CN103720642A - Bioprotein-containing solution for external use and preparation method thereof - Google Patents
Bioprotein-containing solution for external use and preparation method thereof Download PDFInfo
- Publication number
- CN103720642A CN103720642A CN201210391695.1A CN201210391695A CN103720642A CN 103720642 A CN103720642 A CN 103720642A CN 201210391695 A CN201210391695 A CN 201210391695A CN 103720642 A CN103720642 A CN 103720642A
- Authority
- CN
- China
- Prior art keywords
- bioprotein
- sodium
- acid
- growth factor
- externally used
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000002360 preparation method Methods 0.000 title abstract description 28
- 239000000243 solution Substances 0.000 claims abstract description 52
- 239000003223 protective agent Substances 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 17
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims abstract description 12
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims abstract description 12
- 239000000203 mixture Substances 0.000 claims abstract description 8
- 102000019197 Superoxide Dismutase Human genes 0.000 claims abstract description 3
- 108010012715 Superoxide dismutase Proteins 0.000 claims abstract description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 24
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 16
- 239000008215 water for injection Substances 0.000 claims description 16
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 claims description 13
- 235000002949 phytic acid Nutrition 0.000 claims description 13
- 239000007921 spray Substances 0.000 claims description 13
- 239000007853 buffer solution Substances 0.000 claims description 12
- 239000003381 stabilizer Substances 0.000 claims description 12
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 11
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 11
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 11
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 claims description 10
- 229940068041 phytic acid Drugs 0.000 claims description 10
- 239000000467 phytic acid Substances 0.000 claims description 10
- 238000012859 sterile filling Methods 0.000 claims description 9
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 8
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 8
- 229940116977 epidermal growth factor Drugs 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 8
- 238000011068 loading method Methods 0.000 claims description 8
- 238000005374 membrane filtration Methods 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 238000012856 packing Methods 0.000 claims description 8
- 239000004033 plastic Substances 0.000 claims description 8
- 229920003023 plastic Polymers 0.000 claims description 8
- 235000011121 sodium hydroxide Nutrition 0.000 claims description 8
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 7
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 7
- 235000019800 disodium phosphate Nutrition 0.000 claims description 7
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 7
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 7
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 7
- 229920002307 Dextran Polymers 0.000 claims description 4
- 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 claims description 4
- 229960002086 dextran Drugs 0.000 claims description 4
- 108010074060 lecithinized superoxide dismutase Proteins 0.000 claims description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 4
- 229920000053 polysorbate 80 Polymers 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- 239000004094 surface-active agent Substances 0.000 claims description 4
- 102100027992 Casein kinase II subunit beta Human genes 0.000 claims description 3
- 101710158100 Casein kinase II subunit beta Proteins 0.000 claims description 3
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 235000011572 Pyrus ussuriensis Nutrition 0.000 claims description 3
- 244000173166 Pyrus ussuriensis Species 0.000 claims description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 3
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 3
- 239000000194 fatty acid Substances 0.000 claims description 3
- 229930195729 fatty acid Natural products 0.000 claims description 3
- 150000004665 fatty acids Chemical class 0.000 claims description 3
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 3
- 229960000367 inositol Drugs 0.000 claims description 3
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 3
- 239000004114 Ammonium polyphosphate Substances 0.000 claims description 2
- 102000007299 Amphiregulin Human genes 0.000 claims description 2
- 108010033760 Amphiregulin Proteins 0.000 claims description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- 101000737574 Homo sapiens Complement factor H Proteins 0.000 claims description 2
- 108010085895 Laminin Proteins 0.000 claims description 2
- 108010013563 Lipoprotein Lipase Proteins 0.000 claims description 2
- 102100022119 Lipoprotein lipase Human genes 0.000 claims description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 claims description 2
- 235000019826 ammonium polyphosphate Nutrition 0.000 claims description 2
- 229920001276 ammonium polyphosphate Polymers 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 229940069078 citric acid / sodium citrate Drugs 0.000 claims description 2
- 229960000633 dextran sulfate Drugs 0.000 claims description 2
- -1 dextran sulfate Chemical compound 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- BEGBSFPALGFMJI-UHFFFAOYSA-N ethene;sodium Chemical group [Na].C=C BEGBSFPALGFMJI-UHFFFAOYSA-N 0.000 claims description 2
- WPEXVRDUEAJUGY-UHFFFAOYSA-B hexacalcium;(2,3,4,5,6-pentaphosphonatooxycyclohexyl) phosphate Chemical compound [Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])(=O)OC1C(OP([O-])([O-])=O)C(OP([O-])([O-])=O)C(OP([O-])([O-])=O)C(OP([O-])([O-])=O)C1OP([O-])([O-])=O WPEXVRDUEAJUGY-UHFFFAOYSA-B 0.000 claims description 2
- 102000045512 human CFH Human genes 0.000 claims description 2
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 claims description 2
- 239000011777 magnesium Substances 0.000 claims description 2
- 229910052749 magnesium Inorganic materials 0.000 claims description 2
- 229940083542 sodium Drugs 0.000 claims description 2
- WEPNHBQBLCNOBB-FZJVNAOYSA-N sucrose octasulfate Chemical compound OS(=O)(=O)O[C@@H]1[C@H](OS(O)(=O)=O)[C@H](COS(=O)(=O)O)O[C@]1(COS(O)(=O)=O)O[C@@H]1[C@H](OS(O)(=O)=O)[C@@H](OS(O)(=O)=O)[C@@H](OS(O)(=O)=O)[C@@H](COS(O)(=O)=O)O1 WEPNHBQBLCNOBB-FZJVNAOYSA-N 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 29
- 102000004169 proteins and genes Human genes 0.000 abstract description 28
- 208000027418 Wounds and injury Diseases 0.000 abstract description 25
- 206010052428 Wound Diseases 0.000 abstract description 23
- 230000000694 effects Effects 0.000 abstract description 16
- 239000012460 protein solution Substances 0.000 abstract description 7
- 208000014674 injury Diseases 0.000 abstract description 6
- 230000007547 defect Effects 0.000 abstract description 5
- 230000007774 longterm Effects 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 4
- 230000008733 trauma Effects 0.000 abstract description 4
- 230000006378 damage Effects 0.000 abstract description 3
- 230000035876 healing Effects 0.000 abstract description 3
- 208000015181 infectious disease Diseases 0.000 abstract description 3
- 238000003860 storage Methods 0.000 abstract description 3
- 206010040943 Skin Ulcer Diseases 0.000 abstract description 2
- 206010012601 diabetes mellitus Diseases 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 231100000019 skin ulcer Toxicity 0.000 abstract description 2
- 239000004480 active ingredient Substances 0.000 abstract 2
- 230000001684 chronic effect Effects 0.000 abstract 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 abstract 1
- 208000010392 Bone Fractures Diseases 0.000 abstract 1
- 102000018386 EGF Family of Proteins Human genes 0.000 abstract 1
- 108010066486 EGF Family of Proteins Proteins 0.000 abstract 1
- 206010017076 Fracture Diseases 0.000 abstract 1
- 208000002847 Surgical Wound Diseases 0.000 abstract 1
- 208000025865 Ulcer Diseases 0.000 abstract 1
- 238000005469 granulation Methods 0.000 abstract 1
- 230000003179 granulation Effects 0.000 abstract 1
- 229940067606 lecithin Drugs 0.000 abstract 1
- 235000010445 lecithin Nutrition 0.000 abstract 1
- 239000000787 lecithin Substances 0.000 abstract 1
- 201000001245 periodontitis Diseases 0.000 abstract 1
- 239000003755 preservative agent Substances 0.000 abstract 1
- 230000002335 preservative effect Effects 0.000 abstract 1
- 231100000397 ulcer Toxicity 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 26
- 210000004027 cell Anatomy 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- 102000008100 Human Serum Albumin Human genes 0.000 description 8
- 108091006905 Human Serum Albumin Proteins 0.000 description 8
- 229920000669 heparin Polymers 0.000 description 8
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 7
- 229960001008 heparin sodium Drugs 0.000 description 7
- 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 6
- 229930195725 Mannitol Natural products 0.000 description 6
- 239000000594 mannitol Substances 0.000 description 6
- 235000010355 mannitol Nutrition 0.000 description 6
- 238000013094 purity test Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 230000029663 wound healing Effects 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000005138 cryopreservation Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 3
- 206010002198 Anaphylactic reaction Diseases 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 3
- 230000036783 anaphylactic response Effects 0.000 description 3
- 208000003455 anaphylaxis Diseases 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 101100120045 Bos taurus FGF1 gene Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 2
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 206010072170 Skin wound Diseases 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000002421 anti-septic effect Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 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 2
- 239000000126 substance Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 101001051969 Bos taurus Fibroblast growth factor 2 Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 206010063560 Excessive granulation tissue Diseases 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 108010064719 Oxyhemoglobins Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 206010040880 Skin irritation Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 206010053615 Thermal burn Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- QGKAVFXNTADNHB-UHFFFAOYSA-N [Mg].[K].[Ca] Chemical compound [Mg].[K].[Ca] QGKAVFXNTADNHB-UHFFFAOYSA-N 0.000 description 1
- FENRSEGZMITUEF-ATTCVCFYSA-E [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OP(=O)([O-])O[C@@H]1[C@@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H]1OP(=O)([O-])[O-] Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OP(=O)([O-])O[C@@H]1[C@@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H]1OP(=O)([O-])[O-] FENRSEGZMITUEF-ATTCVCFYSA-E 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003026 anti-oxygenic effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000010836 blood and blood product Substances 0.000 description 1
- 229940125691 blood product Drugs 0.000 description 1
- 238000010504 bond cleavage reaction Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000001804 debridement Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000001126 granulation tissue Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 230000002061 histotrophic effect Effects 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 244000309715 mini pig Species 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000003924 normoblast Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 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
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000006318 protein oxidation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000036556 skin irritation Effects 0.000 description 1
- 231100000475 skin irritation Toxicity 0.000 description 1
- 229940083982 sodium phytate Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to a bioprotein-containing solution for external use and a preparation method thereof, in particular to a bioprotein active ingredient-containing solution for external use and a preparation method thereof, and bioprotein active ingredients such as fibroblast growth factors, epidermal growth factors and lecithin superoxide dismutase are used for treating burn injury, promoting healing of burn wounds including a superficial II wound, a deep II wound and a granulation wound, chronic wounds including chronic ulcer and the like and new wounds including trauma, a surgical wound and the like and also possibly treating fracture, periodontitis and diabetic skin ulcer in the future. According to the solution prepared by the method, any preservative or bacteriostat is not added, the infection of the wounds is avoided, the safety is high, a protecting agent in auxiliary materials is a special composition, a protein solution can be stored at low temperature for a long term, the defect that a common protein preparation needs to be freeze-dried and pulverized for stable storage is overcome, the solution is convenient to carry and use by patients, the use safety is improved, and high purity and high activity of protein in the solution can be guaranteed.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, be specifically related to a kind of externally used solution that contains bioprotein active component and preparation method thereof.
Background technology
Due to the development of gene recombination technology, the commercialization of medical protein preparation is produced and is become a reality.Recently existing more than 140 kind of recombinant protein preparation carrying out I phase or clinical trial more than the I phase, has 12 kinds of approvals that obtained U.S. FDA.But, due to the physicochemical property of protein, it is existed when purification, separation, storage and sale exclusive because of difficulty.The degradation pathway of protein can be divided into two types, i.e. chemical instability and physical instability.The former means that protein is by becoming key or scission of link to generate new compound; Physical stability does not relate to the covalent bond variation of protein, but finger protein is changed into more senior structure (secondary or higher), comprises degeneration, surface adsorption, cohesion and precipitation.These physics and chemistry defects of pharmaceutical grade protein, have limited application and the development of its preparation.At present, in order to overcome the unstability of pharmaceutical grade protein, preserve after generally adopting Freeze Drying Technique to make freeze-dried powder, during use, redissolve again.Although lyophilized formulations is stable compared with liquid preparation, but lyophilization is a very complicated process, in first, second drying stage of pre-cooling and storage process, the physicochemical change that the structure of medicine may be regarded highly affects and changes, particularly two of protein and peptide class medicine, tertiary structure is easily damaged, lose activity and affect drug effect.During use, redissolve and also have all multi-risk Systems, as content heterogeneity, separate out impurity, have foreign body, cost is high, use is inconvenient etc.
Skin wound healing is complicated biological process, in this course, has many cells to participate in, and fibroblast is considered to one of main cell of repair in trauma.Recent study is found, various kinds of cell somatomedin, as b-FGF, aFGF, PDGF, TGF, IGF, epidermal growth factor etc. have huge effect in wound healing, their fibrosis, vascularization and re-epithelializations to skin wound have the effect of mediation and regulation and control, wound healing.Wherein, bFGF has to deriving from mesoderm and neuroectodermal cell (as epithelial cell, hypodermal cell, fibroblast, vascular endothelial cell etc.) effect that promotes reparation and regeneration, maintains and damage the aspects such as injury repairing to have significant curative effect in histo-differentiation, normal structure structure and physiological function.Research shows: it directly stimulates the albumen of fibroblast and extracellular matrix synthetic on the one hand, form collagen, on the other hand, can improve local microcirculation and histotrophic nutrition situation, promote into gratified cell, sarcoplast propagation, make granulation tissue ramp, edge of wound epithelium is creeped and wound surface is dwindled and heal to center rapidly.But because fibroblast growth factor, epidermal growth factor etc. is fusion rotein, easily be subject to the impact of various extraneous factors and reduce activity, the stability in preparation is very poor, affects therapeutic effect, high temperature or excessively cold environment also affect its biological activity, and albuminous degeneration easily occurs.
In current prior art; in order to solve the stability of protein formulation and the shortcoming of lyophilized formulations; Chinese patent application 200510036641.3 discloses a kind of externally used solution of recombination human basic fibroblast growth factor; wherein adopting heparin sodium is protective agent, realizes the cryopreservation of active polypeptide solution state.But, the main effectively activity index of ingredient r b-bFGF in externally used solution is not disclosed in this scheme, simultaneously, there is bibliographical information, heparin sodium can produce rare skin irritation as burn feeling, or anaphylaxis is as side effect such as erythra, prurituss, in the points for attention of the description of Hepudiod Cream, clearly write and understand that heparin sodium is not directly applied to and fester on wound and mucosal tissue, the healing of the wound surface of the skin ulcer that inapplicable and severe trauma, burn and scald and for example diabetes of some disease cause.From year January in mid-December, 2007 to 2008, special company of American Manufacturing Company hundred (Baxter International Inc.) receives continuously report and claims, nearly 350 people are after the heparin sodium (heparin) that has used the said firm to produce, produce serious anaphylaxis, wherein four people are dead.This is an in recent years larger heparin sodium Adverse Event.
In addition; Chinese patent application 201110342458.1 discloses bFGF bovine basic fibroblast growth factor externally used solution; wherein adopting human albumin is protein protective agent; in order to keep the stability of solution, a kind of in solution protective agent mannitol and Polyethylene Glycol or the combination of the two have also been added.Although this invention case has avoided employing heparin class material if heparin sodium is as protective agent; but the protective agent human albumin who adopts is blood products, although through processing, some materials wherein may make receiver produce anaphylaxis; or even infection virus, for example HIV (human immunodeficiency virus).For burn wound, particularly contain the wound surface festering and easily produce infection, also have human albumin to produce to shiver, the report of the symptom side effect such as heating, Blushing, erythra, nausea and vomiting.And mannitol is relevant to the protective effect of protein and its concentration, morphosis, the mannitol of crystalline state loses defencive function; 1% or the mannitol of lower concentration by the formation of undefined structure, stop the gathering of protein molecule, but the mannitol of high concentration is easy to form crystallization, can promote the gathering of protein, therefore, it adds can produce very large risk to protein denaturation.Studies show that, adding of human albumin and mannitol or Polyethylene Glycol, can reduce activity and the purity of activated protein after placement.
In view of current protein solution type preparation, especially for thering is burn, wound healing, the difficulty that the externally used solution agent at position such as fester exists and and not enough, the inventor has carried out research with great concentration, be surprised to find that with the form of calcium magnesium potassium salt and be extensively present in the acid of planting in plant seed, to maintaining the stable environment of protein solution agent, the cryopreservation of realizing protein solution agent has important function, planting acid is also present in animal erythroblast, can promote the release of oxygen in HbO2 Oxyhemoglobin, improve erythrocyte function, extend the existence of erythrocyte, the pharmacologically active itself having can play synergism with activated protein, particularly to relating to activated protein for wound healings such as burning as fibroblast growth factor, epidermal growth factor has synergism.Natural phytic acid is fabulous antioxygenic property also, has avoided as maintaining the stable antioxidant that adds other of protein active etc. in protein solution agent.
Summary of the invention
The object of this invention is to provide a kind of externally used solution that contains bioprotein and preparation method thereof; in this externally used solution, do not contain antiseptic and antibacterial; guaranteed with the wound surface of wound and the safety of the wound surface use of festering; avoid side effect and the defect of in prior art, using heparin class material and human albumin to produce as protective agent simultaneously, realized the cryopreservation of protein solution without lyophilizing.The preparation method such as aseptic thin-film filtering, best pH regulator step is simple, favorable reproducibility, is suitable for industrialized great production.Externally used solution agent sterile filling, in spray bottle, facilitates patient to use, and prevents from producing pollution to remaining medicinal liquid in bottle.
In conjunction with existing achievement in research, the present invention seeks to be achieved through the following technical solutions:
An externally used solution that contains bioprotein, volume ratio (g/100ml) is calculated by weight, and the amount that contains bioprotein is that 5-50mg/100mL, buffer system are 0.5-2g/100mL, stabilizing agent, protective agent, and surplus is water for injection.
Wherein, contained bioprotein is any one in fibroblast growth factor, epidermal growth factor, superoxide dismutase, Lecithinized superoxide dismutase, laminin，LN, human complement factor H, lipoprotein lipase, amphiregulin, is wherein preferably fibroblast growth factor, epidermal growth factor, Lecithinized superoxide dismutase.
Wherein, described fibroblast growth factor is acid fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF).
Wherein, contained buffer system is selected from citric acid/sodium citrate system or sodium dihydrogen phosphate/sodium hydrogen phosphate system or phosphate buffer or citric acid/sodium dihydrogen phosphate system or citric acid/sodium hydrogen phosphate system.
Wherein, contained stabilizing agent is the compositions that 5-30mg/100mL surfactant and 10-50mg/100mL are selected from low molecular dextran or ethylenediaminetetraacetic acid or disodiumedetate or sodium ethylene diamine tetracetate calcium any one.
Wherein, surfactant be selected from that Tween 80, amino acid pattern, fatty acid Pyrusussuriensis are smooth, any one in dodecyl sodium sulfate.
Wherein, contained protective agent is that 1-10g/100mL glycerol and 5-50mg/100mL are selected from any one the compositions in sucrose octasulfate sodium, dextran sulfate, phytic acid, matter acid sodium, phytic acid calcium, phytic acid magnesium, Monopotassium phytate, phosvitin, ammonium polyphosphate, inositol six sulfuric esters.
Wherein, prepare the method for bioprotein externally used solution, step is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
The beneficial effect that technical scheme of the present invention produces comprises:
(1) having overcome activated protein preparation needs freeze drying process could stablize the defect of preserving, and has realized activated protein with solution state cryopreservation;
(2) overcome and in prior art, reassembled into the side effect that fibroblast growth factor externally used solution adopts heparin sodium to produce for protective agent;
(3) human albumin who has overcome available technology adopting blood source for activated protein solution protective agent produce batch between poor repeatability, have the defect of irritated risk;
(4), not containing any antiseptic, antibacterial, burn wound, the wound surface that festers are used to safety;
(5) preparation method is simple, has guaranteed the technique of sterile working, and fill is in spray bottle, easy to use, with after do not pollute residue medicinal liquid;
(6) take natural origin, plant acid as protein protective agent, be conducive to maintaining of activated protein stable environment, and phytic acid itself is exactly the nutriment useful to human body, phytic acid hydrolyzate in human body is inositol and phospholipid, the former has anti-aging effects, and the latter is human body cell important component part;
(7) plant acid most metal ions are had to extremely strong complexing power, complexation power is similar to EDTA, but the range of application than EDTA is wider, the ion that adsorbable various containers or filling bottle are separated out due to long-term placement etc., the stability of assurance solution environmental
(8) each phytic acid molecule can provide the electronics that six pairs of hydrogen atoms make free radical to form rock-steady structure, thereby replaces by fresh-keeping thing molecule as for oxygen molecule, can be used as natural antioxidant and avoids activated protein oxidation inactivation, avoids adding more adjuvant.
Accompanying drawing explanation
Fig. 1 is the purity liquid chromatogram that the embodiment of the present invention 1 makes externally used solution bFGF.
Fig. 2 is the purity liquid chromatogram take human albumin as protectant externally used solution bFGF in comparative example 1.
The specific embodiment
The specific embodiment of form by the following examples, is described in further detail content of the present invention.But should not be understood as scope of the present invention and only limit to following examples.All technical schemes realizing based on content of the present invention all belong to scope of the present invention.Obviously, according to content of the present invention, according to ordinary skill knowledge and the customary means of this area, do not departing under the prerequisite of basic fundamental thought of the present invention, can also make modification, replacement and the change of other various ways.
The preparation prescription of embodiment 1:rh-bFGF externally used solution:
Citric acid 2.9g
Sodium citrate 10.7g
Tween80 0.1g
Dextran 0.185g
Glycerol 10g
Phytic acid 0.05g
Water for injection adds to 1L recombination human basic fibroblast growth factor 100mg and makes altogether 400.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test: adopt liquid chromatography to carry out the detection of activated protein purity.
Detection method:
Detect wavelength: 215nm flow velocity 1.0 column temperatures: 40 ℃ of chromatographic columns: C-18 sampling volume: 200 μ l
Mobile phase A: water-0.1% trifluoroacetic acid Mobile phase B: acetonitrile-0.08% trifluoroacetic acid
Gradient:
Time B%
0.1 10
5 20
30 50
40 70
45 10
60 10
Active inspection: the method derives from the 3rd appendix of version Chinese Pharmacopoeia in 2010.
Test principle: the bFGF of debita spissitudo has promotion proliferation function to BALB/c 3T3 cell, MTT can be reduced to quantitatively MTT Formazan(first a ceremonial jade-ladle, used in libation in the mitochondrion of living cells), amount by colorimetric method for determining MTT Formazan can direct representation BALB/c 3T3 cell growth conditions, by the extension rate of 50% ceiling effect point, can convert as tiring in product to be checked, measurement result is proofreaied and correct with determination of activity standard substance.
Test method:
First day:
(1) bed board: digestion and collection BALB/c 3T3 cell, by complete medium 1(20%BCS DMEM culture medium) be made into 8.0 × 10
4the cell suspension of/ml, is inoculated in 96 porocyte culture plates, every hole 100 μ l, 37 ℃, 5%CO
2under condition, cultivate 18-24 hour.
Second day:
(2) hunger: get Tissue Culture Plate prepared by previous step, suck each hole supernatant, add complete medium 2(0.4% BCS DMEM culture medium), every hole 100 μ l.37 ℃, 5%CO
2under condition, cultivate 18-24 hour.
The 3rd day:
(3) prepare sample solution: sample thief is according to its protein concentration, and with complete medium 2, diluting in advance certain multiple to protein concentration is 1 μ g/ml left and right, then in 96 orifice plates, by 4 times, carries out gradient dilution, and each dilution factor does 2-3 multiple hole.
(4) application of sample: get Tissue Culture Plate prepared by the 2nd step, suck each hole supernatant, the sample having diluted is joined in each hole, every hole adds 100 μ l.37 ℃, 5%CO
2under condition, cultivate 64-72 hour.
The 6th day:
(5) add MTT solution and cultivate: every hole adds 20 μ l MTT solution (5.0mg/ml), 37 ℃, 5%CO
2under condition, cultivate 4-5 hour.
(6) add lysate measurement result: suck each hole supernatant, add lysate (DMSO) 100 μ l, after dissolving mixes, with microplate reader mensuration 570/630nm absorbance.
Result is calculated: adopt four parametric methods to carry out matching to result, that calculates respectively each sample partly imitates extension rate, then calculation sample IC50 etc. respectively.
Embodiment 2:
The preparation prescription of aFGF externally used solution:
Sodium dihydrogen phosphate 2.5g
Sodium hydrogen phosphate 2.5g
Dodecyl sodium sulfate 0.05g
EDTA 0.1g
Glycerol 20g
Monopotassium phytate 0.1g
Water for injection adds to 1L
AFGF 100mg makes 400 altogether.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test and the active method checking are with embodiment 1.
Embodiment 3:
The preparation prescription of bFGF externally used solution:
Citric acid 3g
Sodium citrate 7g
Tween80 0.1g
EDTA-2Na 0.215g
Glycerol 50g
Phytic acid 0.15g
Water for injection adds to 1L
BFGF 50mg makes 400 altogether.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test and the active method checking are with embodiment 1.
Embodiment 4:
The preparation prescription of epidermal growth factor (EGF) externally used solution:
Citric acid 8g
Sodium dihydrogen phosphate 7g
Cysteine 0.3g
EDTA-2Na 0.3g
Glycerol 100g
Sodium phytate 0.2g
Water for injection adds to 1L
Epidermal growth factor 300mg makes 400 altogether.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test and the active method checking are with embodiment 1.
Embodiment 5:
The preparation prescription of bFGF externally used solution:
Citric acid 4g
Sodium hydrogen phosphate 16g
The smooth 0.2g of fatty acid Pyrusussuriensis
EDTA 0.42g
Glycerol 80g
Phosvitin 0.3g
Water for injection adds to 1L bFGF 400mg and makes altogether 400.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test and the active method checking are with embodiment 1.
Embodiment 6:
The preparation prescription of aFGF externally used solution:
Sodium dihydrogen phosphate 8g
Sodium hydrogen phosphate 9g
Arginine 0.3g
EDTANaCa 0.5g
Glycerol 30g
Dextran phosphate 0.5g
Water for injection adds to 1L
AFGF 500mg makes 400 altogether.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test and the active method checking are with embodiment 1.
Comparative example
According to the prescription of Chinese patent application 201110342458.1, the externally used solution of preparing take human albumin as protective agent.Adopt liquid chromatography and active method to detect.
Test example: accelerate the comparison of experiment stability
At 25 ℃, lucifuge is placed 60 days results
Embodiment purity actives
Embodiment 1 96.5 % 9.86 × 105U/mg
Embodiment 2 97.1 % 9.69 × 105U/mg
Embodiment 3 98.4 % 1.09 × 106U/mg
Embodiment 4 96.6 % 9.60 × 105U/mg
Embodiment 5 96.0 % 9.12 × 105U/mg
Embodiment 6 96.2 % 9.03 × 105U/mg
Comparative example 77.2 % 5.06 × 104U/mg
Test example: long-term experiment stability comparison
At 4 ℃, lucifuge is placed 360 days results
Embodiment purity actives
Embodiment 1 97.1 % 1.01 × 106U/mg
Embodiment 2 97.7 % 9.72 × 105U/mg
Embodiment 3 97.8 % 1.04 × 106U/mg
Embodiment 4 96.6 % 9.34 × 105U/mg
Embodiment 5 96.8 % 9.14 × 105U/mg
Embodiment 6 96.3 % 8.89 × 105U/mg
Comparative example 76.4 % 9.02 × 103U/mg
From accelerated test and long term test, comparative example purity and active decline obviously, be not suitable for long-term preservation, and embodiment can keep most purity and activity substantially, illustrate that the prescription formation that this patent relates to can keep the stable of active component effectively.
Trauma model efficiency assay
1, animal model is chosen:
Select the Chinese miniature pig of three 25kg to carry out animal experiment, after anesthesia, at each back, cause 8 of the dark 11 ° of burn woundes of diameter 3cm respectively, with the normal saline that does not contain active component, do negative control, with the positive contrast of comparative example.
2, administering mode:
After debridement, the wound surface of positive controls directly sprays comparative example solution.All the other each group is undertaken by double-blind method, in the mode of three parallel tests, respectively by example 1 to the externally used solution of gained in example 6 from wound surface 5cm part spray 5 times, with clean gauze, cover.With same method spray medicine once, share medicine 13 days every day later.
3, wound healing index:
When changing dressings, measure wound surface diameter, diameter is less shows that promoting healing effect is better at every turn.
4, result:
Each sample average wound surface diameter (cm)
Natural law embodiment comparative example negative control
1 2 3 4 5 6
0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0
2 2.6 2.8 2.7 2.8 2.7 2.7 2.8 3.0
4 2.3 2.5 2.1 2.5 2.4 2.4 2.5 2.9
6 1.7 1.5 1.3 1.6 1.8 1.4 2.4 2.5
9 1.1 1.3 0.9 1.2 1.5 1.1 2.0 2.2
13 0.6 0.4 0.1 0.6 0.8 0.5 1.1 2.1
As can be seen from the results, embodiment is better than comparative example, illustrates that the prescription formation that this patent relates to can not affect drug effect, can promote albumen more to play consistently physiological activity, without any stimulation and sensitization.
Claims (8)
1. contain an externally used solution for bioprotein, it is characterized in that volume ratio (g/100ml) is calculated by weight, the amount that contains bioprotein is that 5-50mg/100mL, buffer system are 0.5-2g/100mL, stabilizing agent, protective agent, and surplus is water for injection.
2. a bioprotein externally used solution according to claim 1, it is characterized in that contained bioprotein is any one in fibroblast growth factor, epidermal growth factor, superoxide dismutase, Lecithinized superoxide dismutase, laminin，LN, human complement factor H, lipoprotein lipase, amphiregulin, is wherein preferably fibroblast growth factor, epidermal growth factor, Lecithinized superoxide dismutase.
3. a bioprotein externally used solution according to claim 2, is characterized in that described fibroblast growth factor is acid fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF).
4. a bioprotein externally used solution according to claim 1, is characterized in that contained buffer system is selected from citric acid/sodium citrate system or sodium dihydrogen phosphate/sodium hydrogen phosphate system or citric acid/sodium dihydrogen phosphate system or citric acid/sodium hydrogen phosphate system.
5. a bioprotein externally used solution according to claim 1, is characterized in that contained stabilizing agent is the compositions that 5-30mg/100mL surfactant and 10-50mg/100mL are selected from low molecular dextran or ethylenediaminetetraacetic acid or disodiumedetate or sodium ethylene diamine tetracetate calcium any one.
6. a bioprotein externally used solution according to claim 4, is characterized in that, wherein surfactant be selected from that Tween 80, amino acid pattern, fatty acid Pyrusussuriensis are smooth, any one in dodecyl sodium sulfate.
7. a bioprotein externally used solution according to claim 1, is characterized in that contained protective agent is that 1-10g/100mL glycerol and 5-50mg/100mL are selected from any one the compositions in sucrose octasulfate sodium, dextran sulfate, phytic acid, matter acid sodium, phytic acid calcium, phytic acid magnesium, Monopotassium phytate, phosvitin, ammonium polyphosphate, inositol six sulfuric esters.
8. a method of preparing bioprotein externally used solution according to claim 1, step is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210391695.1A CN103720642B (en) | 2012-10-16 | 2012-10-16 | A kind of externally used solution containing bioprotein and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210391695.1A CN103720642B (en) | 2012-10-16 | 2012-10-16 | A kind of externally used solution containing bioprotein and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103720642A true CN103720642A (en) | 2014-04-16 |
| CN103720642B CN103720642B (en) | 2015-09-16 |
Family
ID=50445109
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210391695.1A Active CN103720642B (en) | 2012-10-16 | 2012-10-16 | A kind of externally used solution containing bioprotein and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103720642B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104645313A (en) * | 2015-01-28 | 2015-05-27 | 南京航空航天大学 | Mussel mucoprotein gel for repairing and reliving itching and preparation method of mussel mucoprotein gel |
| CN112569346A (en) * | 2019-09-29 | 2021-03-30 | 国家卫生健康委科学技术研究所 | Hydrogel for promoting wound healing and preparation method thereof |
| WO2024217067A1 (en) * | 2023-04-21 | 2024-10-24 | 上海腾瑞制药股份有限公司 | Acidic fibroblast growth factor eye drop preparation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5348941A (en) * | 1992-04-01 | 1994-09-20 | Merck & Co., Inc. | Stabilizers for fibroblast growth factors |
| CN1160582A (en) * | 1996-12-27 | 1997-10-01 | 暨南大学生物工程研究所 | External-use composite containing cell growth factor |
-
2012
- 2012-10-16 CN CN201210391695.1A patent/CN103720642B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5348941A (en) * | 1992-04-01 | 1994-09-20 | Merck & Co., Inc. | Stabilizers for fibroblast growth factors |
| CN1160582A (en) * | 1996-12-27 | 1997-10-01 | 暨南大学生物工程研究所 | External-use composite containing cell growth factor |
Non-Patent Citations (1)
| Title |
|---|
| 李国辉: "重组人碱性成纤维细胞生长因子体外稳定性的研究", 《中国优秀博硕士学位论文全文数据库（硕士） 医药卫生科技辑》, 15 January 2005 (2005-01-15), pages 059 - 25 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104645313A (en) * | 2015-01-28 | 2015-05-27 | 南京航空航天大学 | Mussel mucoprotein gel for repairing and reliving itching and preparation method of mussel mucoprotein gel |
| CN112569346A (en) * | 2019-09-29 | 2021-03-30 | 国家卫生健康委科学技术研究所 | Hydrogel for promoting wound healing and preparation method thereof |
| WO2024217067A1 (en) * | 2023-04-21 | 2024-10-24 | 上海腾瑞制药股份有限公司 | Acidic fibroblast growth factor eye drop preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103720642B (en) | 2015-09-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP1180368B1 (en) | Freeze dried hgf preparations | |
| ES2397234T3 (en) | Stabilized thrombin compositions | |
| CN104323994A (en) | Sodium nitroprusside liposome combined medicine and large-scale production process and application thereof | |
| KR19990028819A (en) | HGF lyophilized formulation | |
| CN101249043B (en) | Applications of human growth hormone and human epidermal growth factor combination in beauty treatment | |
| CN108619086A (en) | A kind of Cellular gels preparation for treating tissue damage and application thereof and the active gel solution of holding freeze-stored cell used | |
| CN100503634C (en) | A kind of T4 synthesis product and application thereof | |
| CN103720642B (en) | A kind of externally used solution containing bioprotein and preparation method thereof | |
| CN101020715B (en) | Process of extracting and preparing deer nerve growth factor (DEER NGF) | |
| US10493133B2 (en) | Method for preparing highly concentrated fibrinogen solution and method for preparing fibrin sealant by using thereof | |
| ES2604707T3 (en) | Pharmaceutical composition for use in the treatment of skin scars | |
| CN104586778A (en) | External recombinant bovine basic fibroblast growth factor lyophilized preparation | |
| CN112237571B (en) | A kind of freeze-dried powder for improving the stability of exenatide human serum albumin fusion protein | |
| CN101327172A (en) | Alkaline fibroblast growth factor and use of its composition in preparing beauty treatment cosmetics | |
| CN109836487B (en) | Human fibroblast growth factor 18, and soluble recombinant expression method, preparation and application thereof | |
| US20240189372A1 (en) | Arthrospira for use in the treatment of diseases | |
| CN114469877A (en) | Semetreuptase implant and preparation method thereof | |
| CN102327239A (en) | Salmon calcitonin nano liposome injection and preparation method thereof | |
| US20240139105A1 (en) | Pharmaceutical composition containing ghrp-6 | |
| CN102552890A (en) | Medicinal composition of recombinant carboxypeptidase G2 | |
| WO1992011022A1 (en) | Pharmaceutical preparation for pernasal administration | |
| AU2023259072A1 (en) | Compositions and methods relating to pooled fetal support tissue | |
| KR101322937B1 (en) | Composition comprising auraptene for tissue regeneration | |
| CN116421566A (en) | Preparation containing polyethylene glycol recombinant human growth hormone | |
| CN101502640A (en) | Compound dipeptide pharmaceutical formulations and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |