JP5444544B2 - Angiogenesis inducer and polypeptide used therefor - Google Patents
Angiogenesis inducer and polypeptide used therefor Download PDFInfo
- Publication number
- JP5444544B2 JP5444544B2 JP2008557153A JP2008557153A JP5444544B2 JP 5444544 B2 JP5444544 B2 JP 5444544B2 JP 2008557153 A JP2008557153 A JP 2008557153A JP 2008557153 A JP2008557153 A JP 2008557153A JP 5444544 B2 JP5444544 B2 JP 5444544B2
- Authority
- JP
- Japan
- Prior art keywords
- polypeptide
- angiogenesis
- activity
- seq
- inducing
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 65
- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 55
- 229920001184 polypeptide Polymers 0.000 title claims description 52
- 230000033115 angiogenesis Effects 0.000 title claims description 36
- 239000000411 inducer Substances 0.000 title claims description 8
- 230000000844 anti-bacterial effect Effects 0.000 claims description 18
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 11
- 239000002870 angiogenesis inducing agent Substances 0.000 claims description 10
- 208000027418 Wounds and injury Diseases 0.000 claims description 9
- 206010052428 Wound Diseases 0.000 claims description 8
- 239000004480 active ingredient Substances 0.000 claims description 7
- 201000010099 disease Diseases 0.000 claims description 7
- 208000004210 Pressure Ulcer Diseases 0.000 claims description 5
- 206010040943 Skin Ulcer Diseases 0.000 claims description 5
- 231100000019 skin ulcer Toxicity 0.000 claims description 5
- 200000000007 Arterial disease Diseases 0.000 claims description 4
- 206010003210 Arteriosclerosis Diseases 0.000 claims description 4
- 208000011775 arteriosclerosis disease Diseases 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 229940124597 therapeutic agent Drugs 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims 1
- 241000191967 Staphylococcus aureus Species 0.000 claims 1
- 230000000069 prophylactic effect Effects 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 description 21
- 239000000243 solution Substances 0.000 description 15
- 150000001413 amino acids Chemical group 0.000 description 12
- 238000000034 method Methods 0.000 description 10
- 239000002202 Polyethylene glycol Substances 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- -1 adipic acid ester Chemical class 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 230000017531 blood circulation Effects 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- GLDOVTGHNKAZLK-UHFFFAOYSA-N octadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCCCO GLDOVTGHNKAZLK-UHFFFAOYSA-N 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- POIUWJQBRNEFGX-XAMSXPGMSA-N cathelicidin Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C)C1=CC=CC=C1 POIUWJQBRNEFGX-XAMSXPGMSA-N 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000006641 stabilisation Effects 0.000 description 3
- 238000011105 stabilization Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 2
- 206010053567 Coagulopathies Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 235000021314 Palmitic acid Nutrition 0.000 description 2
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 2
- 235000011037 adipic acid Nutrition 0.000 description 2
- 239000001361 adipic acid Substances 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 2
- 230000035602 clotting Effects 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 2
- GOQYKNQRPGWPLP-UHFFFAOYSA-N n-heptadecyl alcohol Natural products CCCCCCCCCCCCCCCCCO GOQYKNQRPGWPLP-UHFFFAOYSA-N 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 235000019271 petrolatum Nutrition 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- DSEKYWAQQVUQTP-XEWMWGOFSA-N (2r,4r,4as,6as,6as,6br,8ar,12ar,14as,14bs)-2-hydroxy-4,4a,6a,6b,8a,11,11,14a-octamethyl-2,4,5,6,6a,7,8,9,10,12,12a,13,14,14b-tetradecahydro-1h-picen-3-one Chemical compound C([C@H]1[C@]2(C)CC[C@@]34C)C(C)(C)CC[C@]1(C)CC[C@]2(C)[C@H]4CC[C@@]1(C)[C@H]3C[C@@H](O)C(=O)[C@@H]1C DSEKYWAQQVUQTP-XEWMWGOFSA-N 0.000 description 1
- ORFOPKXBNMVMKC-CEZXYXJGSA-N (6S,7S)-7-[[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(2-carboxypropan-2-yloxyimino)acetyl]amino]-8-oxo-3-(pyridin-1-ium-1-ylmethyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate Chemical compound CC(C)(O\N=C(/C(=O)N[C@@H]1[C@@H]2SCC(C[n+]3ccccc3)=C(N2C1=O)C([O-])=O)c1csc(N)n1)C(O)=O ORFOPKXBNMVMKC-CEZXYXJGSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- CMBYOWLFQAFZCP-UHFFFAOYSA-N Hexyl dodecanoate Chemical compound CCCCCCCCCCCC(=O)OCCCCCC CMBYOWLFQAFZCP-UHFFFAOYSA-N 0.000 description 1
- 101000904173 Homo sapiens Progonadoliberin-1 Proteins 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 239000004166 Lanolin Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 1
- 235000021360 Myristic acid Nutrition 0.000 description 1
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000004264 Petrolatum Substances 0.000 description 1
- 108010069381 Platelet Endothelial Cell Adhesion Molecule-1 Proteins 0.000 description 1
- 102100024028 Progonadoliberin-1 Human genes 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 101000996723 Sus scrofa Gonadotropin-releasing hormone receptor Proteins 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000001623 arteriogenic effect Effects 0.000 description 1
- 235000013871 bee wax Nutrition 0.000 description 1
- 239000012166 beeswax Substances 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 102000014509 cathelicidin Human genes 0.000 description 1
