are capable of easily dispersing and dissolving in water can be used. Examples of sugars and sugar alcohols that can be used in this invention are: xylitol, mannitol, sorbitol, arabinose, ribose, xylose, glucose, mannose, galactose, sucrose, lactose and the like. They can be used alone or as a mixture of two or more compounds. The most preferred sugar is mannitol, especially in the lyophilized preparation, and sucrose in the solution of the preparation is also preferred.
Također je nađeno da upotreba QS-21 kao adjuvansa u popravcima iz izuma dolazi u interakciju sa suspendiranim proteinom tako da nastaje vizualno bistra suspenzija. Ova interakcija je poželjna jer je peptid u konformaciji β-nabrane ploče, ali je suspendiran u fazi i vrlo malim česticama i može dovesti do povoljnih svojstava pripravka tako da se poveća stabilnost suspenzije ili poboljša imunogenog svojstva. DPPC (dipalmitoil fosfoatifil kolin) je poznat u struci o očekuje se da stupa u interakciju s Aβ peptidom na sličan način kao QS-21, a da se dobije suspenzija malih čestica, a dozvoljava se upotreba drugih adjuvansa iz izuma, pri čemu nastaje bistra suspenzija. Alternativno se mogu koristiti drugi adjuvansi u smjesi s QS-21. The use of QS-21 as an adjuvant in the repairs of the invention was also found to interact with the suspended protein to form a visually clear suspension. This interaction is desirable because the peptide is in a β-pleated sheet conformation, but is suspended in a phase with very small particles and can lead to favorable properties of the preparation by increasing the stability of the suspension or improving the immunogenic properties. DPPC (dipalmitoyl phosphoatiphil choline) is known in the art to be expected to interact with the Aβ peptide in a similar manner to QS-21 to produce a suspension of small particles, and the use of other adjuvants of the invention is permitted, resulting in a clear suspension. . Alternatively, other adjuvants can be used in admixture with QS-21.
Liofilizacija je tehnika dobro poznata u farmaceutskoj struci, kao što su i tehnike za stabilizaciju peptida tijekom postupka liofilizacije. Stabilizacija proteinskog ili peptidnog liofilizata u aminokiselinama i saharidnoj matrici je poznata stručnjacima. Vidi, primjerice: Lueckel B., et al., Formulations ofsugars with amino acids or mannitol -Influence of concentration ratio on the properities of the freeze-concentrate and the liophilizate, PHARM. DEV. TECHNOL. 3(3) str. 325-336 (1998).The Royal Pharmaceutical Society u GB Symp: fNew Anaytical Approaches to the Characterization of Biotechnology Products' lipanj 19%, predstavljeno od Luckel B., et al: A strategyfor optimizingthelyophilizationofbiotechnoloicalproduts, PHARM. SCI. (UK) 3(1) str. 3-8 (1997). Obje ove reference su ovdje urađene u cijelosti za sve svrhe. Lyophilization is a technique well known in the pharmaceutical art, as are techniques for stabilizing peptides during the lyophilization process. Stabilization of protein or peptide lyophilizate in amino acids and saccharide matrix is known to experts. See, for example: Lueckel B., et al., Formulations of sugars with amino acids or mannitol - Influence of concentration ratio on the properties of the freeze-concentrate and the lyophilizate, PHARM. DEV. TECHNOLOGY. 3(3) p. 325-336 (1998). The Royal Pharmaceutical Society in GB Symp: fNew Anaytical Approaches to the Characterization of Biotechnology Products' June 19%, presented by Luckel B., et al: A strategyfor optimizingthelyophilizationofbiotechnoloicalproduts, PHARM. SCI. (UK) 3(1) p. 3-8 (1997). Both of these references are reproduced here in full for all purposes.
Termin "farmaceutski prihvatljiv" modificira svaki pripravak tako da označuje da nema štetan ili nepovoljan efekt na osobu kojoj je dan i u uvjetima kojim je dano. Termin "farmaceutski prihvatljiv razrjeđivač" odnosi se na spoj koji čuva ili ne mijenja aktivnost aktivnog spoja ( spojeva) i nema štetan ili nepovoljan efekt na osobu kojoj je dan u uvjetima kojim je dano, kao što su netoksična sredstva za podešavanje pH ili puferi ili sredstva za modifikaciju toničnosti ili kelatori i slično. The term "pharmaceutically acceptable" modifies any preparation to indicate that it has no harmful or adverse effect on the person to whom it is administered and under the conditions under which it is administered. The term "pharmaceutically acceptable diluent" refers to a compound that preserves or does not alter the activity of the active compound(s) and has no harmful or adverse effect on the subject under the conditions in which it is administered, such as non-toxic pH adjusting agents or buffers or agents to modify tonicity or chelators and the like.
Pripravci ili postupci koji sadrže "jedan ili više od nabrojenih elemenata mogu uključivati ostale elemente koji nisu specifično nabrojeni. Primjerice, pripravak koji sadrži Aβ peptid obuhvaća Aβ peptid u pripravku kako je navedeno i Aβ peptid kao komponentu pripravka koji ima nenavedene komponente. Preparations or processes that contain "one or more of the listed elements may include other elements that are not specifically listed. For example, a preparation containing Aβ peptide includes Aβ peptide in the preparation as specified and Aβ peptide as a component of the preparation that has unspecified components.
Daljnje definicije su sljedeće Further definitions are as follows
Kratica Definicija Abbreviation Definition
°C stupanj Cesiusa °C degree of Cesius
cc kubični centimetar cc cubic centimeter
C. D. cirkularni dikroizam C. D. circular dichroism
pH log[H], mjera sadržaja vodika i stoga kiselosti ili bazičnosti otopine pH log[H], a measure of the hydrogen content and therefore the acidity or basicity of a solution
p K' konstanta disocijacije kiseline p K' acid dissociation constant
odnosi se na pH prema pH = pK' + [H + akceptor] refers to pH according to pH = pK' + [H + acceptor]
[H + donor] [H + donor]
PS80 polisorbat 80 ili Tween 80® kopolimer polisorbatai etilen-oksida; PS80 polysorbate 80 or Tween 80® copolymer of polysorbate and ethylene oxide;
Merck Idex monorafija br. 7559 (11. izd) Merck Idex monograph no. 7559 (11th ed.)
μm mikron μm micron
min minuta min minutes
mL mililitar mL milliliter
N normalitet - pokazatelj molarnosti otopine N normality - an indicator of the molarity of the solution
M molaritet, vrijednost izražena mol/litru M molarity, value expressed in mol/liter
mM milimol mM millimole
DMSO dimetilsufoksid DMSO dimethylsulfoxide
EDTA etilendiamin-tetraacetat, obično dinatrijeva sol EDTA ethylenediamine tetraacetate, usually the disodium salt
Tris trimetamin, tris(hidroksimetil)aminometan; Merck Idex monorafija br. 9684 (11. izd) Tris trimethamine, tris(hydroxymethyl)aminomethane; Merck Idex monograph no. 9684 (11th ed.)
RP HPLC visokotačna tekućinksa kromatorafija reverzne faze RP HPLC high precision reverse phase liquid chromatography
RPM revolucija po minuti RPM revolutions per minute
CFA/IFA kompletni Freundov adjuvans/nekompletan Freundov adjuvans CFA/IFA complete Freund's adjuvant/incomplete Freund's adjuvant
(Chang et al. Advanced Dru Delivery Reviews 32,173-186 (1998) (Chang et al. Advanced Drug Delivery Reviews 32,173-186 (1998)
MPL 3-O-deacilirani monofosforoilni lipid A (MPL™) (vidi primjerice B 2220211). MPL 3-O-deacylated monophosphoroyl lipid A (MPL™) (see for example B 2220211).
QS 21 triterpen glikozid izoliran iz kore drveća Quillaja Saponaria Molina iz Južne Amerike (vidi Kensil et al. u Vaccine Design: The Subunit and Adjuvant Approach (izd. Powell & Newman, Plenum Press, NY, 1995); US Pat. br. 5,057,540), (Stimulon™QS-21) QS 21 triterpene glycoside isolated from the bark of Quillaja Saponaria Molina trees from South America (see Kensil et al. in Vaccine Design: The Subunit and Adjuvant Approach (ed. Powell & Newman, Plenum Press, NY, 1995); US Pat. No. 5,057,540 ), (Stimulon™QS-21)
FIO samo za informaciju (engl. for information only) FIO for information only
Filtriranje je postupak uklanjanja kontaminata isključivanjem čestica iz tekućine po veličini, a prolazom preko membranskog filtera koji ima jednaku veličinu pora. Mada se čestice veličine mikrona mogu ukloniti i bez membrane, a upotrebom materijala nađenih u fibrilnom mediju, samo membranski filteri koji imaju točno definirane veličine pora mogu osigurati kvantitativno uklanjanje čestica koje imaju manju veličinu. Stoga je membranskom filtracijom je moguće kvantitativno ukloniti bakteriju iz otopine, a kada prolazi kroz mikrofiltere, čime se dobiva efekt sterilizacije. Prethodnim pročišćavanjem je Aβ peptid bio tako netopljiv ili je ostao u agregatima do one mjere da se otopina nije mogla mikrofiltrirati na komercijalnoj skali zbog začepljenja pora membrane i/ili loše izolacije peptida u mikrofiltarskoj otopini. Filtration is the process of removing contaminants by excluding particles from the liquid by size, and passing them through a membrane filter that has the same pore size. Although micron-sized particles can be removed without a membrane, and by using materials found in a fibril medium, only membrane filters that have precisely defined pore sizes can ensure the quantitative removal of particles that have a smaller size. Therefore, with membrane filtration, it is possible to quantitatively remove bacteria from the solution, and when it passes through microfilters, which results in a sterilization effect. By previous purification, the Aβ peptide was so insoluble or remained in aggregates to the extent that the solution could not be microfiltered on a commercial scale due to clogging of the membrane pores and/or poor isolation of the peptide in the microfilter solution.
Preferiran filteri su općenito po definiciji oni koji imaju sposobnost uklanjanja čestica preko 0.2 mikrona. Preferred filters are generally by definition those that have the ability to remove particles over 0.2 microns.
Membranski filteri su uniformno pripravljeni prema supstratu, i općenito mogu separirati na osnovu mase na membranskoj površini. Međutim, membranski filteri površinskog tipa primaju česticu u dubinu strukture i na površini membrane. U separaciji isključivanjem mase, čestice manje od pora membranskog filtera prolaze, dok su veće čestice zadržane na površini membrane. Kako se te definirane veličine pora filtera ne "zasićuju" (tj. kad se filter počinje puniti zadržanim česticama, povećanje tlaka pri protoku dozvoljava prolaz malih količina zadržanih krutina), oni su izabrane naprave za mikrobiološku kontrolu. "Stupanj sterilizacije" membranskih filtera površinskog tipa je dobro poznat u farmaceutskoj industriji. Membrane filters are uniformly prepared according to the substrate, and generally can separate based on the mass on the membrane surface. However, membrane filters of the surface type receive the particle in the depth of the structure and on the surface of the membrane. In mass exclusion separation, particles smaller than the pores of the membrane filter pass, while larger particles are retained on the membrane surface. Since these defined filter pore sizes do not "saturate" (ie, when the filter begins to fill with retained particles, the increase in flow pressure allows the passage of small amounts of retained solids), they are the devices of choice for microbiological control. The "degree of sterilization" of surface-type membrane filters is well known in the pharmaceutical industry.
U svim filtracijskim aplikacijama, permeabilnost fiterskog medija može se mijenjati kemikalijama, molekulskim ili elektrostatskim svojstvima filtrata, međutim, nađeno je da su hidrofilni mikrofilteri korišteni u ovom izumu stabilni pri visokom, odnosno niskom pH okoline, a ovisno o korištenoj metodi i pouzdanosti uklanjanja nepoželjnih čestica tvari bez začepljivanja. Obično će stručnjak biti sposoban odabrati i koristiti hidrofilne mikrofiltere. Specifikacije komercijalno pristupačnih produkata ili "website" kao što je http://millispider.miipore.com/corporate/ditemap.nsf/catalogs (svibanj 1999) omogućuju odabir filtera koji imaju željene karakteristike. In all filtration applications, the permeability of the filter media can be changed by chemicals, molecular or electrostatic properties of the filtrate, however, the hydrophilic microfilters used in this invention were found to be stable at high or low pH environments, depending on the method used and the reliability of removing unwanted particles substances without clogging. Usually the skilled person will be able to select and use hydrophilic microfilters. Commercially available product specifications or websites such as http://millispider.miipore.com/corporate/ditemap.nsf/catalogs (May 1999) allow selection of filters that have the desired characteristics.
Preferirani primjeri hidrofilnih filtera koji se koriste u ovom izumu su Millipore Durapore®, također zvani Millex V, (Millipore Corporation, sjedište u Bedfordu, MA), hidrofilni polimer poliviniliden-fluorid je stabilan i ima karakteristiku slabog vezivanja proteina, te dijametar pora 0.2 μm; te Milex GP™, modificirani polietersulfonski polimer hidrofilne površine, veličine pora 0.22 μm. Preferiraniji je Durapore zbog njegove stabilnosti pri pH 9-9.5. Preferred examples of hydrophilic filters used in this invention are Millipore Durapore®, also called Millex V, (Millipore Corporation, headquartered in Bedford, MA), a hydrophilic polyvinylidene fluoride polymer is stable and has weak protein binding characteristics, and a pore diameter of 0.2 μm ; and Milex GP™, a modified polyethersulfone polymer with a hydrophilic surface, with a pore size of 0.22 μm. Durapore is preferred because of its stability at pH 9-9.5.
Preferirano je da se gotovo sav Aβ peptid izolira nakon filtracije. Gotovo sav izoliran Aβ peptid je definiran tako da je prosjek izoliranog proteina nakon sterilne filtracije veći od oko 50% peptida. Preferirano je više od 80% Aβ peptida izoliranog nakon sterilne filtracije, a najpreferiranije je više od 90% Aβ peptida izolirano nakon sterilne filtracije. It is preferred that almost all of the Aβ peptide is isolated after filtration. Almost all isolated Aβ peptides are defined so that the average isolated protein after sterile filtration is greater than about 50% of the peptides. More than 80% of the Aβ peptide isolated after sterile filtration is preferred, and most preferred more than 90% of the Aβ peptide is isolated after sterile filtration.
Metode Methods
Metode u ovom izumu obuhvaćaju pripravu vodenih pripravaka koncentracije barem 0.01 mg/mL Aβ peptida. Takvi pripravci su priređeni podešavanjem pH vodene otopine tako da se Aβ peptid otopi pri željenoj koncentraciji (npr. pri koncentraciji od oko 0.1 mg/mL do oko 2.0 mg/mL). The methods in this invention include the preparation of aqueous preparations with a concentration of at least 0.01 mg/mL of Aβ peptide. Such preparations are prepared by adjusting the pH of the aqueous solution so that the Aβ peptide dissolves at the desired concentration (eg, at a concentration of about 0.1 mg/mL to about 2.0 mg/mL).
Podešavanje pH vodene otopine postignuto je uobičajenim metodama čiji je tipični primjer dodavanje kiseline ili baze da se postigne željeni pH. Preferirano se koristi farmaceutski prihvatljiva kiselina ii baza. Da bi se pH otopine održavao pri dugotrajnom skladištenju, preferirano je koristiti farmaceutski prihvatljive pufere u pripravku. Odabir pogodnih pufera ovisi o željenom pH i obuhvaćen je umijećem struke. Adjusting the pH of an aqueous solution is achieved by common methods, a typical example of which is the addition of acid or base to achieve the desired pH. A pharmaceutically acceptable acid and base are preferably used. In order to maintain the pH of the solution during long-term storage, it is preferred to use pharmaceutically acceptable buffers in the preparation. The selection of suitable buffers depends on the desired pH and is within the skill of the art.
Preferirano, pH vodenog pripravka podešen je između oko pH 2 do 4 ili oko pH 8.5 do 12. Pri takvom pH Aβ peptid iznenađujuće postaje dobro topljiv. Preferably, the pH of the aqueous preparation is adjusted between about pH 2 to 4 or about pH 8.5 to 12. At such a pH, the Aβ peptide surprisingly becomes well soluble.
Kada se postupak iz izuma provodi pri niskom pH (oko pH 2 do 4) za snižavanje pH otopine na željeni pH se mogu koristiti kiseline kao što su halovodične kiseline (npr. HCl, HBr), fosforna kiselina, limunska kiselina i octena kiselina i ostale farmaceutski prihvatljive kiseline. Odabiri pogodnih kiselina obuhvaćen je umijećem struke. When the process of the invention is carried out at a low pH (around pH 2 to 4), acids such as hydrohalic acids (e.g. HCl, HBr), phosphoric acid, citric acid and acetic acid and others can be used to lower the pH of the solution to the desired pH pharmaceutically acceptable acids. Selection of suitable acids is within the skill of the art.
Kada se postupak iz izuma provodi pri visokom pH (oko pH 8.5 do 12) za povećanje pH otopine na željeni pH mogu se koristiti baze kao što su alkalijski metali, amonijevi hidroksidi (npr. NaOH, NH4OH) i slično. Pri visokom pH je preferirano pH otopine u danom puferu podešen između 8.5 do 11. Preferiranija je razina pH između pH 9 i 10. When the process of the invention is carried out at a high pH (about pH 8.5 to 12), bases such as alkali metals, ammonium hydroxides (eg NaOH, NH4OH) and the like can be used to increase the pH of the solution to the desired pH. At high pH, the pH of the solution in the given buffer is preferably adjusted between 8.5 and 11. A more preferred pH level is between pH 9 and 10.
Prije ili odmah posije podešavanja pH, Aβ peptid pri potrebnoj koncentraciji dodan je u otopinu. Preferirano je, naravno, dodati Aβ peptid nakon podešavanja pH da bi se odmah otopio. Nakon dodavanja, može biti potrebno blago miješanje i zagrijavanje da bi se potpomogao proces otapanja. Before or immediately after pH adjustment, Aβ peptide at the required concentration was added to the solution. It is preferred, of course, to add the Aβ peptide after adjusting the pH so that it dissolves immediately. After addition, gentle stirring and heating may be necessary to aid the dissolution process.
Dodatak u pripravak bilo kojeg aditiva, kao što su farmaceutski prihvatljivi puferi, sredstva za podešavanje toničmosti, adjuvansi itd., može se desiti u bilo kojem pogodnom trenutku prije ili nakon dodatka Aβ peptida. Addition to the composition of any additives, such as pharmaceutically acceptable buffers, tonicity adjusting agents, adjuvants, etc., can occur at any convenient time before or after the addition of the Aβ peptide.
Kad je gore opisana vodena otopina pripravljena, može se izvesti sterilna filtracija i/ili liofilizacija, a prema postupcima dobro poznatim u struci, čiji su primjeri dani dolje. When the aqueous solution described above is prepared, sterile filtration and/or lyophilization can be performed according to procedures well known in the art, examples of which are given below.
Pripravljene su sljedeće otopine pufera za otapanje Aβ peptida u željenoj koncentraciji u rasponu od 0.6 do 2.0 mg/mL peptida: The following buffer solutions were prepared for dissolving Aβ peptide in the desired concentration ranging from 0.6 to 2.0 mg/mL peptide:
Preferirani pripravci aminokiselina Preferred Amino Acid Preparations
10 mM natrijev glicinat, pH 9.0, 9.5 ili 10.0 10 mM sodium glycinate, pH 9.0, 9.5 or 10.0
i/ili s 0.02 do 1.0% (w/v) polisorbata 80 (PS-80) and/or with 0.02 to 1.0% (w/v) polysorbate 80 (PS-80)
može sadržavati jedan ili više modifikatora toničnosti pogodnih za parenteralno davanje may contain one or more tonicity modifiers suitable for parenteral administration
10 mM L-arginin-HCl i 10 mM L-lizinat, oba pri pH 9.0,10.0 10 mM L-arginine-HCl and 10 mM L-lysinate, both at pH 9.0, 10.0
i/ili s 0.02 do 1.0% (w/v) polisorbata 80 (PS-80) and/or with 0.02 to 1.0% (w/v) polysorbate 80 (PS-80)
može sadržavati jedan ili više modifikatora toničnosti pogodnih za parenteralno davanje may contain one or more tonicity modifiers suitable for parenteral administration
Pripravci iz ovog izuma se preferirano čuvaju pri niskim temperaturama da bi se spriječila degradacija peptidnog lanca. Preferirani raspon temperature za skladištenje Aβ peptidnog pripravka je 2 do 8 °C. The compositions of this invention are preferably stored at low temperatures to prevent degradation of the peptide chain. The preferred temperature range for storage of the Aβ peptide preparation is 2 to 8 °C.
Sljedeći primjeri ilustriraju prakticiranje izuma. Ovi primjeri su za ilustrativne svrhe i nije namjera da ograniče obujam prijavljenog izuma. The following examples illustrate the practice of the invention. These examples are for illustrative purposes and are not intended to limit the scope of the claimed invention.
Primjeri Examples
Primjer 1 - Topljvost peptida Example 1 - Peptide solubility
Peptid i reagensi Peptide and reagents
Aβ42 i njena trifluoracetatna sol je dobivena od American Peptide Co., rupe broj M05503T1, M10028T1 su korišteni bez daljnjih modifikacija. Na raspolaganju su i druge soli i uspješno su korištene u procesima iz izuma. Primjere alternativnih protuiona soli Aβ peptida vidi u Tablici 2. Aβ42 and its trifluoroacetate salt were obtained from American Peptide Co., well numbers M05503T1, M10028T1 were used without further modification. Other salts are available and have been used successfully in the processes of the invention. See Table 2 for examples of alternative Aβ peptide salt counterions.
Sve otopine kiselina, baza i pufera su pripravljene iz reagensa laboratorijske čistoće koje su skladištene kao sterilne filtrirane otopine. Polisorbat 80 (Tween 80, PS 80); 4% otopina je pripravljena w/v otapanjem otopine PS 80 s niskim sadržajem peroksida (primjerice, 10% otopina polisorbiran monooleata s niskom razinom peroksida se može pribaviti od Adrich-Sima Chemicals). All acid, base and buffer solutions were prepared from laboratory grade reagents that were stored as sterile filtered solutions. Polysorbate 80 (Tween 80, PS 80); A 4% solution was prepared by dissolving a w/v solution of PS 80 with a low peroxide content (for example, a 10% solution of low peroxide polysorbate monooleate is available from Adrich-Sima Chemicals).
Otapanje peptida: Peptide dissolution:
Približno 500-700 μg Aβ peptida je vagano i otopljeno u odgovarajućoj količini puferne otopine da se dobije teorijska konačna koncentracija Aβ42 od 0.6, 0.8 ili 1.0 mg/mL. Peptid je izravno vagan u Weatonovu staklenu posudu od 4 mL s poklopcem s navojem. Dodana je pogodna količina puferske otopine i peptid je lagano miješan 15 do 30 minuta. Otopina je procijenjena vizualno prema topljivosti na sljedeći način: (+)=loša topivost, mutna suspenzija; (++)= bistra suspenzija s netopljivim česticama; (+++)=bistra otopina. Approximately 500-700 μg of Aβ peptide was weighed and dissolved in an appropriate amount of buffer solution to obtain a theoretical final Aβ42 concentration of 0.6, 0.8, or 1.0 mg/mL. Peptide was weighed directly into a 4 mL Weaton glass screw cap. A suitable amount of buffer solution was added and the peptide was gently mixed for 15 to 30 minutes. The solution was evaluated visually for solubility as follows: (+)=poor solubility, cloudy suspension; (++)= clear suspension with insoluble particles; (+++)=clear solution.
Tablica 1. Vidljiva topljivost u otopini Table 1. Apparent solubility in solution
[image] [image] [image] [image]
Otopine označene kao +++ su bile vizualno bistre pri ciljnim koncentracijama. Raspon pH je od pH 9 do pH 10 za otopine pufera. pH anorganskih otopina kao što je NaOH i NH4OH je bazni, dok je HCl i fosforne kiseline bio jako kiseli. Topljivost peptida je postignuta pri 0.6 mg/mL peptida pri pH vrijednostima pribižno >pH 9. Aditivi kao stoje polisorbat 80, saharoza, manito i EDTA nisu utjecali na topljivost peptida ali su pomogli povećanju toničnosti i izolaciji nakon filtracije, ili mogu djelovati kao kelatori. Kisele otopine (približno pH 4 ili manje) također otapaju Aβ42 peptide pri 0.6 do 1 mg/mL. Otopine označene kao ++ izgledaju djelomično otopljene nakon otapanja peptida pri ciljnoj koncentraciji. Peptid može biti topljiv pri nižim koncentracijama nego stoje ovdje testirano. Otopine označene kao + vizualno nisu postigle potpuno otapanje pri ciljnoj koncentraciji. Solutions marked +++ were visually clear at target concentrations. The pH range is from pH 9 to pH 10 for buffer solutions. The pH of inorganic solutions such as NaOH and NH4OH is basic, while HCl and phosphoric acid were strongly acidic. Peptide solubility was achieved at 0.6 mg/mL peptide at pH values approximately >pH 9. Additives such as polysorbate 80, sucrose, mannitol, and EDTA did not affect peptide solubility but helped increase tonicity and isolation after filtration, or may act as chelators. Acidic solutions (approximately pH 4 or less) also dissolve Aβ42 peptides at 0.6 to 1 mg/mL. Solutions marked ++ appear partially dissolved after dissolving the peptide at the target concentration. The peptide may be soluble at lower concentrations than tested here. Solutions marked + did not visually achieve complete dissolution at the target concentration.
Primjer 2 - Topljivost Aβ peptida u puferiranoj otopini Example 2 - Solubility of Aβ peptide in a buffered solution
Aβ42 (0.6, 1.0, 1.5, 2.0, 3.0 i 3.5 mg/mL) je otopljen u 10 mM natrij-licinatnom puferu, pH 9.0. Svaka otopina je centrifugirana u stolnoj centrifugi (> 10000 RPM, ~10 min) i zamućena suspenzija je opažena za koncentracije 2.0-3.5 mg/mL (otopine 0.6-1.5 mg/mL su vizualno bile bistre). Aβ42 (0.6, 1.0, 1.5, 2.0, 3.0 and 3.5 mg/mL) was dissolved in 10 mM sodium-lycinate buffer, pH 9.0. Each solution was centrifuged in a benchtop centrifuge (> 10000 RPM, ~10 min) and a cloudy suspension was observed for concentrations of 2.0-3.5 mg/mL (0.6-1.5 mg/mL solutions were visually clear).
Aikvot supernatante svake otopine je analiziran RP HPLC. An aliquot of the supernatant of each solution was analyzed by RP HPLC.
Tablica 2 sadrži tablično prikazane veličine peptidnih signala i graf, ukazujući na topljivost Aβ42 u natrij-gicinatnom puferu pri pH 9. Table 2 contains tabulated peptide signal sizes and a graph, indicating the solubility of Aβ42 in sodium glycinate buffer at pH 9.
Tablica 2. Granice topljivosti Aβ42 peptida, TFA soli Table 2. Solubility limits of Aβ42 peptide, TFA salt
[image] [image]
Nadalje, Aβ42 peptid je pročišćen kao trifluoracetatna, amonijeva, kloridna i natrijeva sol. Te soli su otopljene u nekoliko pufera pri koncentraciji Aβ42 peptida 45 mg/mL, korigirano za doprinos protuiona različitih soli. Otopine peptida su filtrirane i izolacija peptida je određena usporedbom veličine signala RP-HPC prije ili nakon filtracije. Sve soli su topljive i lako se filtriraju pri 0.45 mg/mL, kao stoje prikazano na Tabilici3. Furthermore, the Aβ42 peptide was purified as the trifluoroacetate, ammonium, chloride and sodium salts. These salts were dissolved in several buffers at an Aβ42 peptide concentration of 45 mg/mL, corrected for the contribution of counterions from different salts. Peptide solutions were filtered and peptide isolation was determined by comparing the magnitude of the RP-HPC signal before or after filtration. All salts are soluble and easily filtered at 0.45 mg/mL, as shown in Table 3.
Tablica 3. Granice topljivosti Aβ42 peptida, TFA soli Table 3. Solubility limits of Aβ42 peptide, TFA salt
[image] [image]
Primjer 3 - Izolacija otopljenog peptida s predstavnika hidrofobnih filtera Example 3 - Isolation of a dissolved peptide from a representative of hydrophobic filters
Siringni fiteri su testirani (promjer filtera 25 mm, 3.9 cm2 područje filtera): Millex GV, 0.22 μM: hidrofilni poliviniliden difluorid (PVDF, Durapore®) membrana s malom sposobnosti vezanja proteina, Millex GN, 0.20 μM: hidrofilna najlonska membrana s malom sposobnosti vezanja proteina, te Millex GP, 0.22 μM: hidrofilna polietan-sulfonska membrana (PES) modificirane površine s malom sposobnosti vezanja proteina. Syringe filters were tested (filter diameter 25 mm, 3.9 cm2 filter area): Millex GV, 0.22 μM: hydrophilic polyvinylidene difluoride (PVDF, Durapore®) membrane with low protein binding capacity, Millex GN, 0.20 μM: hydrophilic nylon membrane with low protein binding capacity protein binding, and Millex GP, 0.22 μM: a hydrophilic polyethylene-sulfone membrane (PES) with a modified surface with a low protein binding capacity.
Gore navedeni filteri su korišteni kao predstavnici tipova komercijalno pristupačnih hidrofilnih mikrofiltera. Ispitivanja filtracije su izvedena upotrebom sljedećih sustava za otapanje: The above filters were used as representatives of the types of commercially available hydrophilic microfilters. Filtration tests were performed using the following dissolution systems:
1. 0.6 mg/mL Aβ42 u 0.01 M NH4OH 1. 0.6 mg/mL Aβ42 in 0.01 M NH4OH
2. 0.6 mg/mL Aβ42 u 10 mM natrijevom glicinatu, pH 9.0 2. 0.6 mg/mL Aβ42 in 10 mM sodium glycinate, pH 9.0
3. 0.6 mg/mL Aβ42 u 10 mM natrijevom lizinatu, pH 9.0 3. 0.6 mg/mL Aβ42 in 10 mM sodium lysinate, pH 9.0
4. 0.6 mg/mL Aβ42 u 10 mM arinin-HCl, pH 9.0 4. 0.6 mg/mL Aβ42 in 10 mM arinine-HCl, pH 9.0
Volumen od 2 približno mL svake otopine Aβ42 je filtriran preko svakog filtera. Koncentracije peptida su određene kromatografijom reverzne faze (RP HPLC). Regeneracija filtera je određena usporedbom veličine signala peptida prije i nakon mikrofiltriranja, a rezultati su prikazani na donjim Tablicama 3 i 4. A volume of approximately 2 mL of each Aβ42 solution was filtered over each filter. Peptide concentrations were determined by reverse phase chromatography (RP HPLC). Filter regeneration was determined by comparing the magnitude of the peptide signal before and after microfiltration, and the results are shown in Tables 3 and 4 below.
Tablica 3 - Regeneracija filtera Table 3 - Filter regeneration
[image] [image]
Primjer 4 - Filtracija Aβ42 u puferima natrijevog glicinata i natrijevog lizinata sa i bez dodatka ekcipijensa Example 4 - Filtration of Aβ42 in sodium glycinate and sodium lysinate buffers with and without the addition of excipients
U ovim istraživanjima je 0.6 mg/mL Aβ42 otopljeno u 10 mM natrijevog glicinata koji sadrži 0.1% polisorbat 80 (PS80), 0.9% natrijevog klorida i kombinaciju 0.1% PD80, 0.9% NaCl, 5% saharoze, 1% saharoze i/ili 4% manitola. Približno 2-5 mL svake formulacije je filtrirano preko Millex GV filtera. Aikvot filtrata je centrifugiran u stolnoj centrifugi postavljenoj na >10,000 RPM kroz ~3 minuta. Regeneracija filtera je određena usporedbom RP-HPLC veličine signala peptida prije i nakon filtracije. In these studies, 0.6 mg/mL Aβ42 was dissolved in 10 mM sodium glycinate containing 0.1% polysorbate 80 (PS80), 0.9% sodium chloride, and a combination of 0.1% PD80, 0.9% NaCl, 5% sucrose, 1% sucrose, and/or 4 % mannitol. Approximately 2-5 mL of each formulation was filtered through a Millex GV filter. An aliquot of the filtrate was centrifuged in a benchtop centrifuge set at >10,000 RPM for ~3 minutes. Filter regeneration was determined by comparing the RP-HPLC signal magnitude of the peptides before and after filtration.
Tabica 4 - Izolacija fitracijom za različite puferske formulacije Table 4 - Isolation by filtration for different buffer formulations
[image] [image]
Pokazano je da je Millex V filter preferirani filter za Aβ formulaciju zbog dobre izolacije peptida i prihvatljivosti za upotrebu u komercijalnim procesima. Formulacije peptida koje sadrže 0.9% NaCl su vidljivo manje topljive i manja je regeneracije filtera, a ustanovljeno RP HPLC. Da bi se povećala topljivost peptida u prisutnosti anorganskih soli, kao stoje natrijev klorid, može biti preferirano dodati sterilizirani otopinu sredstva za modifikaciju toničnosti u pufersku otopinu, a nakon mikrofiltracije peptida. The Millex V filter has been shown to be the preferred filter for Aβ formulation due to good peptide isolation and acceptability for use in commercial processes. Peptide formulations containing 0.9% NaCl are visibly less soluble and there is less filter regeneration, as established by RP HPLC. To increase the solubility of the peptide in the presence of inorganic salts, such as sodium chloride, it may be preferred to add a sterilized solution of the tonicity modifier to the buffer solution after microfiltration of the peptide.
S druge strane, sredstva za modifikaciju toničnosti koji su saharidi (šećeri) pokazuju različite tipove aktivnosti u otopini, i mogu pomagati održavanju peptidnoe suspenzije. To svojstvo, poznato kao "water-ordering" i ima tendenciju "hidratiranja" peptidnog lanca u otopini, tako da taj poprimi termodinamski stabilniju konformaciju β-nabrane ploče. On the other hand, tonicity modifiers that are saccharides (sugars) show different types of activity in solution, and can help maintain the peptide suspension. This property, known as "water-ordering", tends to "hydrate" the peptide chain in solution, so that it assumes a thermodynamically more stable β-pleated sheet conformation.
Aβ peptid se može filtrirati u rasponu od oko 8.5 do oko 12, preferirano od pH 9 do 10 uz dobru izolaciju. Filtriranje se može izvesti preko 0.2 μm Millex V (Millipore Durapore) membrane koja je prihvatljiva za postupak proizvodnje. Aβ peptide can be filtered in the range of about 8.5 to about 12, preferably pH 9 to 10 with good isolation. Filtration can be performed through a 0.2 μm Millex V (Millipore Durapore) membrane that is acceptable for the manufacturing process.
Za stabilnu biološki aktivnu formulaciju Aβ peptida u fiziološki prihvatljivim uvjetima pogodnim za kliničku upotrebu, početni korak postupka formulacije će preferirano uključivati pH 8.5 do 10 sterilne filtracije peptida. For a stable biologically active formulation of the Aβ peptide under physiologically acceptable conditions suitable for clinical use, the initial step of the formulation process will preferably involve pH 8.5 to 10 sterile filtration of the peptide.
Primjer 5 - Topljive tekuće formulacije Example 5 - Soluble liquid formulations
Testiranje metode Method testing
Tri skupine Aβ peptida su proizvedene od American Peptide Co (APC). Potvrdne formulacije, kemijska i biološka karakterizacija rezultata APC peptida ponovljene su s Aβ42 pripravljenim u California peptide Research. Three groups of Aβ peptides are produced by American Peptide Co (APC). Confirmatory formulations, chemical and biological characterization of APC peptide results were repeated with Aβ42 prepared at California Peptide Research.
Opis formulacije je opisan u donjim Dijelovima 1, 2, i 3. A description of the formulation is described in Parts 1, 2, and 3 below.
Testiranje topljivosti Solubility testing
Opisi karakteristične za formulacije su prikazani u pojedinim donjim dijelovima. Pojedine posudice su analizirane periodično kroz nekoliko mjeseci skladištenja pri 2-8 °C Izgled, pH, koncentracija peptida i čistoća su rutinski praćeni tijekom cijelog istraživanja. HPLC reverzne faze (RP HPLC) je korišten da se kvantitativno odredi koncentracija i područje čistoće Aβ peptida. RP HPLC koristi polimernu reverznu kolonu s amonijevim bikarbonatom (ili tris)/acetonitrilom gradijentom eluiranja peptida i detekciju pri 220 nm. Koncentracija peptida je mjerena prema referentnom standardu, čistoća je izračunata kao postotak površine signala Aβ peptida detektiranog u nastalom integriranom kromatografu. Descriptions characteristic of the formulations are presented in individual lower parts. Individual dishes were analyzed periodically during several months of storage at 2-8 °C. Appearance, pH, peptide concentration and purity were routinely monitored throughout the research. Reverse phase HPLC (RP HPLC) was used to quantitatively determine the concentration and purity range of Aβ peptides. RP HPLC uses a polymeric reverse ammonium bicarbonate (or tris)/acetonitrile gradient elution column for peptides and detection at 220 nm. Peptide concentration was measured according to the reference standard, purity was calculated as the percentage of the Aβ peptide signal area detected in the resulting integrated chromatograph.
Karakterizacija Characterization
Karakterizacija otopine strukture AJ342 dobivena je istraživanjem cirkularnog dikroizma, kao stoje pokazano na Slici 1. Characterization of the AJ342 structure solution was obtained by circular dichroism research, as shown in Figure 1.
Biološka aktivnost Biological activity
Švicarskim Webster miševima (4-8 po grupi) su injektirani različiti Aβ42 u različitim formulacijama i adjuvansima s CFA/IFA, MPL (Corixa Immunochemicals) ili QS 21 (Aquila Pharmaceuticals) pri naznačenim koncentracijama antigen/adjuvansa za određeno istraživanje. Injekcije su rutinski dane dvotjedno, tjedna 0, 2 i 4, osim ako je drugačije naznačeno. Miševima je uzeta krv drugog i četvrtog tjedna od injekcije. Serumi su analizirani standardnom "sendvič" ELISA testom, korištenjem Aβ42 kao antigena i HRP-konjugirani kozji anti-mišji IgG kao prikazano antitijelo. Titrovi antitijela su prikazani točkasto ili kao srednja vrijednost podataka za svaku životinju u svakoj skupini, i općenito su uspoređeni titrovima dobivenim upotrebom agregiranih Aβ42/CFA/IFA kao kontrolnih imunogena. Vidi Tablice 8 i 10 za rezultate titrova. Swiss Webster mice (4-8 per group) were injected with different Aβ42 in different formulations and adjuvants with CFA/IFA, MPL (Corixa Immunochemicals) or QS 21 (Aquila Pharmaceuticals) at the indicated antigen/adjuvant concentrations for the specific study. Injections were routinely given biweekly at weeks 0, 2, and 4, unless otherwise indicated. Blood was taken from the mice on the second and fourth week after the injection. Sera were analyzed by a standard "sandwich" ELISA, using Aβ42 as antigen and HRP-conjugated goat anti-mouse IgG as the antibody shown. Antibody titers are plotted or as mean data for each animal in each group, and are generally compared to titers obtained using aggregated Aβ42/CFA/IFA as control immunogens. See Tables 8 and 10 for titer results.
1. Formulacije 1. Formulations
Aβ peptid je otopljen pri 0.6 mg/mL u 10 mM natrijevom glicinatu, pH pufera 9-9.5 i filtriranje preko Millex GV 0.2 μm filtera. Formulacijama je volumen dopunjen do 0.5 mL u staklenim bočicama od 2 cc (Genisa P/N X34-113-002) i začepljene su sivim butilnim čepovima (Genisa P/N X66-l 13-030) i zataljene. Posudice su čuvane pri 2-8 °C. Aβ peptide was dissolved at 0.6 mg/mL in 10 mM sodium glycinate, pH 9-9.5 buffer and filtered through a Millex GV 0.2 μm filter. Formulations were made up to 0.5 mL in 2 cc glass vials (Genisa P/N X34-113-002) and sealed with gray butyl caps (Genisa P/N X66-1 13-030) and sealed. The dishes were stored at 2-8 °C.
2. Test stabilnosti 2. Stability test
Koncentracija i čistoća Aβ42 u formulacijama je praćena RP HPLC. Topljivi uzorci su analizirani sami ili nakon mikrocentrifugiranja uzoraka. The concentration and purity of Aβ42 in the formulations was monitored by RP HPLC. Soluble samples were analyzed alone or after microcentrifugation of the samples.
Sljedeća tablica (Tablica 5) predstavlja analitičke podatke za topljive tekuće formulacije. Nije viđena razlika između uzoraka koji su bili i koji nisu bili centrifugirani prije analize, ukazujući trajnu topljivost peptida pri željenoj koncentraciji. % čistoće prema površini signala je uspoređen s referentnim standardom: nije zamijećena značajna degradacija peptida. Topljive formulacija je ostala stabilna u periodu od 3 mjeseca, a kada je čuvana pri 2 do 8 °C The following table (Table 5) presents the analytical data for soluble liquid formulations. No difference was seen between samples that were and were not centrifuged prior to analysis, indicating sustained peptide solubility at the desired concentration. % purity by signal area was compared to a reference standard: no significant peptide degradation was observed. The soluble formulation remained stable for a period of 3 months, and when stored at 2 to 8 °C
Tablica 5. Rezultati nakon čuvanja od tri mjeseca Table 5. Results after three months of storage
[image] [image]
3. Karakterizacija 3. Characterization
Karakterizacija Aβ formulacije cirkularnim dikroizmom pokazuje da peptid zauzima konformaciju slučajnog klupka u otopini s karakterističnom negativnom eliptičnom apsorbancijom između 189-205 mn. Characterization of the Aβ formulation by circular dichroism shows that the peptide occupies a random coil conformation in solution with a characteristic negative elliptical absorbance between 189-205 mn.
Biološka aktivnost Biological activity
Topljiva Aβ42 formulacije koja ima pH 9 je povećala titar antitijela švicarskih Webster miševa, a kada je injektirana s adjuvansom. Injekcije u dvotjednim intervalima (0, 2, 4 tjedana) 33 μg Aβ42 s 50 μig MPL ili 25 μg QS21 su izazvali odgovarajući titar odgovora u usporedbi s kontrolom. A soluble Aβ42 formulation having a pH of 9 increased antibody titers in Swiss Webster mice when injected with adjuvant. Injections at biweekly intervals (0, 2, 4 weeks) of 33 μg Aβ42 with 50 μg MPL or 25 μg QS21 elicited a corresponding titer response compared to control.
Primjer 6 - Topljive formulacije suspenzija Formulacje glicin/acetata Example 6 - Soluble suspension formulations Glycine/acetate formulations
Aβ peptid je otopljen pri 0.6 mg/mL u 10 mM natrijevog glicinata, pH 9-9.5 puferu samom ili s različitim ekcipijensima. Otopine peptida su filtrirane preko Millex G V filtera, te su titrirane s 0.1 M octenom kiselinom do pH-6 da nastane suspenzija peptida u puferima. Formulacije su dopunjene do volumena od 0.5 mL u od 2 cc (Genisa P/N X34-113-002) i začepljene su sivim butilnim čepovima (Genisa P/N X66-113-030) i zataljene. Formulacije su čuvane pri 2-8 °C. Aβ peptide was dissolved at 0.6 mg/mL in 10 mM sodium glycinate, pH 9-9.5 buffer alone or with various excipients. Peptide solutions were filtered through a Millex G V filter and titrated with 0.1 M acetic acid to pH-6 to form a peptide suspension in buffers. The formulations were made up to a volume of 0.5 mL in a 2 cc (Genisa P/N X34-113-002) and capped with gray butyl stoppers (Genisa P/N X66-113-030) and sealed. The formulations were stored at 2-8 °C.
Formulacje glicin/citrata Glycine/citrate formulations
0.6 mg/mL Aβ42 formulacije su pripravljene u 10 mM natrijevom glicinatu koji sadrži 0.1% PS-80 samog ili u kombinaciji s 5% saharozom (25 mL), pH 9-9.5 puferu samom ili s različitim ekcipijensima. Otopine peptida su filtrirane preko Millex G V filtera, te su titrirane s 0.1 M limunskom kiselinom do pH~6.0. Može se glicin/citrat/PS 80 nakon titracije dodati 0.9% natrijev klorid. Formulacije su dopunjene do volumena od 0.5 mL u od 2 cc (Genisa P/N X34-113-002) i začepljene su sivim butilnim čepovima (Genisa P/N X66-113-030) i zataljene. Formulacije su čuvane pri 2-8 °C. 0.6 mg/mL Aβ42 formulations were prepared in 10 mM sodium glycinate containing 0.1% PS-80 alone or in combination with 5% sucrose (25 mL), pH 9-9.5 buffer alone or with various excipients. Peptide solutions were filtered through a Millex G V filter and titrated with 0.1 M citric acid to pH~6.0. Glycine/citrate/PS 80 can be added after titration with 0.9% sodium chloride. The formulations were made up to a volume of 0.5 mL in a 2 cc (Genisa P/N X34-113-002) and capped with gray butyl stoppers (Genisa P/N X66-113-030) and sealed. The formulations were stored at 2-8 °C.
Glicin/citratne formulacije su razvijane dalje, da se uključi kapacitet pufera pri pH 6. Nekoliko formulacija Aβ peptida kod 100 mL su pripravljene u 10 mM natreijvom glicinatu pH 9 koji sadrži 5% saharoze sa ili bez 0.1% PS 80. Pripravljene su dodatne formulacije pri 0.1 mg/mL Aβ42 u 10 mM natrijevog glicinata koji sadrži 5% saharoze sa ili bez 0.1% PS 80. Peptidne otopine su filtrirane prema Millex filtera prije podešavanja pH. pH je zatim podešen na pH 6.0, a 10 mM i 20 mM otopinom citratnog pufera koristeći otopinu 1 M natrijevog citrata, pH 5.5. Formulacije su dopunjene do volumena od 0.5 mL u od 2 cc (Genisa P/N X34-113-002) i začepljene su sivim butilnim čepovima (Genisa P/N X66-113-030) i zataljene. Glycine/citrate formulations were further developed to include buffer capacity at pH 6. Several 100 mL formulations of Aβ peptide were prepared in 10 mM sodium glycinate pH 9 containing 5% sucrose with or without 0.1% PS 80. Additional formulations were prepared at 0.1 mg/mL Aβ42 in 10 mM sodium glycinate containing 5% sucrose with or without 0.1% PS 80. Peptide solutions were filtered using Millex filters before pH adjustment. The pH was then adjusted to pH 6.0, and 10 mM and 20 mM citrate buffer solution using 1 M sodium citrate solution, pH 5.5. The formulations were made up to a volume of 0.5 mL in a 2 cc (Genisa P/N X34-113-002) and capped with gray butyl stoppers (Genisa P/N X66-113-030) and sealed.
Ispitivanje stabilnosti Stability test
Koncentracija i čistoća Aβ42 u formulacijama je praćena RP HPLC. Ukupna koncentracija v:v peptida Aβ42 suspenzija je mjerena ponovnim otapanjem peptida s v razrjeđenjem s 2% natrijevom dodecilnim sulfatom (SDS) u 0.01 M NaOH i 1 min. zagrijavanja pri 100 °C prije analize. Alternativno, koncentracija topljivog Aβ42 u formulaciji suspenzije je određena centrifugiranjem testiranog uzorka u mikrocentrifugi pri > 10,000 rpm kroz 3-5 minuta. Alikvoti ponovno otopljenog peptida ili supernatanta su analizirani pomoću RP HPLC. Valja napomenuti da su tijekom ovih istraživanja, poboljšana kromatografska RP HPLC metoda određen relativno prema površini signala referentnog standarda u vremenu analize i kromatogrami su uspoređeni u svakoj vremenskoj točki da se utvrdi degradacija. Također su praćeni pH i izgled tijekom ispitivanja stabilnosti. The concentration and purity of Aβ42 in the formulations was monitored by RP HPLC. The total concentration of the v:v peptide Aβ42 suspension was measured by redissolving the peptide with a v dilution with 2% sodium dodecyl sulfate (SDS) in 0.01 M NaOH and 1 min. heating at 100 °C before analysis. Alternatively, the concentration of soluble Aβ42 in the suspension formulation was determined by centrifugation of the test sample in a microcentrifuge at > 10,000 rpm for 3-5 minutes. Aliquots of redissolved peptide or supernatant were analyzed by RP HPLC. It should be noted that during these investigations, the improved chromatographic RP HPLC method was determined relative to the signal area of the reference standard at the time of analysis and the chromatograms were compared at each time point to determine the degradation. pH and appearance were also monitored during stability testing.
Tablica 6 predstavlja podatke za nekoliko tekućih formulacija suspenzije. Sve suspenzije izgledaju kao suspenzije. Te formulacije su titrirane do pH 6 određenom kiselinom navedenom u tablici. Ovisno o ekscipijensu, suspenzija peptida može nastati odmah ili u periodu od 2 tjedna ili više. Brže nastajanje formulacije suspenzije se može postići dodatkom natrijevog klorida ili zamjenom citrata za acetat u formulaciji peptida. Table 6 presents data for several liquid suspension formulations. All suspensions look like suspensions. These formulations were titrated to pH 6 with the specified acid listed in the table. Depending on the excipient, the peptide suspension can be formed immediately or over a period of 2 weeks or more. A faster formation of the suspension formulation can be achieved by adding sodium chloride or by substituting citrate for acetate in the peptide formulation.
Sve formulacije su dostigle željenu koncentraciju od 0.6 mg/mL peptida. % čistoće prema površini signala za sve formulacije je usporediva s čistoćom referentnog standarda. Nije zamijećen gubitak ili degradacija peptida u periodu od 3 mjeseca, a kada je čuvana pri 2 do 8 °C. pH i izgled ostaju usporedivi podacima prikazanim u Tablici 1. All formulations reached the desired concentration of 0.6 mg/mL peptide. The % purity by signal area for all formulations is comparable to the purity of the reference standard. No loss or degradation of the peptide was observed over a period of 3 months, when stored at 2 to 8 °C. pH and appearance remain comparable to the data shown in Table 1.
0.9% NaCl i 5% saharoza dovode do fiziološke toničnosti za parenteralno davanje. Kao i kod suspenzija, pH 6 je unutar prihvatljivog raspona pH za injekcije. 0.9% NaCl and 5% sucrose lead to physiological tonicity for parenteral administration. As with suspensions, a pH of 6 is within the acceptable pH range for injections.
Tablica 6 Tekuće formulacije suspenzija Table 6 Liquid formulations of suspensions
[image] nd: nije izvedeno [image] nd: not performed
Formulacije iz Tablice 7 su pripravljene s 10 mM ili 20 mM pufernim kapacitetom od citratnog pufera da bi se osiguralo daje dobiven pH 6 tijekom postupka priprave. Ove formulacije odmah tvore suspenziju. Zbog svojstava Aβ peptida, promjena konformacija peptida u suspenziji se događa unutar sterilne jezgre. Upotreba poznatih koncentracija pufera umjesto titracije dozvoljava lako rukovanje u mogućnost reporodukcije operacija unutar sterilne jedinice. The formulations of Table 7 were prepared with a 10 mM or 20 mM buffer capacity of citrate buffer to ensure that a pH of 6 was achieved during the preparation process. These formulations immediately form a suspension. Due to the properties of the Aβ peptide, the conformational change of the peptide in suspension occurs within the sterile core. The use of known buffer concentrations instead of titration allows easy handling and reproducibility of operations within a sterile unit.
Tablica 7. Puferirane suspenzije Table 7. Buffered suspensions
[image] [image]
Ove formulacije su daljnje proširene. Trifuoracetati ili kloridi Aβ42 peptida su otopljeni u 10 mM natrijevom glicinatu, pH 9-9.5. Koncentracije su podešene prema sadržaju soli da se dobije koncentracija Aβ42 od 0.45 mg/mL. Polisorbat 80, pri rasponu koncentracija od 0.02% do 0.5% može biti dodan u otopinu peptida prije filtracije. Slično, formulacijama se mogu dodati saharoza i natrijev klorid, prije ili nakon dodatka kiseline. pH peptidne otopine je podešen citratom ili HCl, kao što je dolje naznačeno. Sve formulacije tvore suspenzije i dobi vena je očekivana koncentracija Aβ42 peptida. These formulations were further expanded. Aβ42 peptide trifluoroacetates or chlorides were dissolved in 10 mM sodium glycinate, pH 9-9.5. The concentrations were adjusted according to the salt content to obtain an Aβ42 concentration of 0.45 mg/mL. Polysorbate 80, at a concentration range of 0.02% to 0.5% can be added to the peptide solution before filtration. Similarly, sucrose and sodium chloride can be added to the formulations, either before or after the addition of the acid. The pH of the peptide solution was adjusted with citrate or HCl, as indicated below. All formulations form suspensions and the age of veins is the expected concentration of Aβ42 peptide.
Tablica 7b Table 7b
[image] [image]
Karakterizacija Characterization
Karakterizacija Aβ42, pH 6 supsenzije cirkularnim dikroizmom i FT-IR pokazuje da peptid zauzima konformaciju beta nabrane ploče i tvori suspenziju. Strukture beta nabrane ploče su usporedive u 0.6 mg/mL suspenzijama, bez obzira na kiselinu za puferiranje (citrat, acetat, fosfat) ili dodani ekscipijens (saharoza, NaCl) dodane u formulacije. Dodatak PS-80 izgleda da generira jednoličnije dispergiranu suspenziju. Characterization of Aβ42, pH 6 suspension by circular dichroism and FT-IR shows that the peptide adopts a beta-pleated sheet conformation and forms a suspension. Beta-pleated plate structures were comparable in 0.6 mg/mL suspensions, regardless of the buffering acid (citrate, acetate, phosphate) or added excipient (sucrose, NaCl) added to the formulations. The addition of PS-80 appears to generate a more uniformly dispersed suspension.
Biološka aktivnost Biological activity
Suspenzija Aβ peptida, a kada je injektirana s adjuvansom povećava titar antitijela švicarskih Webster miševa. Injekcije u dvotjednim intervalima (0, 2, 4 tjedana) kao što je pokazano u Tablici 8 izazivaju adekvatni titar odgovora u usporedbi s kontrolom. Aβ peptide suspension, when injected with adjuvant, increases the antibody titer of Swiss Webster mice. Injections at two-week intervals (0, 2, 4 weeks) as shown in Table 8 elicit an adequate titer response compared to control.
Tablica 8. Odgovor antitijela na formulaciju suspenzije Table 8. Antibody response to suspension formulation
[image] * MPL formulacija koja sadrži trietanolamin [image] * MPL formulation containing triethanolamine
** titrovi izračunati kao jedinice pri 50% max. OD ** titers calculated as units at 50% max. FROM
Primjer 7 - Liofilizirane formulacije Example 7 - Lyophilized formulations
Formulacije Formulations
Četiri kombinacija 0.6 mg/mL Aβ42 u 10 mM glicinatnom ili lizinskom puferu s manitolom ili manitolom i saharozom su pripravljeni i sterilni filtrirani preko Millex V fitera. Jedna formulacija, 0.6 mg/mL Aβ42 u 10 mM natrijevom glicinatu pH 9.5, 4% manitola je smještena u posudicu i začepljena bez titracije na fiziološki pH. Zaostale tri otopine (lizin/citrat/4% manitol; glicinat/citrat/4% manitol; glicinat/HCl/4% manitol/1% saharoza) su zatim titrirane do pH 7.5 Imunksom kiselinom ili HCl, kao stoje naznačeno. Four combinations of 0.6 mg/mL Aβ42 in 10 mM glycinate or lysine buffer with mannitol or mannitol and sucrose were prepared and sterile filtered through a Millex V filter. One formulation, 0.6 mg/mL Aβ42 in 10 mM sodium glycinate pH 9.5, 4% mannitol was placed in a vial and capped without titration to physiological pH. The remaining three solutions (lysine/citrate/4% mannitol; glycinate/citrate/4% mannitol; glycinate/HCl/4% mannitol/1% sucrose) were then titrated to pH 7.5 with Immune acid or HCl as indicated.
Formulacijama su volumeni dopunjeni do 0.5 mL i staklenim bočicama od 2 cc (Genisa P/N X34-113-002) i labavo si začepljene sivim butilnim čepovima za liofilizaciju (Genisa P/NX66-113-030). Formulations were made up to 0.5 mL in 2 cc glass vials (Genisa P/N X34-113-002) and loosely capped with gray butyl caps for lyophilization (Genisa P/NX66-113-030).
Liofilizacija Lyophilization
Formulacije su liofilizirane u Virtis iofiizatoru koji se može programirati (od Genisa Sicor). Manitol je odabran kao primarna matrična komponenta. Da se postigne kristalizacija manitola, formuacije su zamrznute termičkim cikliziranjem (aneliranjem) nakon čega slijedi primarno i sekundarno sušenje kolača. The formulations were lyophilized in a programmable Virtis lyophilizer (from Genisa Sicor). Mannitol was chosen as the primary matrix component. To achieve the crystallization of mannitol, the formations are frozen by thermal cyclization (annealing) followed by primary and secondary cake drying.
Analiza stabilnosti Stability analysis
Koncentracija i čistoća liofiiziranih formulacija je praćena RP HPLC. Svakoj formulaciji je dodano 1.0 mL vode za injekciju (WFT). Formulacije su odmah analizirane nakon rekonstrukcije (sa ili bez centrifugiranja na stolnoj mikrocentrifugi pri >10,000 rpm kroz 3-5 minuta) ili su ponovno otopljene kao stoje ranije opisano. The concentration and purity of the lyophilized formulations was monitored by RP HPLC. 1.0 mL of water for injection (WFT) was added to each formulation. Formulations were analyzed immediately after reconstitution (with or without centrifugation on a benchtop microcentrifuge at >10,000 rpm for 3-5 minutes) or redissolved as described earlier.
Sljedeća tablica (Tablica 9) prikazuje koncentracije četiriju A(342 formulacija nakon liofilizacije i ponovnog otapanja. Kao što se može vidjeti iz podataka, formulacije su bile topljive. Uzorci su analizirani kao ponovo rekonstituirane formulacije i kao "ponovo otopljene" rekonstituirane formulacije, kao stoje opisano za peptidne suspenzije. Nije zamijećena razlika u rekonstituranoj formulacija prema ukupnoj koncentraciji peptida (ponovo otopljeni uzorak). Nije zamijećena degradacija ili gubitak peptida u periodu od 3 mjeseca, a kada je čuvana pri 2 do 8 °C, što je određeno RP HPLC kao u testu stabilnosti u Primjeru 5. The following table (Table 9) shows the concentrations of the four A(342 formulations after lyophilization and redissolution. As can be seen from the data, the formulations were soluble. The samples were analyzed as reconstituted formulations and as "redissolved" reconstituted formulations, as described for peptide suspensions. No difference was observed in the reconstituted formulation in terms of total peptide concentration (redissolved sample). No degradation or loss of peptide was observed over a period of 3 months, when stored at 2 to 8 °C, as determined by RP HPLC as in the stability test in Example 5.
Tablica 9. Liofilizirane formulacije Table 9. Lyophilized formulations
[image] [image]
Karakterizacija Characterization
Karakterizacija Aβ42, pH 7.5 liofiliziranih formulacija cirkularnim dikroizmom i FT-IR pokazuje da peptid zauzima konformaciju slučajnog klupka tijekom liofilizacije i rekonstrukcije. Ovo opažanje je u skladu s formulacijom Aβ42 natrijevog glicinata pH 9 koja je također topljiva, može se filtrirati i zauzima konformaciju slučajnog klupka u otopini. Characterization of Aβ42, pH 7.5 lyophilized formulations by circular dichroism and FT-IR shows that the peptide adopts a random coil conformation during lyophilization and reconstitution. This observation is consistent with the sodium glycinate pH 9 formulation of Aβ42 which is also soluble, filterable, and assumes a random coil conformation in solution.
Biološka aktivnost Biological activity
Liofilizirana formulacija Aβ42, 0.6 mg/mL Aβ42 u 10 mM natrijevom glicinat/HCl/4% manitol/1% saharozi, pH 7.5 je izabrana kao predstavnik liofilizirane formulacije. Taj produkt, a kada je miješan s MPL ili QS21 adjuvansom, povećava titar antitijela švicarskih Webster miševa. Geometrijske sredine titara su prikazane u Tablici 10 u usporedbi s kontrolom. A lyophilized formulation of Aβ42, 0.6 mg/mL Aβ42 in 10 mM sodium glycinate/HCl/4% mannitol/1% sucrose, pH 7.5 was chosen as a representative lyophilized formulation. This product, when mixed with MPL or QS21 adjuvant, increases the antibody titer of Swiss Webster mice. The geometric means of the titers are shown in Table 10 compared to the control.
Tablica 10. Odgovor antitijela na liofiliziranu formulaciju Table 10. Antibody response to the lyophilized formulation
[image] * MPL formulacija koja sadrži tnetanolamm [image] * MPL formulation containing tnetanolamm
** titrovi izračunati kao jedinice pri 50% max. OD ** titers calculated as units at 50% max. FROM
Primjer 8 - Priprava na povećanoj skali upotrebom GMP standarda priprave Example 8 - Preparation on an increased scale using GMP preparation standards
Suspenzija peptida je na povećanoj skali do 1.5 litara pripravljena i punjena je kod proizvođača lijeka. Punjene su dvije koncentracije: 0.6 mg/mL i 0.1 mg/mL. Obje koncentracije su uspješno pripravljene na povećanoj skali, punjene i ostale su stabilne pri 2-8 ° i pri 25 °C nakon dva mjeseca. The peptide suspension was prepared on an enlarged scale of up to 1.5 liters and was filled by the drug manufacturer. Two concentrations were filled: 0.6 mg/mL and 0.1 mg/mL. Both concentrations were successfully scaled up, filled and remained stable at 2-8 °C and at 25 °C after two months.
Aβ peptid je otopljen pri koncentracijama od 0.6 mg/mL i 0.1 mg/mL u 10 mM otopini glicinatnog pufera pH 9 koji sadrži 5% saharozu. Otopina peptida je sterilno filtrirana preko Molopak 20 Mollopore Durapore 0.2 fim sterilizirajućeg flltera u septičkoj jezgri. RP HPLC analiza nije pokazala značajan gubitak peptida tijekom postupka filtracije. 1M natrijevog citrata pH 5.5 je dodan spoj i sterilno je filtrirana preko 0.2 μm Durapor filtera u aseptičku jezgru. Peptidna otopina je vagana i odgovarajuća količina natrij-citratnog pufera je dodana da se dobije 20 mM citratna formulacija, pH 6. Aβ peptide was dissolved at concentrations of 0.6 mg/mL and 0.1 mg/mL in a 10 mM solution of glycinate buffer pH 9 containing 5% sucrose. The peptide solution was sterile filtered through a Molopak 20 Mollopore Durapore 0.2 fim sterilizing filter in the septic core. RP HPLC analysis showed no significant loss of peptide during the filtration process. 1M sodium citrate pH 5.5 was added to the compound and it was sterile filtered through a 0.2 μm Durapor filter into an aseptic core. The peptide solution was weighed and an appropriate amount of sodium citrate buffer was added to make a 20 mM citrate formulation, pH 6.
Pri koncetraciji od 0.6 mg/mL, peptid je odmah stvorio suspenziju nakon dodatka citratnog pufera, a tijekom mjerenja pH je bio 6.4. Peptidna suspenzija je konstantno miješana, punjena po 1.2 mL u borsilikatne staklene bočice od 2 cc koje su začepljene West 4416 čepovima i zataljene. Pri koncentraciji od 0.1 mg/mL peptid je u bočicama ostao otopljen (i punjenje otopljen) nekoliko sati, a prije tvorbe suspenzije sljedećeg dana. At a concentration of 0.6 mg/mL, the peptide immediately formed a suspension after the addition of citrate buffer, and during the measurement the pH was 6.4. The peptide suspension was constantly mixed, filled 1.2 mL into 2 cc borosilicate glass vials, which were closed with West 4416 stoppers and sealed. At a concentration of 0.1 mg/mL, the peptide remained dissolved in the vials (and the filling dissolved) for several hours, before forming a suspension the next day.
Tablica 11 predstavlja podatke dobivene za tu suspenziju od 0.6 mg/mL Aβ42: Table 11 presents the data obtained for this suspension of 0.6 mg/mL Aβ42:
Tablica 11. Table 11.
Aβ peptid suspenzija 0.6 mg/mL u 10 mM glicin, 20 mM citrat 5% saharoza, pH 6 Aβ peptide suspension 0.6 mg/mL in 10 mM glycine, 20 mM citrate 5% sucrose, pH 6
[image] [image]
Primjer 9 - Puferirane suspenzije Aβ 1-42 s QS1 adjuvansom Example 9 - Buffered suspensions of Aβ 1-42 with QS1 adjuvant
Za formulacije u jednoj bočici u kojima je Aβ1-42 i adjuvans su ispitivani upotrebom QS-21 i triterpenog glukozida koji imaju stimulirajuću imunu aktivnost, je iznenađujuće nađeno da rezultiraju tvorbom vizualno bistre formulacije peptidne suspenzije. Single-vial formulations in which Aβ1-42 and adjuvant were tested using QS-21 and a triterpene glucoside having immune-stimulating activity were surprisingly found to result in the formation of a visually clear peptide suspension formulation.
1. Formulacija Aβ1-42/QS-21 u jednoj bočici 1. Aβ1-42/QS-21 formulation in one vial
Otopljen je i liofiliziran QS-21 s otopinom Aβ1-42 u 10 mM natrijevog glicinata pri pH 9.0. U svakom sljedećem primjeru je liofilizirani QS-21 otopljen s Aβ1-42 (TFA sol 0.45 mg/mL) otopinom u 10 mM glicinu (Gly), pH 9.0. Pripravak sadrži otopljeni QS-21, te je pH podešen na 6.0 dodatkom citratnog pufera (Cit) da se stvori vizualno bistra suspenzija. Mjerenja zamućenosti su određena spektrofotometrom postavljenim na valnu duljinu 405 nm da se mjeri bistrina nastale suspenzije. Rezultati pokazuju da su veličine čestica suspendiranog peptida manje od valne duljine reflektirane svjetlosti. Te formulacije su pokazale stabilnost kad su čuvane pri 2-8 °C, a pri praćenju stabilnost i je nađeno da sve formulacije sadrže Aβ1-42 predominantno u konformaciji (3-nabrane ploče; nijedna formulacija nije pokazala zamućenje pri spektrofotometrijskoj analizi na 405 nm. Formulacija od 0.45 mg/mL Aβ1-42, 0.2 mg/mL QS-21, 10 mM Cit, 5% saharoze, pH 6.0 je testirana u testu mišjeg titra, pri čemu nastaje veći titar odgovora. QS-21 was dissolved and lyophilized with a solution of Aβ1-42 in 10 mM sodium glycinate at pH 9.0. In each subsequent example, lyophilized QS-21 was dissolved with Aβ1-42 (TFA salt 0.45 mg/mL) solution in 10 mM glycine (Gly), pH 9.0. The preparation contains dissolved QS-21, and the pH is adjusted to 6.0 with the addition of citrate buffer (Cit) to create a visually clear suspension. Turbidity measurements were taken with a spectrophotometer set at a wavelength of 405 nm to measure the clarity of the resulting suspension. The results show that the particle sizes of the suspended peptide are smaller than the wavelength of the reflected light. These formulations showed stability when stored at 2-8 °C, and when monitoring stability, all formulations were found to contain Aβ1-42 predominantly in conformation (3-pleated plates; none of the formulations showed turbidity when spectrophotometrically analyzed at 405 nm. A formulation of 0.45 mg/mL Aβ1-42, 0.2 mg/mL QS-21, 10 mM Cit, 5% sucrose, pH 6.0 was tested in a mouse titer assay, producing a higher titer response.
Tablica 12A Table 12A
[image] 1 nađeno da ima odovor s visokom razinom titra u misu 2. Aβ42titracijesQS-21 [image] 1 found to have a high titer in mass 2. Aβ42titrationsQS-21
Aβ42 (2 mg/mL) je otopljen u 10 mM glicinu, pH 9.5. pH je podešen na 9.5 dodatkom 1 M NaOH. Otopina Aβ42 je zatim filtrirana preko Millex GV filtera. QS-21 (5 mg/mL) je otopljen u 10 mM citratu, pH 6.0, te je filtrirana preko Millex GV filtera. Alikvot Aβ42 je pomiješan s alikvotom QS-21 da nastane željeni omjer, miješan i razrijeđen 10 mM glicinom, pH 9.5, te 1 M citratom pH 5.2 da se dobije konačna koncentracija naznačena u donjoj Tablici 12. Aβ42 (2 mg/mL) was dissolved in 10 mM glycine, pH 9.5. The pH was adjusted to 9.5 by adding 1 M NaOH. The Aβ42 solution was then filtered through a Millex GV filter. QS-21 (5 mg/mL) was dissolved in 10 mM citrate, pH 6.0, and filtered through a Millex GV filter. An aliquot of Aβ42 was mixed with an aliquot of QS-21 to form the desired ratio, mixed and diluted with 10 mM glycine, pH 9.5, and 1 M citrate pH 5.2 to obtain the final concentration indicated in Table 12 below.
Tablica 12B. Početna AN1792/QS21 Co-formulacija Table 12B. Initial AN1792/QS21 Co-formulation
[image] [image] [image] [image]
3. Dodatneformuacije Aβ42 i QS-21 3. Additional formulations of Aβ42 and QS-21
Aβ42 i korid su otopljeni pri 1 mg/mL u 20 natrijevog glicinata, pH 9.0-9.5 sa ili bez saharoze, 0.1% PS-80 ili 0.4% PS-80. Otopina peptida je sterilno filtrirana preko Millex GV siringnog filtera. Liofilizat QS-21 je otopljen u 5 mg/mL u 10 mM citratu, pH 6.0 i sterilno je filtriran preko Millex GV siringnog filtera. Aβ42 and corrid were dissolved at 1 mg/mL in 20 sodium glycinate, pH 9.0-9.5 with or without sucrose, 0.1% PS-80, or 0.4% PS-80. The peptide solution was sterile filtered through a Millex GV syringe filter. Lyophilisate QS-21 was dissolved at 5 mg/mL in 10 mM citrate, pH 6.0 and sterile filtered through a Millex GV syringe filter.
Odgovarajući volumeni otopina Aβ42 i QS-21 otopina su spojeni da se dobije konačna koncentracija Aβ42 i QS-21 naznačena u formulacijama u donjoj Tablici 12C. Konačno, pH je smanjen 1 M citratnim puferom, pH 5.4 da se dobije konačni pH 6 u 20 mM citratnom puferu. Vizualni izgled formulacije je procijenjen kao bistro do mutnog (+ do +++). Promjenom koncentracijskih omjera Aβ42 i QS-21 se može podesiti izgled formulacija. Nadalje, šećeri i površinski aktivna sredstva su prihvatljivi ekscipijerisi formulacija. Appropriate volumes of Aβ42 and QS-21 solutions were combined to obtain the final concentration of Aβ42 and QS-21 indicated in the formulations in Table 12C below. Finally, the pH was adjusted with 1 M citrate buffer, pH 5.4 to give a final pH of 6 in 20 mM citrate buffer. The visual appearance of the formulation was evaluated as clear to cloudy (+ to +++). By changing the concentration ratios of Aβ42 and QS-21, the appearance of the formulations can be adjusted. Furthermore, sugars and surfactants are acceptable formulation excipients.
Tablica 12C. Dodatne formulacije A(342 i QS-21 Table 12C. Additional formulations A(342 and QS-21
[image] [image]
Primjer 10 - Mjerenje i interpretacija C. D. spektara Aβ42 formulacija Example 10 - Measurement and interpretation of C.D. spectra of Aβ42 formulations
Podaci o ciruarnom dikroizmu su sakupljeni upotrebom Aviv modela 62-DS spektropolarimetra (Lakewood, NJ). Odgovarajući uzorci su pripravljeni za sakupljanje bliskog UV i dalekog UV spektra, i naneseni su u kvarcnu ćeliju od 1 mm. Držač uzoraka je održavan pri temperaturi od točno 25 °C. Podaci su sakupljeni u intervalima od 0.5 nm upotrebom prosječnog vremena od 4 sekundi. U dalekoj UV regiji (λ,~1250-350 nm) dobiva se potpuno drugačija informacija, u toj regiji primarni signali rastu zbog aromatskog bočnog lanca (Phe, Tyr i Trp). Oznaka i veličina signala pokazuju stupanj fleksibilnosti na svakom mjestu kao i orijentaciju bočnog lanca relativno prema peptidnom skeletu. Kako je broj tih kromofora manji od broja amidnih skupina, i zato isto su kromofori razdijeljeni kroz cijelu molekulu, oni daju informacije samo o lokalnoj strukturi. Surgical dichroism data were collected using an Aviv model 62-DS spectropolarimeter (Lakewood, NJ). Appropriate samples were prepared to collect the near UV and far UV spectra, and were loaded into a 1 mm quartz cell. The sample holder was maintained at a temperature of exactly 25 °C. Data were collected at 0.5 nm intervals using an averaging time of 4 seconds. In the far UV region (λ, ~1250-350 nm) completely different information is obtained, in that region the primary signals increase due to the aromatic side chain (Phe, Tyr and Trp). The label and magnitude of the signal indicate the degree of flexibility at each site as well as the orientation of the side chain relative to the peptide backbone. As the number of these chromophores is smaller than the number of amide groups, and therefore the chromophores are distributed throughout the entire molecule, they provide information only about the local structure.
Očekuje se da brojne modifikacije i varijacije u izumu, kao što su je gore opisane u ilustrativnim primjerima, izvedu stručnjaci, pa stoga je ograničenje samo ono koje proizlazi iz priloženih zahtjeva. Prema tome, namjera zahtjeva je da pokriju takve ekvivalente varijacija koji ulaze u obujam izuma, a kako je prijavljen. It is expected that numerous modifications and variations in the invention, such as those described above in the illustrative examples, will be made by those skilled in the art, and therefore the limitation is only that which results from the appended claims. Accordingly, the claims are intended to cover such equivalent variations as fall within the scope of the invention as claimed.
Claims (47)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13704799P | 1999-06-01 | 1999-06-01 | |
PCT/US2000/015302 WO2000072870A1 (en) | 1999-06-01 | 2000-06-01 | Compositions of a-beta peptide and processes for producing same |
Publications (1)
Publication Number | Publication Date |
---|---|
HRP20010901A2 true HRP20010901A2 (en) | 2003-04-30 |
Family
ID=22475602
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
HR20010901A HRP20010901A2 (en) | 1999-06-01 | 2001-12-04 | Compositions of a-beta peptide and processes for producing same |
Country Status (25)
Country | Link |
---|---|
EP (1) | EP1181040A1 (en) |
JP (1) | JP2003519620A (en) |
KR (1) | KR20020016813A (en) |
CN (1) | CN1353615A (en) |
AR (1) | AR024558A1 (en) |
AU (1) | AU5726100A (en) |
BG (1) | BG106249A (en) |
BR (1) | BR0011251A (en) |
CA (1) | CA2374897A1 (en) |
CZ (1) | CZ20014150A3 (en) |
EE (1) | EE200100649A (en) |
HK (1) | HK1045938A1 (en) |
HR (1) | HRP20010901A2 (en) |
IL (1) | IL146575A0 (en) |
IS (1) | IS6182A (en) |
MX (1) | MXPA01012355A (en) |
NO (1) | NO20015859L (en) |
NZ (1) | NZ515744A (en) |
PE (1) | PE20010212A1 (en) |
PL (1) | PL352575A1 (en) |
RU (1) | RU2001135800A (en) |
SK (1) | SK17012001A3 (en) |
TR (1) | TR200103476T2 (en) |
WO (1) | WO2000072870A1 (en) |
ZA (1) | ZA200109704B (en) |
Families Citing this family (36)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7964192B1 (en) | 1997-12-02 | 2011-06-21 | Janssen Alzheimer Immunotherapy | Prevention and treatment of amyloidgenic disease |
US6761888B1 (en) | 2000-05-26 | 2004-07-13 | Neuralab Limited | Passive immunization treatment of Alzheimer's disease |
TWI239847B (en) | 1997-12-02 | 2005-09-21 | Elan Pharm Inc | N-terminal fragment of Abeta peptide and an adjuvant for preventing and treating amyloidogenic disease |
US6787523B1 (en) | 1997-12-02 | 2004-09-07 | Neuralab Limited | Prevention and treatment of amyloidogenic disease |
US7790856B2 (en) | 1998-04-07 | 2010-09-07 | Janssen Alzheimer Immunotherapy | Humanized antibodies that recognize beta amyloid peptide |
US6905686B1 (en) | 1997-12-02 | 2005-06-14 | Neuralab Limited | Active immunization for treatment of alzheimer's disease |
US6750324B1 (en) | 1997-12-02 | 2004-06-15 | Neuralab Limited | Humanized and chimeric N-terminal amyloid beta-antibodies |
US20080050367A1 (en) | 1998-04-07 | 2008-02-28 | Guriq Basi | Humanized antibodies that recognize beta amyloid peptide |
US20030147882A1 (en) | 1998-05-21 | 2003-08-07 | Alan Solomon | Methods for amyloid removal using anti-amyloid antibodies |
US6787637B1 (en) | 1999-05-28 | 2004-09-07 | Neuralab Limited | N-Terminal amyloid-β antibodies |
UA81216C2 (en) | 1999-06-01 | 2007-12-25 | Prevention and treatment of amyloid disease | |
CA2414772C (en) | 2000-07-07 | 2011-06-28 | Jan Naslund | Prevention and treatment of alzheimer's disease |
WO2002025279A2 (en) * | 2000-09-19 | 2002-03-28 | Evotec Neurosciences Gmbh | Use of amyloid precursor protein for treating brain amyloidosis |
US7700751B2 (en) | 2000-12-06 | 2010-04-20 | Janssen Alzheimer Immunotherapy | Humanized antibodies that recognize β-amyloid peptide |
MY144532A (en) | 2001-08-20 | 2011-09-30 | Lundbeck & Co As H | Novel method for down-regulation of amyloid |
MY139983A (en) | 2002-03-12 | 2009-11-30 | Janssen Alzheimer Immunotherap | Humanized antibodies that recognize beta amyloid peptide |
DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
TWI374893B (en) | 2003-05-30 | 2012-10-21 | Janssen Alzheimer Immunotherap | Humanized antibodies that recognize beta amyloid peptide |
SE0401601D0 (en) | 2004-06-21 | 2004-06-21 | Bioarctic Neuroscience Ab | Protofibril specific antibodies and uses thereof |
EP1838348B1 (en) | 2004-12-15 | 2013-06-26 | Janssen Alzheimer Immunotherapy | Humanized amyloid beta antibodies for use in improving cognition |
KR101434935B1 (en) | 2005-11-30 | 2014-10-01 | 애브비 인코포레이티드 | Monoclonal antibodies against amyloid beta protein and uses thereof |
JP5475994B2 (en) | 2005-11-30 | 2014-04-16 | アッヴィ・インコーポレイテッド | Anti-Aβ globulomer antibody, antigen-binding portion thereof, corresponding hybridoma, nucleic acid, vector, host cell, method for producing said antibody, composition comprising said antibody, use of said antibody and method of using said antibody. |
NO347079B1 (en) | 2006-03-23 | 2023-05-08 | Bioarctic Neuroscience Ab | Improved protofibril-selective antibodies and use thereof |
US8784810B2 (en) | 2006-04-18 | 2014-07-22 | Janssen Alzheimer Immunotherapy | Treatment of amyloidogenic diseases |
US8455626B2 (en) | 2006-11-30 | 2013-06-04 | Abbott Laboratories | Aβ conformer selective anti-aβ globulomer monoclonal antibodies |
US20100311767A1 (en) | 2007-02-27 | 2010-12-09 | Abbott Gmbh & Co. Kg | Method for the treatment of amyloidoses |
US8003097B2 (en) | 2007-04-18 | 2011-08-23 | Janssen Alzheimer Immunotherapy | Treatment of cerebral amyloid angiopathy |
PL2182983T3 (en) | 2007-07-27 | 2014-10-31 | Janssen Alzheimer Immunotherap | Treatment of amyloidogenic diseases with humanised anti-abeta antibodies |
JO3076B1 (en) | 2007-10-17 | 2017-03-15 | Janssen Alzheimer Immunotherap | Immunotherapy regimes dependent on apoe status |
US9067981B1 (en) | 2008-10-30 | 2015-06-30 | Janssen Sciences Ireland Uc | Hybrid amyloid-beta antibodies |
CN104744591B (en) | 2010-04-15 | 2022-09-27 | Abbvie德国有限责任两合公司 | Amyloid beta binding proteins |
CA2808187A1 (en) | 2010-08-14 | 2012-02-23 | Abbvie Inc. | Amyloid-beta binding proteins |
GB201113570D0 (en) | 2011-08-05 | 2011-09-21 | Glaxosmithkline Biolog Sa | Vaccine |
PL3166970T3 (en) | 2014-07-10 | 2021-09-13 | Bioarctic Ab | Improved a-beta protofibril binding antibodies |
IL293979B2 (en) | 2016-01-08 | 2024-08-01 | Ascendis Pharma Growth Disorders As | Controlled-release cnp agonists with increased nep stability |
CN114957438B (en) * | 2022-06-28 | 2024-04-02 | 福建亿彤生物科技有限公司 | Human Abeta 1-42 epitope polypeptide for detecting Alzheimer disease and preparation method thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2081482C (en) * | 1990-04-27 | 2000-11-21 | Ellis L. Kline | Method and composition for treatment of central nervous system disease states associated with abnormal amyloid beta protein molecular organization |
TWI239847B (en) * | 1997-12-02 | 2005-09-21 | Elan Pharm Inc | N-terminal fragment of Abeta peptide and an adjuvant for preventing and treating amyloidogenic disease |
-
2000
- 2000-05-31 PE PE2000000521A patent/PE20010212A1/en not_active Application Discontinuation
- 2000-05-31 AR ARP000102690A patent/AR024558A1/en unknown
- 2000-06-01 EP EP00942668A patent/EP1181040A1/en not_active Withdrawn
- 2000-06-01 CA CA002374897A patent/CA2374897A1/en not_active Abandoned
- 2000-06-01 TR TR2001/03476T patent/TR200103476T2/en unknown
- 2000-06-01 RU RU2001135800/14A patent/RU2001135800A/en unknown
- 2000-06-01 WO PCT/US2000/015302 patent/WO2000072870A1/en not_active Application Discontinuation
- 2000-06-01 IL IL14657500A patent/IL146575A0/en unknown
- 2000-06-01 AU AU57261/00A patent/AU5726100A/en not_active Abandoned
- 2000-06-01 EE EEP200100649A patent/EE200100649A/en unknown
- 2000-06-01 SK SK1701-2001A patent/SK17012001A3/en unknown
- 2000-06-01 NZ NZ515744A patent/NZ515744A/en unknown
- 2000-06-01 BR BR0011251-8A patent/BR0011251A/en not_active Application Discontinuation
- 2000-06-01 MX MXPA01012355A patent/MXPA01012355A/en unknown
- 2000-06-01 CZ CZ20014150A patent/CZ20014150A3/en unknown
- 2000-06-01 PL PL00352575A patent/PL352575A1/en not_active Application Discontinuation
- 2000-06-01 CN CN00808374A patent/CN1353615A/en active Pending
- 2000-06-01 JP JP2001511317A patent/JP2003519620A/en not_active Withdrawn
- 2000-06-01 KR KR1020017015474A patent/KR20020016813A/en not_active Application Discontinuation
-
2001
- 2001-11-26 ZA ZA200109704A patent/ZA200109704B/en unknown
- 2001-11-29 IS IS6182A patent/IS6182A/en unknown
- 2001-11-30 NO NO20015859A patent/NO20015859L/en not_active Application Discontinuation
- 2001-12-04 HR HR20010901A patent/HRP20010901A2/en not_active Application Discontinuation
- 2001-12-20 BG BG106249A patent/BG106249A/en unknown
-
2002
- 2002-08-23 HK HK02106241.2A patent/HK1045938A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
NO20015859D0 (en) | 2001-11-30 |
EE200100649A (en) | 2003-02-17 |
NZ515744A (en) | 2004-04-30 |
BR0011251A (en) | 2002-03-05 |
AU5726100A (en) | 2000-12-18 |
IS6182A (en) | 2001-11-29 |
HK1045938A1 (en) | 2002-12-20 |
NO20015859L (en) | 2002-02-01 |
IL146575A0 (en) | 2002-07-25 |
MXPA01012355A (en) | 2003-06-24 |
CZ20014150A3 (en) | 2002-05-15 |
JP2003519620A (en) | 2003-06-24 |
RU2001135800A (en) | 2003-08-20 |
SK17012001A3 (en) | 2002-06-04 |
PE20010212A1 (en) | 2001-02-22 |
EP1181040A1 (en) | 2002-02-27 |
KR20020016813A (en) | 2002-03-06 |
PL352575A1 (en) | 2003-08-25 |
TR200103476T2 (en) | 2002-04-22 |
CA2374897A1 (en) | 2000-12-07 |
BG106249A (en) | 2002-08-30 |
ZA200109704B (en) | 2003-02-26 |
CN1353615A (en) | 2002-06-12 |
AR024558A1 (en) | 2002-10-16 |
WO2000072870A1 (en) | 2000-12-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
HRP20010901A2 (en) | Compositions of a-beta peptide and processes for producing same | |
JP6438599B2 (en) | Anti-CGRP antibody preparation | |
EP2676677B1 (en) | Highly concentrated anti-cd40 antibody pharmaceutical preparation | |
KR102546471B1 (en) | liquid pharmaceutical composition | |
EP2198007B1 (en) | Pharmaceutical compositions containing clostridium difficile toxoids a and b | |
EP2088997B1 (en) | Liquid anti-rabies antibody formulations | |
JP2012531431A (en) | Stabilized alkyl glycoside composition and method thereof | |
KR20190027878A (en) | Stabilizing excipients for therapeutic protein preparations | |
MX2012005195A (en) | Formulation for hgh and rhigf-1 combination. | |
KR20220020819A (en) | Pharmaceutical parenteral composition of dual GLP1/2 agonist | |
JP2023539476A (en) | Annexin A1 N-terminal peptide formulation and method | |
CA2743725A1 (en) | Stabilizing alkylglycoside compositions and methods thereof | |
CN107028897B (en) | Immune regulator thymopentin powder injection medicine composition and quality control method | |
CN111375057A (en) | Pharmaceutical formulation comprising anti-Her 2 monoclonal antibody | |
CN116077646A (en) | Antibody preparation for resisting coronavirus S protein, preparation method and application thereof | |
JP2010536906A (en) | Liquid formulation of G-CSF | |
EA033742B1 (en) | FORMULATION FOR SUBCUTANEOUS ADMINISTRATION COMPRISING A SOLUBLE Fc RECEPTOR, PHARMACEUTICAL COMPOSITION COMPRISING A FORMULATION AND USE OF THE FORMULATION AND THE PHARMACEUTICAL COMPOSITION FOR TREATMENT AND PREVENTION OF AUTOIMMUNE DISEASES | |
JP2022543264A (en) | Processes for Preparing Compositions Containing Protein D Polypeptides | |
US20180313853A1 (en) | Chiral conversion of amyloid proteins associated with diseases | |
Winter et al. | Formulation Strategies for Recombinant Protein and Related Biotech Drugs | |
WO2022034545A1 (en) | Etelcalcetide formulations for parenteral use | |
BE1022254B1 (en) | FORMULATION OF SUCROSE VACCINE | |
JP2024503462A (en) | Ophthalmic composition containing tampanacept, stable without the use of stabilizers | |
TW202339788A (en) | Pharmaceutical formulations comprising a cyclodextrin | |
NZ714292B2 (en) | HIGHLY CONCENTRATED FORMULATIONS OF SOLUBLE Fc RECEPTORS |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A1OB | Publication of a patent application | ||
AIPI | Request for the grant of a patent on the basis of a substantive examination of a patent application | ||
ODRP | Renewal fee for the maintenance of a patent |
Payment date: 20040603 Year of fee payment: 5 |
|
OBST | Application withdrawn |