- 108060001132 cathelicidin Proteins 0.000 description 1
- 229960000484 ceftazidime Drugs 0.000 description 1
- 150000001782 cephems Chemical class 0.000 description 1
- 229960000541 cetyl alcohol Drugs 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 229960003405 ciprofloxacin Drugs 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 208000037765 diseases and disorders Diseases 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 108010085662 ecarin Proteins 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- XLXSAKCOAKORKW-UHFFFAOYSA-N gonadorelin Chemical compound C1CCC(C(=O)NCC(N)=O)N1C(=O)C(CCCN=C(N)N)NC(=O)C(CC(C)C)NC(=O)CNC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 XLXSAKCOAKORKW-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000001631 haemodialysis Methods 0.000 description 1
- 230000000322 hemodialysis Effects 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- BXWNKGSJHAJOGX-UHFFFAOYSA-N hexadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCO BXWNKGSJHAJOGX-UHFFFAOYSA-N 0.000 description 1
- OIKBVOIOVNEVJR-UHFFFAOYSA-N hexadecyl 6-methylheptanoate Chemical compound CCCCCCCCCCCCCCCCOC(=O)CCCCC(C)C OIKBVOIOVNEVJR-UHFFFAOYSA-N 0.000 description 1
- 229940100463 hexyl laurate Drugs 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 239000004026 insulin derivative Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 235000019388 lanolin Nutrition 0.000 description 1
- 229940039717 lanolin Drugs 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 229960002260 meropenem Drugs 0.000 description 1
- DMJNNHOOLUXYBV-PQTSNVLCSA-N meropenem Chemical compound C=1([C@H](C)[C@@H]2[C@H](C(N2C=1C(O)=O)=O)[C@H](O)C)S[C@@H]1CN[C@H](C(=O)N(C)C)C1 DMJNNHOOLUXYBV-PQTSNVLCSA-N 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 229960003085 meticillin Drugs 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- KSCKTBJJRVPGKM-UHFFFAOYSA-N octan-1-olate;titanium(4+) Chemical compound [Ti+4].CCCCCCCC[O-].CCCCCCCC[O-].CCCCCCCC[O-].CCCCCCCC[O-] KSCKTBJJRVPGKM-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229940066842 petrolatum Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 108010039231 polyethyleneglycol-hirudin Proteins 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- LISFMEBWQUVKPJ-UHFFFAOYSA-N quinolin-2-ol Chemical compound C1=CC=C2NC(=O)C=CC2=C1 LISFMEBWQUVKPJ-UHFFFAOYSA-N 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
- 150000004044 tetrasaccharides Chemical class 0.000 description 1
- NLVFBUXFDBBNBW-PBSUHMDJSA-N tobramycin Chemical compound N[C@@H]1C[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N NLVFBUXFDBBNBW-PBSUHMDJSA-N 0.000 description 1
- 229960000707 tobramycin Drugs 0.000 description 1
- 239000012929 tonicity agent Substances 0.000 description 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003871 white petrolatum Substances 0.000 description 1
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 1
- 229960001600 xylazine Drugs 0.000 description 1
- 239000011787 zinc oxide Substances 0.000 description 1
- 150000003952 β-lactams Chemical class 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Description
本発明は、血管新生誘導剤及びそれに用いられるポリペプチドに関する。 The present invention relates to an angiogenesis inducer and a polypeptide used therefor.
褥瘡、創傷、皮膚潰瘍、閉塞性動脈疾患及び閉塞性動脈硬化症等の種々の疾患又は傷害の治療においては、血管の新生が有効である。幾つかの血管新生誘導活性を有する血管新生誘導剤が知られている。
血管新生誘導活性を有するポリペプチドとしては、LL−37が知られている。LL−37は、血管新生活性のみならず、抗菌活性および動脈形成活性を有するペプチドである(非特許文献1および2)。Angiogenesis is effective in the treatment of various diseases or injuries such as pressure ulcers, wounds, skin ulcers, occlusive arterial disease and occlusive arteriosclerosis. Angiogenesis-inducing agents having several angiogenesis-inducing activities are known.
LL-37 is known as a polypeptide having angiogenesis-inducing activity. LL-37 is a peptide having not only angiogenic activity but also antibacterial activity and arteriogenic activity (Non-patent Documents 1 and 2).
本願発明者らは、先に血管内皮増殖活性、ひいては血管新生誘導活性を有するポリペプチドを発明し、さらに該ペプチドがLL−37より高い血管新生誘導活性を有していることを見いだし、これを特許出願した(特許文献1)。特許文献1には、該ポリペプチドが抗菌活性を有することも示唆されている。 The inventors of the present application previously invented a polypeptide having vascular endothelial growth activity, and thus angiogenesis-inducing activity, and further found that the peptide has a higher angiogenesis-inducing activity than LL-37. A patent application was filed (Patent Document 1). Patent Document 1 also suggests that the polypeptide has antibacterial activity.
本発明の目的は、優れた血管新生誘導活性を有する新規な血管新生誘導剤及びその有効成分として用いられる新規なポリペプチドを提供することである。 An object of the present invention is to provide a novel angiogenesis-inducing agent having excellent angiogenesis-inducing activity and a novel polypeptide used as an active ingredient thereof.
本願発明者らは、鋭意研究の結果、特許文献1に記載されたポリペプチドを修飾することにより、該ポリペプチドよりも高い血管新生誘導活性を有するか又は該ポリペプチドと血管新生誘導活性は同等であるがより高い抗菌活性を有するポリペプチドが得られることを見出し本発明を完成した。 As a result of intensive studies, the inventors of the present application have a higher angiogenesis-inducing activity than the polypeptide by modifying the polypeptide described in Patent Document 1, or the polypeptide and angiogenesis-inducing activity are equivalent. However, the present invention was completed by finding that a polypeptide having higher antibacterial activity can be obtained.
すなわち、本発明は、配列番号1ないし4のいずれかで示されるアミノ酸配列から成るポリペプチドを有効成分として含有する血管新生誘導剤を提供する。 That is, the present invention provides an angiogenesis-inducing agent containing, as an active ingredient, a polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 4 .
また、本発明は、上記ポリペプチドを有効成分として含有する、褥瘡、創傷、皮膚潰瘍、閉塞性動脈疾患及び閉塞性動脈硬化症から成る群から選択される疾患の処置のための予防、改善又は治療剤を提供する。 The present invention also provides prevention, amelioration or treatment for treatment of a disease selected from the group consisting of pressure ulcers, wounds, skin ulcers, occlusive arterial disease and occlusive arteriosclerosis, comprising the polypeptide as an active ingredient. Provide a therapeutic agent.
本発明により、優れた血管新生誘導活性を有するポリペプチド系の新規な血管新生誘導剤が提供された。また、この血管新生誘導剤の有効成分として用いられる新規なポリペプチドが提供された。 According to the present invention, a novel angiogenesis-inducing agent based on a polypeptide having excellent angiogenesis-inducing activity has been provided. Moreover, the novel polypeptide used as an active ingredient of this angiogenesis inducer was provided.
特許文献1に記載されているF2ペプチド(本願明細書において「AG-30」と呼ぶ)のアミノ酸配列を配列番号5に示す。AG-30は、特許文献1に記載され、また、下記実施例において具体的に記載するように抗菌活性及び血管新生誘導活性を有する。AG-30の種々の修飾体を作製し、その血管新生誘導活性及び抗菌活性を調べた。その結果、上記した本発明のポリペプチドが、血管新生誘導活性を有することが明らかになった。下記実施例に具体的に記載するように、本発明の実施例になるポリペプチドは、AG-30よりも血管新生誘導活性が高いか、又はAG-30と血管新生誘導活性が同等の場合には、AG-30よりも抗菌活性が高かった。 The amino acid sequence of the F2 peptide (referred to herein as “AG-30”) described in Patent Document 1 is shown in SEQ ID NO: 5. AG-30 is described in Patent Document 1 and has antibacterial activity and angiogenesis-inducing activity as specifically described in the following examples. Various modified forms of AG-30 were prepared and their angiogenesis-inducing activity and antibacterial activity were examined. As a result, it was revealed that the above-described polypeptide of the present invention has angiogenesis-inducing activity. As specifically described in the following examples, the polypeptide according to the examples of the present invention has an angiogenesis-inducing activity higher than that of AG-30, or has a similar angiogenesis-inducing activity to AG-30. Had higher antibacterial activity than AG-30.
一般に、ポリペプチドから成る医薬において、生体内でのポリペプチドの安定性を高めるために、ポリペプチドに糖鎖やポリエチレングリコール(PEG)鎖を付加したり、ポリペプチドを構成するアミノ酸の少なくとも一部としてD体アミノ酸を用いたりする技術が広く知られており、用いられている。糖鎖やPEG鎖を付加したり、ポリペプチドを構成するアミノ酸の少なくとも一部としてD体アミノ酸を用いたりすることにより、生体内でのペプチダーゼによる分解を受けにくくなり、生体内におけるポリペプチドの半減期が長くなる。本発明のポリペプチドは、抗菌活性を有する限り、生体内安定化のためのこれらの公知の修飾を施したものであってもよく、本明細書及び特許請求の範囲における「ポリペプチド」という語は、文脈上そうでないことが明らかな場合を除き、生体内安定化のための修飾を施したものも包含する意味で用いている。 In general, in a pharmaceutical comprising a polypeptide, a sugar chain or a polyethylene glycol (PEG) chain is added to the polypeptide or at least a part of the amino acids constituting the polypeptide in order to increase the stability of the polypeptide in vivo. A technique using a D-form amino acid is widely known and used. By adding sugar chains or PEG chains, or by using D-form amino acids as at least part of the amino acids that make up the polypeptide, it is less susceptible to degradation by peptidases in vivo, reducing the polypeptide in vivo by half. The period becomes longer. As long as the polypeptide of the present invention has antibacterial activity, it may be those subjected to these known modifications for in vivo stabilization, and the term “polypeptide” in the present specification and claims. Is used in the sense of including those that have been modified for in vivo stabilization, unless otherwise apparent from the context.
ポリペプチドに対する糖鎖付加は周知であり、例えば、Sato M, Furuike T, Sadamoto R, Fujitani N, Nakahara T, Niikura K, Monde K, Kondo H, Nishimura S., "Glycoinsulins: dendritic sialyloligosaccharide-displaying insulins showing a prolonged blood-sugar-lowering activity.",J Am Chem Soc. 2004 Nov 3;126(43):14013-22やSato M, Sadamoto R, Niikura K, Monde K, Kondo H, Nishimura S,"Site-specific introduction of sialic acid into insulin.", Angew Chem Int Ed Engl. 2004 Mar 12;43(12):1516-20等に記載されている。糖鎖は、N末端、C末端又はそれらの間のアミノ酸に結合可能であるが、ポリペプチドの活性を阻害しないためにN末端又はC末端に結合することが好ましい。また、付加する糖鎖の個数は、1個又は2個が好ましく、1個が好ましい。糖鎖は、単糖から4糖が好ましく、さらには2糖又は3糖が好ましい。糖鎖は、ポリペプチドの遊離のアミノ基又はカルボキシル基に直接又は例えば炭素数1〜10程度のメチレン鎖等のスペーサー構造を介して結合することができる。 Glycosylation to polypeptides is well known, for example, Sato M, Furuike T, Sadamoto R, Fujitani N, Nakahara T, Niikura K, Monde K, Kondo H, Nishimura S., "Glycoinsulins: dendritic sialyloligosaccharide-displaying insulins showing a prolonged blood-sugar-lowering activity. ", J Am Chem Soc. 2004 Nov 3; 126 (43): 14013-22 and Sato M, Sadamoto R, Niikura K, Monde K, Kondo H, Nishimura S," Site- specific introduction of sialic acid into insulin. ", Angew Chem Int Ed Engl. 2004 Mar 12; 43 (12): 1516-20. The sugar chain can be bound to the N-terminus, C-terminus, or an amino acid therebetween, but is preferably bound to the N-terminus or C-terminus so as not to inhibit the activity of the polypeptide. The number of sugar chains to be added is preferably 1 or 2, and preferably 1. The sugar chain is preferably monosaccharide to tetrasaccharide, more preferably disaccharide or trisaccharide. The sugar chain can be bound to a free amino group or carboxyl group of the polypeptide directly or via a spacer structure such as a methylene chain having about 1 to 10 carbon atoms.
ポリペプチドに対するPEG鎖の付加も周知であり、例えば、Ulbricht K, Bucha E, Poschel KA, Stein G, Wolf G, Nowak G., "The use of PEG-Hirudin in chronic hemodialysis monitored by the Ecarin Clotting Time: influence on clotting of the extracorporeal system and hemostatic parameters.", Clin Nephrol. 2006 Mar;65(3):180-90.やDharap SS, Wang Y, Chandna P, Khandare JJ, Qiu B, Gunaseelan S, Sinko PJ, Stein S, Farmanfarmaian A, Minko T., "Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide.", Proc Natl Acad Sci USA. 2005 Sep 6;102(36):12962-7."等に記載されている。PEG鎖は、N末端、C末端又はそれらの間のアミノ酸に結合可能であり、通常、1個又は2個のPEG鎖が、ポリペプチド上の遊離のアミノ基やカルボキシル基に結合される。PEG鎖の分子量は、特に限定されないが、通常3000〜7000程度、好ましくは5000程度のものが用いられる。 Addition of PEG chains to polypeptides is also well known, e.g. Ulbricht K, Bucha E, Poschel KA, Stein G, Wolf G, Nowak G., "The use of PEG-Hirudin in chronic hemodialysis monitored by the Ecarin Clotting Time: influence on clotting of the extracorporeal system and hemostatic parameters. ", Clin Nephrol. 2006 Mar; 65 (3): 180-90. and Dharap SS, Wang Y, Chandna P, Khandare JJ, Qiu B, Gunaseelan S, Sinko PJ, Stein S, Farmanfarmaian A, Minko T., "Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide.", Proc Natl Acad Sci USA. 2005 Sep 6; 102 (36): 12962-7. The PEG chain can be attached to the N-terminus, C-terminus, or the amino acid between them, and usually one or two PEG chains are attached to a free amino group or carboxyl group on the polypeptide. The molecular weight of the PEG chain is not particularly limited, but is usually about 3000 to 7000, preferably about 5000.
ポリペプチドを構成するアミノ酸の少なくとも一部をD体とする方法も周知であり、例えば、Brenneman DE, Spong CY, Hauser JM, Abebe D, Pinhasov A, Golian T, Gozes I., "Protective peptides that are orally active and mechanistically nonchiral.", J Pharmacol Exp Ther. 2004 Jun;309(3):1190-7やWilkemeyer MF, Chen SY, Menkari CE, Sulik KK, Charness ME., "Ethanol antagonist peptides: structural specificity without stereospecificity.", J Pharmacol Exp Ther. 2004 Jun;309(3):1183-9.等に記載されている。ポリペプチドを構成するアミノ酸の一部をD体としてもよいが、ポリペプチドの活性をできるだけ阻害しないため、ポリペプチドを構成するアミノ酸の全てをD体アミノ酸とすることが好ましい。 A method in which at least a part of amino acids constituting a polypeptide is in D form is also well known. For example, Brenneman DE, Spong CY, Hauser JM, Abebe D, Pinhasov A, Golian T, Gozes I., “Protective peptides that are orally active and mechanistically nonchiral. ", J Pharmacol Exp Ther. 2004 Jun; 309 (3): 1190-7, Wilkemeyer MF, Chen SY, Menkari CE, Sulik KK, Charness ME.," Ethanol antagonist peptides: structural specificity without stereospecificity ., J Pharmacol Exp Ther. 2004 Jun; 309 (3): 1183-9. Although some of the amino acids constituting the polypeptide may be D-form, it is preferable that all of the amino acids constituting the polypeptide are D-form amino acids in order not to inhibit the polypeptide activity as much as possible.
本発明の血管新生誘導剤の有効成分であるポリペプチドは、市販のペプチド合成機を用いた化学合成等の常法により容易に製造することができる。また、上記安定化修飾も、上記各文献に記載されているような周知の方法により容易に行なうことができる。 The polypeptide which is an active ingredient of the angiogenesis inducer of the present invention can be easily produced by a conventional method such as chemical synthesis using a commercially available peptide synthesizer. The stabilization modification can also be easily performed by a known method as described in each of the above documents.
本発明のポリペプチドは、高い血管新生誘導活性を有するので、血管新生誘導剤として用いることができる。血管新生誘導剤としての使用方法は公知のポリペプチド系の血管新生誘導剤と同様であり、溶液剤、乳剤、懸濁剤、粉剤、散剤、顆粒剤、ゲル、軟膏、または経皮パッチとして、特に好ましくは溶液剤、粉剤、あるいは経皮パッチとして投与することができる。溶液剤として好ましくは緩衝液、特に好ましくは生理食塩緩衝液等の水系媒体に溶解した溶液を投与することができる。溶液中のポリペプチドの濃度は、特に限定されないが、通常、0.01mg /mL〜100mg/mL程度、好ましくは0.1mg /mL〜100mg/mL程度、特に好ましくは1mg/mL〜10mg/mL程度である。投与経路は、通常、血管新生が必要な部位への塗布や注射等の局所投与である。投与量は、症状や患部の大きさ等に応じて適宜選択されるが、通常、ポリペプチド量として0.01mg〜100mg程度、好ましくは0.1mg〜100mg程度、特に好ましくは0.1mg〜10mg程度であるが、もちろん、これらの範囲に限定されるものではない。なお、本発明の血管新生誘導剤を製剤する際に用いられる薬剤的に許容できる担体としては、上記した水系媒体の他にも、製剤分野で常用されている担体を用いることができ、例えば、軟膏のような外用剤の場合には、炭化水素類(親水ワセリン、白色ワセリン、精製ラノリン、流動パラフィン等)、酸化亜鉛、高級脂肪酸及びそのエステル(アジピン酸、ミリスチン酸、パルミチン酸、ステアリン酸、オレイン酸、アジピン酸エステル、ミリスチン酸エステル、パルミチン酸エステル、セバシン酸ジエチル、ラウリン酸ヘキシル、イソオクタン酸セチル等)、ロウ類(鯨ロウ、ミツロウ、セレシン等)、高級アルコール(セタノール、ステアリルアルコール、セトステアリルアルコール等)などが挙げられる。溶液剤の場合には、例えば、水、生理食塩水、リン酸緩衝生理食塩水等を挙げることができ、経口剤の場合には、乳糖、デンプン等を挙げることができる。その他、必要に応じ、乳化剤、界面活性剤、等張化剤、pH調整剤等の各種医薬添加剤を配合することもできる。なお、これらの薬剤的に許容できる担体及び医薬添加剤は、製剤分野において周知であり、広く用いられているものである。 Since the polypeptide of the present invention has high angiogenesis-inducing activity, it can be used as an angiogenesis-inducing agent. The method of use as an angiogenesis inducer is the same as that of known polypeptide-based angiogenesis inducers, and as a solution, emulsion, suspension, powder, powder, granule, gel, ointment, or transdermal patch, Particularly preferably, it can be administered as a solution, a powder, or a transdermal patch. As the solution, a solution dissolved in an aqueous medium such as a buffer solution, particularly preferably a physiological saline buffer can be preferably administered. The concentration of the polypeptide in the solution is not particularly limited, but is usually about 0.01 mg / mL to 100 mg / mL, preferably about 0.1 mg / mL to 100 mg / mL, particularly preferably about 1 mg / mL to 10 mg / mL. is there. The route of administration is usually local administration such as application or injection to a site requiring angiogenesis. The dose is appropriately selected according to the symptom and the size of the affected area, but is usually about 0.01 mg to 100 mg, preferably about 0.1 mg to 100 mg, particularly preferably about 0.1 mg to 10 mg as the amount of polypeptide. However, of course, it is not limited to these ranges. As the pharmaceutically acceptable carrier used in formulating the angiogenesis-inducing agent of the present invention, in addition to the aqueous medium described above, a carrier commonly used in the pharmaceutical field can be used. In the case of an external preparation such as an ointment, hydrocarbons (hydrophilic petrolatum, white petrolatum, purified lanolin, liquid paraffin, etc.), zinc oxide, higher fatty acids and esters thereof (adipic acid, myristic acid, palmitic acid, stearic acid, Oleic acid, adipic acid ester, myristic acid ester, palmitic acid ester, diethyl sebacate, hexyl laurate, cetyl isooctanoate, etc., waxes (whale wax, beeswax, ceresin, etc.), higher alcohols (cetanol, stearyl alcohol, ceto) Stearyl alcohol, etc.). In the case of a solution, for example, water, physiological saline, phosphate buffered saline and the like can be mentioned, and in the case of an oral preparation, lactose, starch and the like can be mentioned. In addition, various pharmaceutical additives such as an emulsifier, a surfactant, a tonicity agent, and a pH adjuster can be blended as necessary. These pharmaceutically acceptable carriers and pharmaceutical additives are well known and widely used in the pharmaceutical field.
生体に投与する場合の具体的な疾患及び障害の例として、褥瘡、創傷、皮膚潰瘍、閉塞性動脈疾患及び閉塞性動脈硬化症等を挙げることができるがこれらに限定されるものではない。本発明の血管新生誘導剤は、これらの疾患又は障害に対する予防、改善又は治療剤として用いることができる。なお、上記の通り、上記(2)のポリペプチドは、高い血管新生誘導活性を有する一方、抗菌活性を有しているので、抗菌活性を併有することが望まれる疾患又は障害の予防、改善又は治療剤として特に適しており、このような疾患又は障害として、上記の疾患又は障害のうち褥瘡、創傷、皮膚潰瘍等を挙げることができる。 Specific examples of diseases and disorders when administered to a living body include, but are not limited to, pressure ulcers, wounds, skin ulcers, occlusive arterial diseases, and occlusive arteriosclerosis. The angiogenesis-inducing agent of the present invention can be used as a preventive, ameliorating or therapeutic agent for these diseases or disorders. As described above, the polypeptide of the above (2) has a high angiogenesis-inducing activity, while having an antibacterial activity. Therefore, the prevention or improvement of a disease or disorder desired to have an antibacterial activity, or Particularly suitable as a therapeutic agent, examples of such diseases or disorders include pressure ulcers, wounds, skin ulcers and the like among the above diseases or disorders.
本発明の血管新生誘導剤は、単独で使用することもできるし、抗菌性が望まれる場合には他の抗菌剤又は抗生物質と併用することもできる。これらの抗菌剤又は抗生物質の例として、セフェム系、カルバペネム系、アミノグリコシト系、ニューキノロン系、β-ラクタム系、ペニシリン系及びグリコペプチド系抗生物質等を挙げることができ、より具体的には、セフタジジム、メロペネム、トブラマイシン、シプロフロキサシン、メチシリン、アンピシリン及びバンコマイシン等を挙げることができるがこれらに限定されるものではない。 The angiogenesis-inducing agent of the present invention can be used alone, or can be used in combination with other antibacterial agents or antibiotics when antibacterial properties are desired. Examples of these antibacterial agents or antibiotics include cephem, carbapenem, aminoglycosito, new quinolone, β-lactam, penicillin and glycopeptide antibiotics, and more specifically , Ceftazidime, meropenem, tobramycin, ciprofloxacin, methicillin, ampicillin, vancomycin and the like, but are not limited thereto.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
実施例1〜4、比較例1
1. ポリペプチドの作製
市販のペプチド合成機を用いて、tBoc法、Fmoc法のいずれかを用い固相合成法でポリペプチドを化学合成した。Fmoc法には Applied Biosystems社 433A 型全自動固相合成機を、tBoc法には Applied Biosystems社 430A 型全自動固相合成機をそれぞれ使用した。化学合成したポリペプチドは、AG-30(配列番号5、比較例1)、AG30-5C(配列番号1、実施例1)、AG30-5Hb(配列番号2、実施例2)、AG30-2C(配列番号3、実施例3)、AG30-2Hb(配列番号4、実施例4)であった。Examples 1-4, Comparative Example 1
1. Preparation of polypeptide Using a commercially available peptide synthesizer, a polypeptide was chemically synthesized by solid phase synthesis using either tBoc method or Fmoc method. Applied Biosystems 433A fully automatic solid phase synthesizer was used for the Fmoc method, and Applied Biosystems 430A fully automatic solid phase synthesizer was used for the tBoc method. The chemically synthesized polypeptides are AG-30 (SEQ ID NO: 5, Comparative Example 1), AG30-5C (SEQ ID NO: 1, Example 1), AG30-5Hb (SEQ ID NO: 2, Example 2), AG30-2C ( SEQ ID NO: 3, Example 3) and AG30-2Hb (SEQ ID NO: 4, Example 4).
2. ポリペプチドの血管新生誘導活性
AG-30(比較例1)、AG30-5C(実施例1)、AG30-5Hb(実施例2)及びAG30-2C(実施例3)、AG30-2Hb(実施例4)の血管新生誘導活性を調べた。クラボウの血管新生キット(Angiogenesis Kit, KZ-1000, 倉敷紡績株式会社)を用いて管腔形成を調べた。陰性対照として、ブランク(NC、ペプチドなし)を用いた。2. Angiogenesis-inducing activity of polypeptides
The angiogenesis-inducing activity of AG-30 (Comparative Example 1), AG30-5C (Example 1), AG30-5Hb (Example 2), AG30-2C (Example 3), and AG30-2Hb (Example 4) Examined. Luminal formation was examined using an angiogenesis kit from Kurabo Industries (Angiogenesis Kit, KZ-1000, Kurashiki Boseki Co., Ltd.). A blank (NC, no peptide) was used as a negative control.
血管新生専用培地(倉敷紡績(株)KZ-1400)中へ、各ポリペプチド10μg/Lの濃度になるように添加して培養した。培養は37℃、5%CO2インキュベーターにて行った。培地は、培養4日目、7日目および9日目に同じ添加物を含有するものと交換した。培養開始11日目に培地を除き、管腔染色キット(CD31抗体染色用:倉敷紡績(株)KZ-1225)を用いて以下の手順に従い染色した。Each angiogenesis medium (Kurashikibo KZ-1400) was added to each polypeptide to a concentration of 10 μg / L and cultured. The culture was performed in a 37 ° C., 5% CO 2 incubator. The medium was changed to one containing the same additive on days 4, 7 and 9 of culture. On day 11 after the start of culture, the medium was removed, and staining was performed using a luminal staining kit (for CD31 antibody staining: Kurashikibo KZ-1225) according to the following procedure.
CD31(PECAM-1)染色1次抗体(マウス抗ヒトCD31抗体)をブロッキング液(1%-BSAを含むダルベッコリン酸緩衝液(PBS(-))で4,000倍希釈した。各ウェルにこの1次抗体溶液0.5mlを添加し、60分間37℃でインキュベートした。インキュベート終了後、1mlのブロッキング液で各ウエルを計3回洗浄した。 CD31 (PECAM-1) -stained primary antibody (mouse anti-human CD31 antibody) was diluted 4,000 times with a blocking solution (Dulbecco's phosphate buffer (PBS (-)) containing 1% -BSA). 0.5 ml of the antibody solution was added and incubated for 60 minutes at 37 ° C. After the incubation, each well was washed 3 times with 1 ml of blocking solution.
次いでブロッキング液で500倍希釈した2次抗体溶液(ヤギ抗マウスIgGアルカリホスファターゼ複合体)0.5mlを各ウエルに添加し、60分間37℃でインキュベートした後、1mlの蒸留水で3回洗浄した。その間に、BCIP/NBTの錠剤2錠を蒸留水20mlに溶解し、ポアサイズ0.2μmのフィルターで濾過して基質溶液を準備した。調製したBCIP/NBT基質溶液0.5mlを各ウエルに添加し、管腔が深紫色になるまで(通常5〜10分間)37℃でインキュベートした。インキュベート終了後、蒸留水1mlで、各ウエルを3回洗浄した後洗浄液を吸引除去し、自然乾燥するため静置した。乾燥後、顕微鏡下で各ウエルを観察した。 Next, 0.5 ml of a secondary antibody solution (goat anti-mouse IgG alkaline phosphatase complex) diluted 500-fold with a blocking solution was added to each well, incubated at 37 ° C. for 60 minutes, and then washed 3 times with 1 ml of distilled water. Meanwhile, two BCIP / NBT tablets were dissolved in 20 ml of distilled water and filtered through a filter having a pore size of 0.2 μm to prepare a substrate solution. 0.5 ml of the prepared BCIP / NBT substrate solution was added to each well and incubated at 37 ° C. until the lumen became deep purple (usually 5-10 minutes). After completion of the incubation, each well was washed three times with 1 ml of distilled water, and then the washing solution was removed by suction and left to stand for natural drying. After drying, each well was observed under a microscope.
各ウエルを顕微鏡で倍率40倍で観察し、写真を撮影した。得られた各画像を血管新生定量ソフトウェア(KSW-5000U, 倉敷紡績株式会社)を用い定量化した。様々な指標でコンピューター解析をして、このスケールをもとに各視野中に形成された管腔の面積(図左欄)、長さ(図右欄)を測定した。 Each well was observed with a microscope at a magnification of 40 times, and a photograph was taken. Each image obtained was quantified using angiogenesis quantification software (KSW-5000U, Kurashiki Boseki Co., Ltd.). Computer analysis was performed with various indices, and the area (left column in the figure) and length (right column in the figure) of the lumen formed in each visual field were measured based on this scale.
結果を図1に示す。図1に示されるように、本発明のポリペプチドは、いずれも血管新生誘導活性を有しており、特に、AG30-5C(実施例1)及びAG30-5Hb(実施例2)は、AG-30(比較例1)よりも血管新生誘導活性が高かった。 The results are shown in FIG. As shown in FIG. 1, all of the polypeptides of the present invention have angiogenesis-inducing activity. In particular, AG30-5C (Example 1) and AG30-5Hb (Example 2) are AG- The angiogenesis-inducing activity was higher than that of 30 (Comparative Example 1).
3. ポリペプチドの抗菌活性
AG-30(比較例1)、AG30-5C(実施例1)、AG30-5Hb(実施例2)及びAG30-2C(実施例3)、AG30-2Hb(実施例4)の抗菌活性を調べた。抗菌活性は、米国National Committee for Clinical Laboratory Standard(NCCL Documents M7-A3)に従って評価した。すなわち、マイクロタイタープレートあるいは試験管を用いて、菌の増殖を阻害するペプチドの最小濃度を求めた。対象菌株には、黄色ブドウ球菌ATCC29213 (S. aureus ATCC29213)を用いた。細菌を培地で16時間培養した後、A600における吸光度を測定した。予め求めた濁度とcolony forming unit(CFU)との相関から、規定のCFUになるように培地で希釈した。各菌株を1×105CFU/ml(最終濃度)程度になるようにミューラー・ヒントン・ブロス(Mueller-Hinton broth:MHB)に懸濁し、添加した。各ペプチドは任意の濃度に調製し、そこから段階希釈を行った。菌液を分注したマイクロプレートあるいは試験管に各濃度段階のポリペプチドを添加した。ペプチド無添加のものを陰性対照とした。プレートを37℃で16時間培養し、菌の増殖を阻害する最小濃度(最小発育阻止濃度:MIC)を求めた。結果を下記表1に示す。3. Antimicrobial activity of polypeptides
The antibacterial activity of AG-30 (Comparative Example 1), AG30-5C (Example 1), AG30-5Hb (Example 2), AG30-2C (Example 3), and AG30-2Hb (Example 4) was examined. . Antibacterial activity was evaluated according to the US National Committee for Clinical Laboratory Standard (NCCL Documents M7-A3). That is, using a microtiter plate or a test tube, the minimum concentration of the peptide that inhibits bacterial growth was determined. As a target strain, S. aureus ATCC29213 was used. After culturing for 16 hours the bacteria in a medium, the absorbance was measured at A 600. Based on the correlation between the turbidity obtained in advance and the colony forming unit (CFU), it was diluted with a medium so as to become a prescribed CFU. Each strain was suspended in Mueller-Hinton broth (MHB) so as to be about 1 × 10 5 CFU / ml (final concentration) and added. Each peptide was prepared at an arbitrary concentration, and serial dilution was performed therefrom. Polypeptides at each concentration step were added to microplates or test tubes into which the bacterial solution was dispensed. A negative control was added without peptide. The plate was cultured at 37 ° C. for 16 hours, and the minimum concentration (minimum growth inhibitory concentration: MIC) that inhibits bacterial growth was determined. The results are shown in Table 1 below.
表1に示されるように、AG30-5C(実施例1)は、抗菌活性もAG-30(比較例1)よりも優れていた。従って、AG30-5Cは、血管新生誘導活性も抗菌活性もAG-30よりも優れており、抗菌活性も併せて望まれる用途において優れた性能を発揮する。また、AG30-2C(実施例3)及びAG30-2Hb(実施例4)は、血管新生誘導活性はAG-30と同等(図1)であったが、抗菌活性はAG-30よりも優れていた。 As shown in Table 1, AG30-5C (Example 1) was also superior in antibacterial activity to AG-30 (Comparative Example 1). Therefore, AG30-5C is superior to AG-30 in both angiogenesis-inducing activity and antibacterial activity, and exhibits superior performance in applications where antibacterial activity is desired. AG30-2C (Example 3) and AG30-2Hb (Example 4) had angiogenesis-inducing activity equivalent to that of AG-30 (FIG. 1), but antibacterial activity was superior to that of AG-30. It was.
実施例5: ポリペプチドの血流量増加活性
AG30-5C(実施例1)の血流量増加活性を調べた。Example 5: Blood flow increasing activity of polypeptide
The blood flow increasing activity of AG30-5C (Example 1) was examined.
動物:C57Bl/6マウスおよび糖尿病マウス(db/db)はオリエンタルバイオサービスより購入した。8〜10週齢の雄のマウスを用いた。 Animals: C57Bl / 6 mice and diabetic mice (db / db) were purchased from Oriental Bioservice. 8-10 week old male mice were used.
方法:マウスの尾の背面側の先端部に、滅菌した#10ゲージのメス(ベクトンディッキンソン)を用いて0.5〜1.0cm長の傷をつけた。出血は、圧迫することによって止めた。AG30-5Cポリペプチドをそれぞれ1, 10, 50μg /mlになるようPBSに溶解し、サンプル溶液とした。創傷部位に当該サンプル溶液を50μlずつ塗布した。コントロールとしては、PBSのみを同量塗布した。マウスは1群に3匹を用いた(n=3)。術2日後、マウスにケタミン(80mg/kg)およびキシラジン(12mg/kg)を筋肉内投与し、麻酔した。レーザードップラー流量計(Moor Instruments (Devon, United Kingdom))を用いて創傷部の血流量を測定し、得られたデータを相対値に換算することにより各サンプルの効果を判定した。 Method: A 0.5-1.0 cm long wound was made on the tip of the back of the mouse's tail using a sterilized # 10 gauge scalpel (Becton Dickinson). The bleeding was stopped by pressing. AG30-5C polypeptide was dissolved in PBS at 1, 10, 50 μg / ml, respectively, to prepare a sample solution. 50 μl of the sample solution was applied to the wound site. As a control, the same amount of PBS alone was applied. Three mice were used per group (n = 3). Two days after the operation, the mice were anesthetized by intramuscular administration of ketamine (80 mg / kg) and xylazine (12 mg / kg). The blood flow of the wound was measured using a laser Doppler flow meter (Moor Instruments (Devon, United Kingdom)), and the effect of each sample was determined by converting the obtained data into a relative value.
結果を下記表2及び表3に示す。 The results are shown in Tables 2 and 3 below.
何れのマウスにおいてもAG30-5C 投与群において有意に血流量の増加が認められた。 In any mouse, a significant increase in blood flow was observed in the AG30-5C administration group.
Claims (5)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008557153A JP5444544B2 (en) | 2007-02-09 | 2008-02-07 | Angiogenesis inducer and polypeptide used therefor |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2007029945 | 2007-02-09 | ||
JP2007029945 | 2007-02-09 | ||
JP2008557153A JP5444544B2 (en) | 2007-02-09 | 2008-02-07 | Angiogenesis inducer and polypeptide used therefor |
PCT/JP2008/052022 WO2008096816A1 (en) | 2007-02-09 | 2008-02-07 | Angiogenesis inducer and polypeptide for use in the angiogenesis inducer |
Publications (2)
Publication Number | Publication Date |
---|---|
JPWO2008096816A1 JPWO2008096816A1 (en) | 2010-05-27 |
JP5444544B2 true JP5444544B2 (en) | 2014-03-19 |
Family
ID=39681720
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2008557153A Active JP5444544B2 (en) | 2007-02-09 | 2008-02-07 | Angiogenesis inducer and polypeptide used therefor |
Country Status (2)
Country | Link |
---|---|
JP (1) | JP5444544B2 (en) |
WO (1) | WO2008096816A1 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5691049B2 (en) * | 2008-11-28 | 2015-04-01 | アンジェスMg株式会社 | Novel polypeptide having angiogenesis-inducing activity and antibacterial activity and pharmaceutical use thereof |
WO2010101237A1 (en) | 2009-03-06 | 2010-09-10 | アンジェスMg株式会社 | Polypeptides and antibacterial or antiseptic use of same |
WO2010137594A1 (en) * | 2009-05-25 | 2010-12-02 | アンジェスMg株式会社 | Polypeptide having antibacterial activity and angiogenesis-inducing activity and wound-healing drug containing said polypeptide |
EP2481751A1 (en) * | 2011-01-26 | 2012-08-01 | PharmaSurgics in Sweden AB | Human lactoferrin derived peptides |
KR102498220B1 (en) | 2014-09-26 | 2023-02-08 | 고꾸리쯔 다이가꾸 호우징 오사까 다이가꾸 | Novel peptide and use thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005090564A1 (en) * | 2004-03-19 | 2005-09-29 | Genomidea Inc. | Gene promoting vascular endothelial cell growth |
-
2008
- 2008-02-07 JP JP2008557153A patent/JP5444544B2/en active Active
- 2008-02-07 WO PCT/JP2008/052022 patent/WO2008096816A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005090564A1 (en) * | 2004-03-19 | 2005-09-29 | Genomidea Inc. | Gene promoting vascular endothelial cell growth |
Also Published As
Publication number | Publication date |
---|---|
JPWO2008096816A1 (en) | 2010-05-27 |
WO2008096816A1 (en) | 2008-08-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20200308219A1 (en) | Compositions containing hc-ha/ptx3 complexes and methods of use thereof | |
JP6033781B2 (en) | Selective targeting of CD40L / Mac-1 interaction by small peptide inhibitors and its use for the treatment of inflammation and atherogenesis | |
JP2002532068A (en) | Protein binding to angiogenesis inhibitory protein and composition and method using the same | |
EP2076290A1 (en) | Multimeric tie 2 agonists and uses thereof in stimulating angiogenesis | |
JP5444544B2 (en) | Angiogenesis inducer and polypeptide used therefor | |
JP5384948B2 (en) | Novel polypeptide and antibacterial agent containing it as an active ingredient | |
JP2013506681A (en) | Hematopoietic stem cells for use in the treatment of kidney injury | |
JP2023179739A (en) | Compositions and methods for prevention and treatment of corneal haze and scarring | |
JP6416240B2 (en) | Tetrapeptide derived from human C—X—C chemokine useful for treatment of various skin conditions | |
JP5691049B2 (en) | Novel polypeptide having angiogenesis-inducing activity and antibacterial activity and pharmaceutical use thereof | |
US9695219B2 (en) | Polypeptide having antibacterial activity and angiogenesis-inducing activity and wound-healing drug containing said polypeptide | |
Yin et al. | SDF-1α in glycan nanoparticles exhibits full activity and reduces pulmonary hypertension in rats | |
WO2016104436A1 (en) | Cytokine storm inhibitor | |
JP2018510870A (en) | Isolated peptides derived from the B7 ligand dimer interface and their use | |
JP6713148B2 (en) | Composition for the treatment of peritonitis | |
JP6042106B2 (en) | Novel polypeptide containing methionine sulfoxide | |
US20190216889A1 (en) | Methods and compositions for treatment of fibrosis | |
TWI445543B (en) | Medication for treating choroidal neovascularization | |
DE60008749T2 (en) | THERAPEUTIC PEPTIDES FROM BSE IN-SEQUENCES | |
JP2019031486A (en) | Novel polypeptide and application thereof | |
CA2642494A1 (en) | Novel phosphopeptides and uses thereof in preventing kidney stone formation | |
WO2014164669A1 (en) | Improved medical device |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20101208 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20121023 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20121225 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20130416 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20130716 |
|
A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20130731 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20130827 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20131025 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20131126 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20131202 |
|
R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 Ref document number: 5444544 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |