WO2008127066A1 - Peptide inhibitors of receptor activator of nf-kb, and pharmaceutical composition including the same - Google Patents
Peptide inhibitors of receptor activator of nf-kb, and pharmaceutical composition including the same Download PDFInfo
- Publication number
- WO2008127066A1 WO2008127066A1 PCT/KR2008/002133 KR2008002133W WO2008127066A1 WO 2008127066 A1 WO2008127066 A1 WO 2008127066A1 KR 2008002133 W KR2008002133 W KR 2008002133W WO 2008127066 A1 WO2008127066 A1 WO 2008127066A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polypeptide
- rank
- amino acid
- osteoclast
- seq
- Prior art date
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 154
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 34
- 239000003112 inhibitor Substances 0.000 title abstract description 6
- 101710178436 Tumor necrosis factor receptor superfamily member 11A Proteins 0.000 title description 2
- 102100028787 Tumor necrosis factor receptor superfamily member 11A Human genes 0.000 title description 2
- 210000002997 osteoclast Anatomy 0.000 claims abstract description 121
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 106
- 229920001184 polypeptide Polymers 0.000 claims abstract description 83
- 230000004069 differentiation Effects 0.000 claims abstract description 73
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 66
- 210000004027 cell Anatomy 0.000 claims abstract description 49
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 22
- 201000010099 disease Diseases 0.000 claims abstract description 21
- 230000004913 activation Effects 0.000 claims abstract description 15
- 239000013598 vector Substances 0.000 claims abstract description 13
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 11
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 11
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 53
- 210000000988 bone and bone Anatomy 0.000 claims description 45
- 230000006378 damage Effects 0.000 claims description 36
- 208000001132 Osteoporosis Diseases 0.000 claims description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 7
- 241000700605 Viruses Species 0.000 claims description 7
- 108091023040 Transcription factor Proteins 0.000 claims description 6
- 102000040945 Transcription factor Human genes 0.000 claims description 6
- 206010003246 arthritis Diseases 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 230000004568 DNA-binding Effects 0.000 claims description 4
- 208000029725 Metabolic bone disease Diseases 0.000 claims description 4
- 208000034578 Multiple myelomas Diseases 0.000 claims description 4
- 206010028980 Neoplasm Diseases 0.000 claims description 4
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 4
- 230000004570 RNA-binding Effects 0.000 claims description 4
- 235000009697 arginine Nutrition 0.000 claims description 4
- 208000032839 leukemia Diseases 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 210000004899 c-terminal region Anatomy 0.000 claims description 3
- 101100107610 Arabidopsis thaliana ABCF4 gene Proteins 0.000 claims description 2
- 239000004475 Arginine Substances 0.000 claims description 2
- 241000724256 Brome mosaic virus Species 0.000 claims description 2
- 101710132601 Capsid protein Proteins 0.000 claims description 2
- 101710094648 Coat protein Proteins 0.000 claims description 2
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 claims description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 2
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 claims description 2
- 101001050288 Homo sapiens Transcription factor Jun Proteins 0.000 claims description 2
- 108700020121 Human Immunodeficiency Virus-1 rev Proteins 0.000 claims description 2
- 241000714259 Human T-lymphotropic virus 2 Species 0.000 claims description 2
- 101710125418 Major capsid protein Proteins 0.000 claims description 2
- 101710141454 Nucleoprotein Proteins 0.000 claims description 2
- 101710083689 Probable capsid protein Proteins 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 101100068078 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GCN4 gene Proteins 0.000 claims description 2
- 102100023132 Transcription factor Jun Human genes 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 150000001484 arginines Chemical class 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 2
- 239000002502 liposome Substances 0.000 claims description 2
- 108010011110 polyarginine Proteins 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims 1
- 102000014128 RANK Ligand Human genes 0.000 description 54
- 108010025832 RANK Ligand Proteins 0.000 description 54
- 230000000694 effects Effects 0.000 description 32
- 230000004927 fusion Effects 0.000 description 29
- 230000005764 inhibitory process Effects 0.000 description 24
- 241000699666 Mus <mouse, genus> Species 0.000 description 22
- 239000002158 endotoxin Substances 0.000 description 18
- 229920006008 lipopolysaccharide Polymers 0.000 description 18
- 239000002243 precursor Substances 0.000 description 15
- 102000007469 Actins Human genes 0.000 description 13
- 108010085238 Actins Proteins 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 12
- 150000001413 amino acids Chemical class 0.000 description 12
- 238000002347 injection Methods 0.000 description 12
- 239000007924 injection Substances 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 11
- 238000010521 absorption reaction Methods 0.000 description 10
- 210000002540 macrophage Anatomy 0.000 description 10
- 238000007363 ring formation reaction Methods 0.000 description 10
- 102000007591 Tartrate-Resistant Acid Phosphatase Human genes 0.000 description 9
- 108010032050 Tartrate-Resistant Acid Phosphatase Proteins 0.000 description 9
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 8
- 101710176384 Peptide 1 Proteins 0.000 description 8
- 210000000963 osteoblast Anatomy 0.000 description 8
- 210000005220 cytoplasmic tail Anatomy 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 210000002798 bone marrow cell Anatomy 0.000 description 6
- 210000001185 bone marrow Anatomy 0.000 description 5
- 230000007910 cell fusion Effects 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 101000648503 Homo sapiens Tumor necrosis factor receptor superfamily member 11A Proteins 0.000 description 4
- 101000830602 Mus musculus Tumor necrosis factor ligand superfamily member 11 Proteins 0.000 description 4
- 102000008108 Osteoprotegerin Human genes 0.000 description 4
- 108010035042 Osteoprotegerin Proteins 0.000 description 4
- 102000003714 TNF receptor-associated factor 6 Human genes 0.000 description 4
- 108090000009 TNF receptor-associated factor 6 Proteins 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 102000053530 human TNFRSF11A Human genes 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- XXUPLYBCNPLTIW-UHFFFAOYSA-N octadec-7-ynoic acid Chemical compound CCCCCCCCCCC#CCCCCCC(O)=O XXUPLYBCNPLTIW-UHFFFAOYSA-N 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 210000004443 dendritic cell Anatomy 0.000 description 3
- 238000011813 knockout mouse model Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108091006084 receptor activators Proteins 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 206010065687 Bone loss Diseases 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 102000008889 TNF receptor-associated factor TRAF Human genes 0.000 description 2
- 108050000808 TNF receptor-associated factor TRAF Proteins 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000011164 ossification Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 210000002536 stromal cell Anatomy 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 102000003390 tumor necrosis factor Human genes 0.000 description 2
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000006386 Bone Resorption Diseases 0.000 description 1
- 208000020084 Bone disease Diseases 0.000 description 1
- 206010055113 Breast cancer metastatic Diseases 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 241000723754 Flock house virus Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100046557 Mus musculus Tnfrsf11a gene Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 101000933967 Pseudomonas phage KPP25 Major capsid protein Proteins 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 1
- HUXIAXQSTATULQ-UHFFFAOYSA-N [6-bromo-3-[(2-methoxyphenyl)carbamoyl]naphthalen-2-yl] dihydrogen phosphate Chemical compound COC1=CC=CC=C1NC(=O)C1=CC2=CC(Br)=CC=C2C=C1OP(O)(O)=O HUXIAXQSTATULQ-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 102000035181 adaptor proteins Human genes 0.000 description 1
- 108091005764 adaptor proteins Proteins 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- 210000004979 bone marrow derived macrophage Anatomy 0.000 description 1
- 230000010072 bone remodeling Effects 0.000 description 1
- 230000024279 bone resorption Effects 0.000 description 1
- 229960005069 calcium Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229960001714 calcium phosphate Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 229960001251 denosumab Drugs 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- 229960002986 dinoprostone Drugs 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002159 nanocrystal Substances 0.000 description 1
- 208000002865 osteopetrosis Diseases 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 230000004526 pharmaceutical effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 102000035025 signaling receptors Human genes 0.000 description 1
- 108091005475 signaling receptors Proteins 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000001433 sodium tartrate Substances 0.000 description 1
- 229960002167 sodium tartrate Drugs 0.000 description 1
- 235000011004 sodium tartrates Nutrition 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000036346 tooth eruption Effects 0.000 description 1
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 1
- 235000005282 vitamin D3 Nutrition 0.000 description 1
- 239000011647 vitamin D3 Substances 0.000 description 1
- 229940021056 vitamin d3 Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
Definitions
- the present invention relates to peptide inhibitors having receptor activator of NF- k
- B (RANK) inhibiting activity a pharmaceutical composition including the peptides, a method of inhibiting osteoclast differentiation in an individual using the pharmaceutical composition, a method of treating or preventing diseases caused by osteoclast differentiation and activation using the pharmaceutical composition, a nucleic acid encoding the peptides, a vector including the nucleic acid, and a host cell transformed with the vector.
- the skeleton functions to support the human body and protects body organs.
- the skeleton stores calcium, a critical ion for a variety of metabolic processes, and it is the site of hematopoiesis.
- the skeleton is a dynamic organ that is constantly remodeling. To accomplish this function there must be a tight coupling between bone resorption by osteoclasts and bone formation by osteoblasts.
- Osteoblasts are derived from a mesenchymal progenitor cell that is multipotential and can also differentiate into adipocytes and stromal cells. Osteoblasts initially produce an osteoid matrix that is calcified extracellularly.
- the major structural protein and mineral crystal of bone are type I collagen and hydroxyappatite, which is a calcium-phosphate salt, respectively.
- Osteoclasts are specialized multinucleated giant cells that resorb bone. They are hematopoietic in origin and derived from myeloid precursors that also gives rise to macrophages.
- RANK tumor necrosis factor
- osteoclast differentiation referst to a processes whereby bone marrow-derived monocyte/macrophages (osteoclast precursors) acquire the specialized features of a os- teoclast cell.
- osteoclast precursors bone marrow-derived monocyte/macrophages
- osteoprotegerin As a soluble decoy-like factor, capable of inhibiting osteoclast differentiation in vitro. Then, they over- expressed OPG in a mouse and found that bone density was increased. They also cloned a ligand of OPG named as OPGL.
- the OPGL was the same cytokine as RANKL that has previously cloned by researchers of Immunex Co. Accordingly, osteoclast differentiation can be achieved using an OPGL treatment even when osteoclast precursors exist alone not when osteoblasts and osteoclast precursors are co-cultured.
- RANK the signaling receptor for RANKL
- RANK belongs to a TNF receptor family that is similar to CD40.
- RANK is expressed in the spleen, lymph nodes, dendritic cells, activated T lymphocyte, osteoclast precursors, mature osteoclast, and chondrocyte. Since there is no known signaling motif in a RANK cytoplasmic tail domain, it can be assumed that an adaptor protein may be involved in signal delivering, and in fact, a TRAF (TNF receptor-associated factor) family protein is bound, specifically, TRAF6 plays an important role in delivering an osteoclast differentiation signal to the downstream of the receptor.
- TRAF TRAF
- bone destruction dominates over bone formation, and in the case of post-menopause females, one third of them experiences serious osteoporosis because an estrogen hormone is not secreted any longer.
- chronic inflammation resulting from disorders of the immune system is a primary cause of rheumatoid arthritis causing destruction of cartilage and bones.
- Bone loss (destruction) occurring in these disorders described above is mainly caused by osteoclast, and RANKL and a receptor of RANKL J that is, RANK, necessarily operates in osteoclast differentiation. Accordingly, the key strategy in developing a therapeutic agent for bone disorders such as arthritis and osteoporosis is to find a method of regulating osteoclast differentiation.
- Inventors of the present invention provide a method of regulating osteoclast differentiation by development of peptide inhibitors targeting RANK that is essential for osteoclasts differentiation and activation.
- peptides can inhibit osteoclast differentiation induced by RANKL, inhibit bone destruction due to osteoclasts activated by RANKL, inhibit cell fusion of osteoclast precursors due to RANKL, inhibit actin ring- formation in osteoclast due to RANKL, inhibit bone destruction due to RANKL injected to a mouse, and inhibit bone destruction due to lipopolysaccharide (LPS) injection.
- LPS lipopolysaccharide
- the present invention provides novel RANK inhibitor peptides.
- the present invention also provides a pharmaceutical composition for treating or preventing diseases caused by bone destruction, including the RANK inhibiting peptide.
- the present invention also provides a method of inhibiting osteoclast differentiation in an individual using the pharmaceutical composition, and a method of treating and preventing diseases caused by osteoclast differentiation and osteoclast activation using the pharmaceutical composition.
- the present invention also provides a nucleic acid encoding the RANK inhibiting peptide, a vector including the nucleic acid, and a host cell transformed with the vector.
- a polypeptide having a RANK (receptor activator of NF- k B) inhibiting activity comprising a 6-12 contiguous amino acid sequence selected from an amino acid sequence set forth in SEQ D NO: 1 and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1.
- the polypeptide having RANK inhibiting activity may be selected from the group consisting of polypeptides having amino acid sequences set forth in SEQ D NOS: 1-4.
- polypeptides having the amino acid sequences set forth in SEQ D NOS: 1-4 includes 12 amino acids, 10 amino acids, 8 amino acids, and 6 amino acids, respectively. Each polypeptide includes 4-9 positions (HVVYV: SEQ D NO: 4) of the amino acid sequence set forth in SEQ D NO: 1. As described in Examples of the present application, polypeptides having an activity inhibiting an RANK-induced activity commonly have IIVVYV (SEQ D NO: 4) domains (for example, SEQ D NOS: 1-4, and SEQ D NOS: 8-11).
- an IVVYV (SEQ D NO: 5) sequence composed of five amino acids and an IIVVY (SEQ D NO: 6) sequence composed of five amino acids prepared by removing a single amino acid from the IIVVYV (SEQ D NO: 4) domain, and polypeptides (SEQ D NOS: 12 and 13) respective prepared by connecting a cell permeable sequence to IVVYV (SEQ D NO: 5) sequence and IIVVY (SEQ D NO: 6) sequence do not have an activity inhibiting an RANK-induced activity.
- polypeptides prepared by substituting IV that are 2 and 3 positions of the IIVVYV (SEQ D NO: 4) domain from the IIVVYV (SEQ D NO: 4) domain or an amino acid sequence having the IIVVYV (SEQ D NO: 4) domain to LA do not have an activity inhibiting an RANK-induced activity either.
- a polypeptide according to the present invention can be any polypeptide having a 6-12 contiguous amino acid sequence selected from the amino acid sequence set forth in SEQ D NO: 1 and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1.
- Examples of the present application disclose only polypeptides respectively composed of 6,8,10 and 12 amino acids, a person having ordinary skill in the art may easily understand that a polypeptide having a 6-12 contiguous amino acid sequence selected from an amino acid sequence set forth in SEQ D NO: 1 and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1, which is not disclosed in Examples of the present application, also has the same activity as the polypeptides disclosed in Examples of the present application.
- the polypeptide having a RANK inhibiting activity according to the present invention may or may not be connected to a cell permeable polypeptide.
- the cell permeable polypeptide may be connected to an N-terminal or C-terminal of the polypeptide having a RANK inhibiting activity, for example, the cell permeable polypeptide may be connected to an N-terminal of the polypeptide having a RANK inhibiting activity.
- the term 'cell permeable polypeptide' refers to a polypeptide having a cell permeable capability to allow a fusion partner with which the polypeptide is fused to enter inside of a cell.
- a cell permeable polypeptide is well known in the art, and can be used in embodiments of the present invention, for example, the cell permeable polypeptide can be a leader signal sequence.
- the leader signal sequence may be selected from the group consisting of Kaposi fibroblast growth factor signal sequence, HIV-I tat (48-60), D-amino acid-substituted HIV-I tat (48-60), arginine-substituted HIV-I tat (48-60), Drosophila Antennapaedia(43-58), 7 or more amino acid-containing virus RNA binding peptide, 7 or more arginine-containing DNA binding peptide, and 6-8 arginines -containing polyarginine polypeptide.
- the leader signal sequence is not limited thereto.
- the virus RNA binding peptide may be selected from the group consisting of of HIV-I rev (34-50), HTLV-II rev (4-16), a brome mosaic virus Gag (7-25) and a flock house virus coat protein (35-49).
- the DNA binding peptide may be selected from the group consisting of human c-Ibs (139-164), human c-Jun (252-279), and a yeast transcription factor GCN4 (231-252).
- 'peptide' or 'polypeptide' are used interchangeably and refers to a polypeptide having two or more amino acids.
- the cell permeable polypeptide can be a human transcription factor Hph-1 having an amino acid sequence set forth in SEQ D NO: 7.
- a polypeptide having an RANK inhibiting activity according to the present invention may have such a structure that a human transcription factor Hph-1 set forth in SEQ D NO: 7 that is a cell permeable polypeptide is connected to an N-terminal of a polypeptide having an RANK inhibiting activity having a 6-12 contiguous amino acid sequence selected from an amino acid sequence set forth in SEQ D NO: 1 and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1.
- the polypeptide having an RANK inhibiting activity according to the present invention may be selected from the group consisting of polypeptides having amino acid sequences set forth in SEQ D NOS: 8-11.
- a pharmaceutical composition for treating or preventing a disease caused by osteoclast differentiation and osteoclast activation comprising the polypeptide having a RANK in- hibiting activity according to the present invention.
- a polypeptide having a RANK inhibiting activity according to the present invention has been described above.
- the polypeptide is derived from a RANK cytoplasmic tail domain of a human or mouse.
- the polypeptide may include an HVVYV (SEQ D NO: 4) sequence of 546-551 of a human RANK amino acid sequence.
- a pharmaceutical composition according to the present invention inhibits osteoclast differentiation due to RANKL J inhibits bone destruction due to osteoclast activated by RANKLj inhibits cell fusion of osteoclast precursors due to RANKL, inhibits actin ring-formation in osteoclast due to RANKL, inhibits bone destruction due to RANKL injected to a mouse, and inhibits bone destruction due to lipopolysaccharide (LPS) injection.
- a pharmaceutical composition according to the present invention can be used for treating or preventing a disease caused by osteoclast differentiation and osteoclast activation.
- a pharmaceutically acceptable carrier can be a diluting agent, an excipient, a dis- integrant, a binder, or a lubricant, but is not limited thereto, for example, when the pharmaceutical composition according to the present invention is used through an injection, the carrier can be a sterilized water or a buffer suitable for physiological conditions, such as PBS.
- the pharmaceutical composition can be formulated to be used for oral administration or non-oral administration.
- the carrier can be a material facilitating the transport of the RANK inhibiting peptides across the cellular barrier such as, cell membrane, a cell wall, organellar membrane, and the like.
- 'pharmaceutically acceptable refers to a condition that when the pharmaceutical composition is administrated to an individual, allergies or unwanted reactions do not occur.
- a method of preparing a protein-containing aqueous composition that is an active component is well known in the art.
- any person having ordinary skill in the art may easily determine a suitable amount of the pharmaceutical composition according to the present invention to be administered and a suitable administration method without undue experimentation.
- a therapeutically effective dose of the pharmaceutical composition that is, a suitable amount of the pharmaceutical composition inhibiting RANKL-mediated osteoclast differentiation or osteoclast activity is administered to an animal or human.
- the amount of the pharmaceutical composition according to the present invention may be in the range from 1 nM to 1 M per individual, preferably from 1 ⁇ M to 1 mM, and more preferably from 0. ImM to 0.2mM, based on the amount of the polypeptide having an RANK inhibiting activity that is an active component.
- the amount of the pharmaceutical composition is not limited thereto.
- the pharmaceutical composition can be formulated to be used for oral administration or non-oral administration, for example, the pharmaceutical composition can be administrated to an individual through an injection administration.
- a disease caused by osteoclast differentiation and osteoclast activation can be selected from the group consisting of osteoporosis, arthritis, bone destruction caused by cancers, metabolic bone diseases, leukemia, and multiple myeloma.
- the disease is not limited thereto.
- the polypeptide according to the present invention may be encapsulated by a liposome or exists in a form of a virus or vector comprising a gene encoding the polypeptide.
- a method of inhibiting osteoclast differentiation of an individual comprising administering the pharmaceutical composition according to the present invention to an individual so as to inhibit a RANK-meditated signal delivery.
- the pharmaceutical composition according to the present invention used in the method may have the same pharmaceutical effect, be used in the same administration dose, and be used using the same administration method, as the pharmaceutical composition according to the present invention described above.
- the pharmaceutical composition can inhibit a RANK-mediated signal delivery, osteoclast differentiation due to RANKL (RANK ligand) is inhibited, bone destruction due to osteoclast activated by RANKL is inhibited, cell fusion of osteoclast precursors due to RANKL is inhibited, actin ring-formation in osteoclast due to RANKL is inhibited, bone destruction due to RANKL injected to a mouse is inhibited, and bone destruction due to lipopolysaccharide (LPS) injection is inhibited.
- RANKL RANK ligand
- the inhibition of RANK-mediated signal delivery may result in treatment and prevention of diseases caused by osteoclast differentiation and osteoclast activation.
- a method of treating or preventing a disease caused by osteoclast differentiation and osteoclast activation comprising administering the pharmaceutical composition according to the present invention to an individual so as to inhibit the RANK- meditated signal delivery.
- the disease can be selected from the group consisting of osteoporosis, arthritis, bone destruction caused by cancers, metabolic bone diseases, leukemia, and multiple myeloma. However, the disease is not limited thereto.
- a nucleic acid encoding the polypeptide having a RANK inhibiting activity according to the present invention there is provided a vector including the nucleic acid encoding the polypeptide having a RANK inhibiting activity according to the present invention.
- a host cell transformed with the vector including the nucleic acid encoding the polypeptide having a RANK inhibiting activity according to the present invention.
- the host cell can be a prokaryote cell or a eukaryotic cell.
- a polypeptide having a RANK inhibition activity according to the present invention has a RANK inhibition activity, so that osteoclast differentiation due to
- RANKL(RANK-ligand) can be inhibited, bone destruction due to osteoclast activated by RANKL can be inhibited, cell fusion of osteoclast precursors due to RANKL can be inhibited, actin protein ring-formation in osteoclast due to RANKL can be inhibited, bone destruction due to RANKL injected to a mouse can be inhibited, and bone destruction due to lipopolysaccharide (LPS) injection can be inhibited.
- a pharmaceutical composition according to the present invention can be used to treat or prevent a disease caused by osteoclast differentiation and osteoclast activation.
- a method of inhibiting osteoclast differentiation of an individual according to the present invention can inhibit osteoclast differentiation of an individual.
- a disease caused by osteoclast differentiation and osteoclast activation occurring in an individual can be treated or prevented using a method of treating or preventing a disease according to the present invention .
- FIG. 1 illustrates domains showing a RANK inhibiting activity in RANK cytoplasmic tail regions of human and mouse (A) and fusion peptides connected to a cell permeable sequence (B).
- FIG. 1 illustrates domains showing a RANK inhibiting activity in RANK cytoplasmic tail regions of human and mouse (A) and fusion peptides connected to a cell permeable sequence (B).
- FIGS. 2 are images illustrating osteoclasts stained with a tartrate resistant acid phosphatase (TRAP) showing an effect of a RANK inhibiting peptide according to a concentration of the RANK inhibiting peptide when an osteoclast precursor separated from a mouse bone marrow cell is treated with RANKL to cause osteoclast differentiation (A) and a graph showing degree of differentiation (B).
- a degree of differentiation was obtained by performing three separate experiments to obtain the number of osteoclasts containing over three nuclei and then measuring a mean value thereof.
- FIG. 3 is a view illustrating test group fusion peptides 1-6 (A) and control group fusion peptides 1-6 (B);
- FIG. 4 are images of osteoclasts stained with TRAP showing a degree of osteoclast differentiation according to fusion peptides 1-6 of a test group and fusion peptides 1-6 of a control group on (A) and a graph showing degree of differentiation (B).
- A fusion peptides 1-6
- B degree of differentiation
- FIG. 5 are images illustrating that a bone absorption activity of an activated osteoclast is inhibited by a cell permeable RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NOS: 8 and 14(A) and a graph showing degree of differentiation in quantification for the peptide having an amino acid sequence set forth in SEQ D NO: 8 and 14 (B).
- a degree of absorption was obtained by measuring a ratio of an absorbed area ratio to the entire area of OAASTM (Oscotec Inc.: Korea) that is a cell culture container in three separate experiments and then obtaining a mean value of the three experimental results and a standard deviation thereof.
- C denotes a control group
- Mt denotes a peptide having an amino acid sequence set forth in SEQ D NO: 14
- WTde denotes a having an amino acid sequence set forth in SEQ D NO: 8.
- FIG. 6 are images illustrating a cell permeable RANK inhibiting peptide inhibiting an actin ring formation in osteoclasts.
- Mtand WT denote peptides having an amino acid sequence of SEQ D NO: 14 and SEQ D NO: 8, respectively;
- FIG. 7 are images illustrating RANKL or LPS injection-induced bone destruction inhibition effect of a cell permeable RANK inhibiting peptide in a mouse (A) and a graph showing degree of bone destruction inhibition in quantification form (B).
- A a ratio of an absorbed area to the entire area in a sectional view of the calvaria was measured to quantify the degree of bone destruction inhibition.
- the absorbed area refers to a bone marrow cavity in calvaria, and an increase or decrease in the absorbed area was compared to the control group to quantify the degree of bone destruction inhibition.
- An average value was obtained from six kinds of samples and a standard deviation thereof was obtained.
- the relative absored area of control group was set to 1.
- a fusion polypeptide composed of 11 amino acids in which a human transcription factor Hph-1 that has a cell permeable sequence YAR VRRRGPRR (SEQ D NO: 7) is connected to an N-terminal of a human RANK cytoplasmic tail domain- derived polypeptide.
- the human RANK cytoplasmic tail domain-derivd polypeptide is a polypeptide fragment having a 5-12 contiguous amino acid sequence selected from an amino acid sequence set forth in SEQ D NO: 1 that has an amino acid sequence of KGDIIVVYVSQT corresponding to an amino acid sequence 543-554 of a human RANK (corresponding to an amino acid sequence 531-542 of a mouse RANK) and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1.
- Polypeptide fragments used in the current example have amino acid sequences set forth in SEQ D NOS: 1-6, and fusion polypeptides (hereinafter referred to as test group fusion peptides 1-6) in which a cell permeable sequence set forth in SEQ D NO: 7 is connected to an N-terminal of each of the polypeptide fragments, have amino acid sequences set forth in SEQ D NOS: 8-13.
- test group fusion peptides 1-6 fusion polypeptides in which a cell permeable sequence set forth in SEQ D NO: 7 is connected to an N-terminal of each of the polypeptide fragments, have amino acid sequences set forth in SEQ D NOS: 8-13.
- test group fusion peptides 1-6 fusion polypeptides in which a cell permeable sequence set forth in SEQ D NO: 7 is connected to an N-terminal of each of the polypeptide fragments
- control group fusion peptides 1-6 an IV sequence of the amino acid sequences set forth in
- polypeptides shown in Table 1 were synthesized by Peptron, Inc. (Korea).
- FIG. 1 illustrates domains showing RANK inhibiting activity in RANK cytoplasmic tail regions of humans and mouse (A) and fusion peptides connected to a cell permeable sequence (B).
- a bone marrow cell collected from a five- week old mouse (C57BL/6J) was loaded onto a 48-well plate at a density of 4xlO 4 cells/well, and then cultured for three days using 30 ng/ml of hM-CSF (R&D system Inc.) that is a macrophage differentiation factor-containing differentiation medium ( ⁇ -MEM (Invitrogen. co.,), 10%(v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 100 ⁇ g /ml streptomycin) at 37 0 C under 95%(v/v) air and 5% (v/v) CO 2 .
- ⁇ -MEM fetal bovine serum
- a mouse RANKL extracellular domain fragment (position 150 to 316) that is an active osteoclast differentiation factor comparable to a full length mouse RANKL (SEQ D NO: 22) (Lacey et al. Cell, 93: 165, 1998) which had been expressed in an E.coli and then purified was added to the obtained macrophage cell and the resultant macrophage cell was cultured in the same conditions as described above to cause osteoclast differ- entiation.
- the 158aa mouse RANKL extracellular domain fragment was cloned into Sail-Not I site of pET28b(+) vector (Novagen), which is a commonly used bacterial expression vector and containing a His tag sequence for the purification of the expressed protein.
- test group fusion peptide 1 according to the present invention and the control group fusion peptide 1 were respectively added thereto while the concentration of the test group fusion peptide 1 and the control group fusion peptide 1 differed at l ⁇ M, 2.5 ⁇ M, and 5 ⁇ M and the culturing was performed.
- the differentiation medium was exchanged every other day. As a result, osteoclast differentiation occurred when the control group fusion peptide 1 was used, and osteoclast differentiation did not occur when the test group fusion peptide 1 was used.
- FIG. 2 are images illustrating osteoclast stained with tartrate resistant acid phosphatase (TRAP) showing an effect of a RANK inhibiting peptide according to a concentration of the RANK inhibiting peptide when an osteoclast precursor separated from a mouse bone marrow cell is treated with RANKL to cause osteoclast differentiation (A) and a graph showing degree of differentiation (B).
- FIG. 2 shows results of such an experiment that bone marrow -derived macrophage/monocyte cells (osteoclast precursors) were cultured using the test group fusion peptide 1 and the control group fusion peptide 1 for three days, respectively, and then on the fourth day, the cultured product was stained with a TRAP and identified using an optical microscope.
- TRAP tartrate resistant acid phosphatase
- the TRAP staining was performed such that the cultured cell was fixed using 3.7% formaldehyde for 15 minutes at room temperature, washed twice, and then a staining solution prepared by mixing acetate, a fast gargnet GBC base, a naphthol AS-BI phosphate, sodium nitrite, and tartrate in a mixture ratio described in the specification of an acid phosphatase, Leukocyte (TRAP) kitTM (Sigma Co.) was added to a 200 ⁇ Jl /well and then reacted together at 37 0 C for 20 minutes.
- TRAP Leukocyte
- RANK inhibiting peptide Specifically, an osteoclast differentiation inhibition effect was identified using test group fusion peptides 1-6 having amino acid sequences set forth in SEQ D NO: 8 to 13 in which some amino acids were removed. At this time, control group fusion peptides 1-6 having amino acid sequences set forth in SEQ D NO: 14-19 were used as a control group.
- FIG. 3 is a view illustrating test group fusion peptides 1-6 and control group fusion peptides 1-6.
- FIG. 4 are images of osteoclasts stained with TRAP showing an osteoclast differentiation inhibiting effect of the test group fusion peptides 1-6 and the control group fusion peptides 1-6 (A) and a graph showing degree of differentiation (B).
- a bone marrow cell collected from a five- week old mouse (C57BL/6J) was loaded onto a OAASTM 48-well plate at a density of 4xlO 4 cells/well, and then cultured for three days using 30 ng/ml of hM-CSF (R&D system Inc.) that is a macrophage differentiation factor-containing differentiation medium ( ⁇ -MEM (Invitrogen. co.), 10%(v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 100 ⁇ g /ml streptomycin) at 37 0 C under 95%(v/v) air and 5% (v/v) CO 2 .
- hM-CSF R&D system Inc.
- ⁇ -MEM macrophage differentiation factor-containing differentiation medium
- FBS fetal bovine serum
- penicillin 100 units/ml bovine serum
- streptomycin 100 ⁇ g /ml streptomycin
- a macrophage derived from a bone marrow cell was obtained. Then, in a ⁇ tion to 30 ng/ml of hM- CSF, 100 ng/ml of a mouse RANKL that is an osteoclast differentiation factor which had been expressed in an E.coli and then purified was added to the obtained macrophage cell and the resultant macrophage cell was cultured in the same conditions as described above to cause osteoclast differentiation.
- the medium used was altered to a new differentiation medium.
- the osteoclast cell was treated with each of polypeptides having amino acid sequences set forth in SEQ D NOS: 8 and 14 having a concentration of 5 ⁇ M and then bone absorption was identified for 48 hours.
- the OAASTM plate was washed using distilled water to remove cells attached to the OAASTM plate after the cell culturing and then a 4-5% sodium hypochlorite solution was added thereto and the resultant solution was left to sit for 5 minutes. Then, the resultant OAASTM plate was washed and dried, and bone absorption was identified.
- the osteoclast absorption and tissue measurement were identified on the basis of images obtained using Axiovert 200 (Carl Zeiss, Gottingen, Germany), that is an optical microscope including a CCD camera therein.
- the obtained images were analyzed using Image-pro Plus4.5 (Media Cybernetics, Inc.) that is a surface measuring program.
- FIG. 5 are images illustrating that a bone absorption activity of an activated osteoclast is inhibited by a cell permeable RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NOS: 8 and 14 (A) and a graph showing degree of differentiation in quantification for the peptide having an amino acid sequence set for th in SEQ D NO: 8 and 14(B).
- osteoclast differentiation was caused in the 48-well plate as described in Example 2 and then, RANKL was not supplied for 6 hours to stop a cell signal delivery due to RANKL.
- the RANKL, the RNAK inhibiting peptide (SEQ D NO: 8), and the control group peptide (SEQ D NO: 14) were used and then the culturing was performed for 36 hours. After the culturing, cells were fixed using 3.7% (v/v) formaldehyde. A per- meabilization was made in the fixed cells using 0.1% (v/v) TritonX-lOQ and then the resultant cells were treated with 2 ⁇ g/ml of Phalloidin-FITC (Sigma Chem. co., U.S.A) at room temperature for 1.5 hours. Then, actin ring formation was identified using a fluorescence microscope.
- FIG. 6 are images illustrating that a cell permeable RANK inhibiting peptide inhibits an actin ring formation in osteoclast. Referring to FIG. 6, when treated with a control group, complete actin rings were formed and sustained. On the other hand, when treated with a RANK inhibiting peptide (SEQ D NO: 8 , incomplete and abnormal actin rings were formed.
- a RANKL or LPS-induced bone destruction inhibition effect was identified using a mouse. Specifically, the calvaria of a 7 week-old mouse was treated only with 10 mg/ kg of a control group peptide, the calvaria of a 7 week-old mouse was treated with 12.5 mg/kg of LPS and thenl ⁇ mg/kg of a control group peptide, and the calvaria of a 7 week-old mouse was treated with 2mg/kg of RANKL and then 10 mg/kg of a control group peptide (3 control group experiments).
- the calvaria of a 7 week-old mouse was treated only with 10mg/kg of a RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NO: 8
- the calvaria of a 7 week-old mouse was treated with 12.5 mg/kg of LPS and then 10mg/kg of a RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NO: 8
- the calvaria of a 7 week-old mouse was treated with 2mg/kg of RANKL and then 10 mg/kg of a RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NO: 8 (3 experiment groups).
- the treatments according each of the six separate experiment group were conducted every day for four days. On the fifth day, the calvarias of the mice were collected and sections thereof were stained with H&E. A degree of bone destruction inhibition was identified using an image-pro Plus4.5 (Media Cybernetics, Inc.).
- FIG. 7 are images illustrating RANKL or LPS injection-induced bone destruction inhibition effect of a cell permeable RANK inhibiting peptide in a mouse (A) and a graph showing degree of bone destruction inhibition in a quantification form (B).
- a polypeptide having a RANK inhibition activity according to the present invention has a RANK inhibition activity, so that osteoclast differentiation due to RANKL(RANK-ligand) can be inhibited, bone destruction due to osteoclast activated by RANKL can be inhibited, cell fusion of osteoclast precursors due to RANKL can be inhibited, actin protein ring-formation in osteoclast due to RANKL can be inhibited, bone destruction due to RANKL injected to a mouse can be inhibited, and bone destruction due to lipopolysaccharide (LPS) injection can be inhibited.
- LPS lipopolysaccharide
- a pharmaceutical composition according to the present invention can be used to treat or prevent a disease caused by osteoclast differentiation and osteoclast activation.
- a method of inhibiting osteoclast differentiation of an individual according to the present invention can inhibit osteoclast differentiation of an individual.
- a disease caused by osteoclast differentiation and osteoclast activation occurring in an individual can be treated or prevented using a method of treating or preventing a disease according to the present invention .
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Provided are a peptide inhibitor having a RANK inhibiting activity, a pharmaceutical composition including the polypeptide, a method of inhibiting osteoclast differentiation in an individual using the pharmaceutical composition, a method of treating or preventing a disease caused by osteoclast differentiation and osteoclast activation, a nucleic acid encoding the polypeptide, a vector including the nucleic acid, and a host cell transformed with the vector.
Description
Description
PEPTIDE INHIBITORS OF RECEPTOR ACTIVATOR OF NF- kB, AND PHARMACEUTICAL COMPOSITION INCLUDING
THE SAME
Technical Field
[1] The present invention relates to peptide inhibitors having receptor activator of NF- k
B (RANK) inhibiting activity, a pharmaceutical composition including the peptides, a method of inhibiting osteoclast differentiation in an individual using the pharmaceutical composition, a method of treating or preventing diseases caused by osteoclast differentiation and activation using the pharmaceutical composition, a nucleic acid encoding the peptides, a vector including the nucleic acid, and a host cell transformed with the vector. Background Art
[2] The skeleton functions to support the human body and protects body organs.
Moreover, the skeleton stores calcium, a critical ion for a variety of metabolic processes, and it is the site of hematopoiesis. The skeleton is a dynamic organ that is constantly remodeling. To accomplish this function there must be a tight coupling between bone resorption by osteoclasts and bone formation by osteoblasts.
[3] Osteoblasts are derived from a mesenchymal progenitor cell that is multipotential and can also differentiate into adipocytes and stromal cells. Osteoblasts initially produce an osteoid matrix that is calcified extracellularly. The major structural protein and mineral crystal of bone are type I collagen and hydroxyappatite, which is a calcium-phosphate salt, respectively. Osteoclasts are specialized multinucleated giant cells that resorb bone. They are hematopoietic in origin and derived from myeloid precursors that also gives rise to macrophages.
[4] Researchers of Imunex Co. have initially discovered a member of a tumor necrosis factor (TNF) receptor family, named as receptor activator of NF- k B (RANK) through a large-scale analysis of genes expressed in dendritic cells. Furthermore, RANK ligand (RANKL) was identified as a ligand of RANK, capable of supporting survival of dendritic cells.
[5] Investigators studying bone biology have long-lasting demanding problems, one of which is to find a factor of osteoclast differentiation. In the present application, the term 'osteoclast differentiation' referst to a processes whereby bone marrow-derived monocyte/macrophages (osteoclast precursors) acquire the specialized features of a os-
teoclast cell.' At the beginning of the 1990s, a Japanese Suda group found that osteoclast differentiation occurs due to stimulation of vitamin D3 and prostaglandin E2 when osteoblasts which are separated from calvaria and an osteoclast precursor are co- cultured. Such results led to an assumption that the factor of osteoclasts differentiation can be derived from osteoblasts. Meanwhile, in 1997, researchers of U. S Amgen Co. identified a novel TNF receptor family, osteoprotegerin (OPG) as a soluble decoy-like factor, capable of inhibiting osteoclast differentiation in vitro. Then, they over- expressed OPG in a mouse and found that bone density was increased. They also cloned a ligand of OPG named as OPGL. The OPGL was the same cytokine as RANKL that has previously cloned by researchers of Immunex Co. Accordingly, osteoclast differentiation can be achieved using an OPGL treatment even when osteoclast precursors exist alone not when osteoblasts and osteoclast precursors are co-cultured.
[6] Experiments on RANKL which is a factor of osteoclast differentiation were performed using a knock-out mouse, and as a result, serious osteopetrosis occurred, tooth eruption did not occur, and growth of the knock-out mouse was inhibited. Such results can also be obtained using a knock-out mouse of a RANKL receptor, that is, RANK, which demonstrates an essential role of an interaction between RANKL made in osteoblast/stromal cells and a receptor (RANK) existing in an osteoclast precursor in osteoclast differentiation.
[7] RANK, the signaling receptor for RANKL, belongs to a TNF receptor family that is similar to CD40. RANK is expressed in the spleen, lymph nodes, dendritic cells, activated T lymphocyte, osteoclast precursors, mature osteoclast, and chondrocyte. Since there is no known signaling motif in a RANK cytoplasmic tail domain, it can be assumed that an adaptor protein may be involved in signal delivering, and in fact, a TRAF (TNF receptor-associated factor) family protein is bound, specifically, TRAF6 plays an important role in delivering an osteoclast differentiation signal to the downstream of the receptor.
[8] Bone remodeling based on balance between bone-resorbing osteoclasts and bone- forming osteoblasts loses its homeostasis due to aging, hormonal disorders, or inflammation. As a result, in the case of adults in their 40's and over, bone destruction dominates over bone formation, and in the case of post-menopause females, one third of them experiences serious osteoporosis because an estrogen hormone is not secreted any longer. In aάϊtion, it is generally known that chronic inflammation resulting from disorders of the immune system is a primary cause of rheumatoid arthritis causing destruction of cartilage and bones. Bone loss (destruction) occurring in these disorders
described above is mainly caused by osteoclast, and RANKL and a receptor of RANKLJ that is, RANK, necessarily operates in osteoclast differentiation. Accordingly, the key strategy in developing a therapeutic agent for bone disorders such as arthritis and osteoporosis is to find a method of regulating osteoclast differentiation.
[9] US Amgen Co. have developed Denosumab that is a humanized monoclonal antibody with respect to RANKL and is now performing clinical trials on bone loss caused by osteoporosis, arthritis, or hormone disorders or bone destruction caused by metastatic breast cancer. In aάϊtion, US Patent No. 6,998,383 discloses a polypeptide inhibiting signaling pathway mediated by TRAF6 (TNF receptor- associated factor 6) including a TRAF6 binding domain.
[10] Inventors of the present invention provide a method of regulating osteoclast differentiation by development of peptide inhibitors targeting RANK that is essential for osteoclasts differentiation and activation. Such peptides can inhibit osteoclast differentiation induced by RANKL, inhibit bone destruction due to osteoclasts activated by RANKL, inhibit cell fusion of osteoclast precursors due to RANKL, inhibit actin ring- formation in osteoclast due to RANKL, inhibit bone destruction due to RANKL injected to a mouse, and inhibit bone destruction due to lipopolysaccharide (LPS) injection.
Disclosure of Invention Technical Problem
[11] The present invention provides novel RANK inhibitor peptides.
[12] The present invention also provides a pharmaceutical composition for treating or preventing diseases caused by bone destruction, including the RANK inhibiting peptide.
[13] The present invention also provides a method of inhibiting osteoclast differentiation in an individual using the pharmaceutical composition, and a method of treating and preventing diseases caused by osteoclast differentiation and osteoclast activation using the pharmaceutical composition.
[14] The present invention also provides a nucleic acid encoding the RANK inhibiting peptide, a vector including the nucleic acid, and a host cell transformed with the vector. Technical Solution
[15] The present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown.
[16] According to an aspect of the present invention, there is provided a polypeptide
having a RANK (receptor activator of NF- k B) inhibiting activity, comprising a 6-12 contiguous amino acid sequence selected from an amino acid sequence set forth in SEQ D NO: 1 and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1.
[17] The polypeptide having RANK inhibiting activity may be selected from the group consisting of polypeptides having amino acid sequences set forth in SEQ D NOS: 1-4.
[18] The polypeptides having the amino acid sequences set forth in SEQ D NOS: 1-4 includes 12 amino acids, 10 amino acids, 8 amino acids, and 6 amino acids, respectively. Each polypeptide includes 4-9 positions (HVVYV: SEQ D NO: 4) of the amino acid sequence set forth in SEQ D NO: 1. As described in Examples of the present application, polypeptides having an activity inhibiting an RANK-induced activity commonly have IIVVYV (SEQ D NO: 4) domains (for example, SEQ D NOS: 1-4, and SEQ D NOS: 8-11). Meanwhile, an IVVYV (SEQ D NO: 5) sequence composed of five amino acids and an IIVVY (SEQ D NO: 6) sequence composed of five amino acids prepared by removing a single amino acid from the IIVVYV (SEQ D NO: 4) domain, and polypeptides (SEQ D NOS: 12 and 13) respective prepared by connecting a cell permeable sequence to IVVYV (SEQ D NO: 5) sequence and IIVVY (SEQ D NO: 6) sequence do not have an activity inhibiting an RANK-induced activity. In aάϊtion, polypeptides (SEQ D NOS: 14-19) prepared by substituting IV that are 2 and 3 positions of the IIVVYV (SEQ D NO: 4) domain from the IIVVYV (SEQ D NO: 4) domain or an amino acid sequence having the IIVVYV (SEQ D NO: 4) domain to LA do not have an activity inhibiting an RANK-induced activity either. Accordingly, a polypeptide according to the present invention can be any polypeptide having a 6-12 contiguous amino acid sequence selected from the amino acid sequence set forth in SEQ D NO: 1 and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1. Although Examples of the present application disclose only polypeptides respectively composed of 6,8,10 and 12 amino acids, a person having ordinary skill in the art may easily understand that a polypeptide having a 6-12 contiguous amino acid sequence selected from an amino acid sequence set forth in SEQ D NO: 1 and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1, which is not disclosed in Examples of the present application, also has the same activity as the polypeptides disclosed in Examples of the present application.
[19] The polypeptide having a RANK inhibiting activity according to the present invention may or may not be connected to a cell permeable polypeptide. The cell permeable polypeptide may be connected to an N-terminal or C-terminal of the
polypeptide having a RANK inhibiting activity, for example, the cell permeable polypeptide may be connected to an N-terminal of the polypeptide having a RANK inhibiting activity.
[20] In the present application, the term 'cell permeable polypeptide' refers to a polypeptide having a cell permeable capability to allow a fusion partner with which the polypeptide is fused to enter inside of a cell. Such a cell permeable polypeptide is well known in the art, and can be used in embodiments of the present invention, for example, the cell permeable polypeptide can be a leader signal sequence. The leader signal sequence may be selected from the group consisting of Kaposi fibroblast growth factor signal sequence, HIV-I tat (48-60), D-amino acid-substituted HIV-I tat (48-60), arginine-substituted HIV-I tat (48-60), Drosophila Antennapaedia(43-58), 7 or more amino acid-containing virus RNA binding peptide, 7 or more arginine-containing DNA binding peptide, and 6-8 arginines -containing polyarginine polypeptide. However, the leader signal sequence is not limited thereto. The virus RNA binding peptide may be selected from the group consisting of of HIV-I rev (34-50), HTLV-II rev (4-16), a brome mosaic virus Gag (7-25) and a flock house virus coat protein (35-49). The DNA binding peptide may be selected from the group consisting of human c-Ibs (139-164), human c-Jun (252-279), and a yeast transcription factor GCN4 (231-252).
[21] In the present application, the term 'peptide' or 'polypeptide' are used interchangeably and refers to a polypeptide having two or more amino acids.
[22] According to the present invention, the cell permeable polypeptide can be a human transcription factor Hph-1 having an amino acid sequence set forth in SEQ D NO: 7.
[23] A polypeptide having an RANK inhibiting activity according to the present invention may have such a structure that a human transcription factor Hph-1 set forth in SEQ D NO: 7 that is a cell permeable polypeptide is connected to an N-terminal of a polypeptide having an RANK inhibiting activity having a 6-12 contiguous amino acid sequence selected from an amino acid sequence set forth in SEQ D NO: 1 and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1. R>r example, the polypeptide having an RANK inhibiting activity according to the present invention may be selected from the group consisting of polypeptides having amino acid sequences set forth in SEQ D NOS: 8-11.
[24] According to another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing a disease caused by osteoclast differentiation and osteoclast activation, comprising the polypeptide having a RANK in-
hibiting activity according to the present invention.
[25] A polypeptide having a RANK inhibiting activity according to the present invention has been described above. The polypeptide is derived from a RANK cytoplasmic tail domain of a human or mouse. The polypeptide may include an HVVYV (SEQ D NO: 4) sequence of 546-551 of a human RANK amino acid sequence. A pharmaceutical composition according to the present invention inhibits osteoclast differentiation due to RANKLJ inhibits bone destruction due to osteoclast activated by RANKLj inhibits cell fusion of osteoclast precursors due to RANKL, inhibits actin ring-formation in osteoclast due to RANKL, inhibits bone destruction due to RANKL injected to a mouse, and inhibits bone destruction due to lipopolysaccharide (LPS) injection. Accordingly, a pharmaceutical composition according to the present invention can be used for treating or preventing a disease caused by osteoclast differentiation and osteoclast activation.
[26] A pharmaceutically acceptable carrier can be a diluting agent, an excipient, a dis- integrant, a binder, or a lubricant, but is not limited thereto, for example, when the pharmaceutical composition according to the present invention is used through an injection, the carrier can be a sterilized water or a buffer suitable for physiological conditions, such as PBS. The pharmaceutical composition can be formulated to be used for oral administration or non-oral administration. In an example embodiment of the present invention, the carrier can be a material facilitating the transport of the RANK inhibiting peptides across the cellular barrier such as, cell membrane, a cell wall, organellar membrane, and the like. The term 'pharmaceutically acceptable' refers to a condition that when the pharmaceutical composition is administrated to an individual, allergies or unwanted reactions do not occur. A method of preparing a protein-containing aqueous composition that is an active component is well known in the art.
[27] Any person having ordinary skill in the art may easily determine a suitable amount of the pharmaceutical composition according to the present invention to be administered and a suitable administration method without undue experimentation. When the pharmaceutical composition is used in vivo for treatments, a therapeutically effective dose of the pharmaceutical composition, that is, a suitable amount of the pharmaceutical composition inhibiting RANKL-mediated osteoclast differentiation or osteoclast activity is administered to an animal or human.
[28] The amount of the pharmaceutical composition according to the present invention may be in the range from 1 nM to 1 M per individual, preferably from 1 μM to 1 mM, and more preferably from 0. ImM to 0.2mM, based on the amount of the polypeptide
having an RANK inhibiting activity that is an active component. However, the amount of the pharmaceutical composition is not limited thereto. The pharmaceutical composition can be formulated to be used for oral administration or non-oral administration, for example, the pharmaceutical composition can be administrated to an individual through an injection administration.
[29] A disease caused by osteoclast differentiation and osteoclast activation can be selected from the group consisting of osteoporosis, arthritis, bone destruction caused by cancers, metabolic bone diseases, leukemia, and multiple myeloma. However, the disease is not limited thereto.
[30] The polypeptide according to the present invention may be encapsulated by a liposome or exists in a form of a virus or vector comprising a gene encoding the polypeptide.
[31] According to another embodiment of the present invention, there is provided a method of inhibiting osteoclast differentiation of an individual, comprising administering the pharmaceutical composition according to the present invention to an individual so as to inhibit a RANK-meditated signal delivery.
[32] The pharmaceutical composition according to the present invention used in the method, may have the same pharmaceutical effect, be used in the same administration dose, and be used using the same administration method, as the pharmaceutical composition according to the present invention described above.
[33] Since the pharmaceutical composition can inhibit a RANK-mediated signal delivery, osteoclast differentiation due to RANKL (RANK ligand) is inhibited, bone destruction due to osteoclast activated by RANKL is inhibited, cell fusion of osteoclast precursors due to RANKL is inhibited, actin ring-formation in osteoclast due to RANKL is inhibited, bone destruction due to RANKL injected to a mouse is inhibited, and bone destruction due to lipopolysaccharide (LPS) injection is inhibited. Accordingly, the inhibition of RANK-mediated signal delivery may result in treatment and prevention of diseases caused by osteoclast differentiation and osteoclast activation.
[34] According to another embodiment of the present invention, there is provided a method of treating or preventing a disease caused by osteoclast differentiation and osteoclast activation, comprising administering the pharmaceutical composition according to the present invention to an individual so as to inhibit the RANK- meditated signal delivery.
[35] The disease can be selected from the group consisting of osteoporosis, arthritis, bone destruction caused by cancers, metabolic bone diseases, leukemia, and multiple
myeloma. However, the disease is not limited thereto. [36] According to another embodiment of the present invention, there is provided a nucleic acid encoding the polypeptide having a RANK inhibiting activity according to the present invention. [37] According to another embodiment of the present invention, there is provided a vector including the nucleic acid encoding the polypeptide having a RANK inhibiting activity according to the present invention. [38] According to another embodiment of the present invention, there is provided a host cell transformed with the vector including the nucleic acid encoding the polypeptide having a RANK inhibiting activity according to the present invention. The host cell can be a prokaryote cell or a eukaryotic cell.
Advantageous Effects [39] A polypeptide having a RANK inhibition activity according to the present invention has a RANK inhibition activity, so that osteoclast differentiation due to
RANKL(RANK-ligand) can be inhibited, bone destruction due to osteoclast activated by RANKL can be inhibited, cell fusion of osteoclast precursors due to RANKL can be inhibited, actin protein ring-formation in osteoclast due to RANKL can be inhibited, bone destruction due to RANKL injected to a mouse can be inhibited, and bone destruction due to lipopolysaccharide (LPS) injection can be inhibited. [40] A pharmaceutical composition according to the present invention can be used to treat or prevent a disease caused by osteoclast differentiation and osteoclast activation. [41] A method of inhibiting osteoclast differentiation of an individual according to the present invention can inhibit osteoclast differentiation of an individual. [42] A disease caused by osteoclast differentiation and osteoclast activation occurring in an individual can be treated or prevented using a method of treating or preventing a disease according to the present invention .
Description of Drawings [43] The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which: [44] FIG. 1 illustrates domains showing a RANK inhibiting activity in RANK cytoplasmic tail regions of human and mouse (A) and fusion peptides connected to a cell permeable sequence (B). [45] FIG. 2 are images illustrating osteoclasts stained with a tartrate resistant acid phosphatase (TRAP) showing an effect of a RANK inhibiting peptide according to a
concentration of the RANK inhibiting peptide when an osteoclast precursor separated from a mouse bone marrow cell is treated with RANKL to cause osteoclast differentiation (A) and a graph showing degree of differentiation (B). A degree of differentiation was obtained by performing three separate experiments to obtain the number of osteoclasts containing over three nuclei and then measuring a mean value thereof.
[46] FIG. 3 is a view illustrating test group fusion peptides 1-6 (A) and control group fusion peptides 1-6 (B);
[47] FIG. 4 are images of osteoclasts stained with TRAP showing a degree of osteoclast differentiation according to fusion peptides 1-6 of a test group and fusion peptides 1-6 of a control group on (A) and a graph showing degree of differentiation (B). In order to obtain a degree of differentiation, a sample was treated with fusion peptides 1-6 and each experiment was performed three times. The number of osteoclast having a complete actin protein ring in the sample was measured and then a mean value thereof was obtained. The average variation of the obtained mean values is shown in a bar diagram.
[48] FIG. 5 are images illustrating that a bone absorption activity of an activated osteoclast is inhibited by a cell permeable RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NOS: 8 and 14(A) and a graph showing degree of differentiation in quantification for the peptide having an amino acid sequence set forth in SEQ D NO: 8 and 14 (B). A degree of absorption was obtained by measuring a ratio of an absorbed area ratio to the entire area of OAAS™ (Oscotec Inc.: Korea) that is a cell culture container in three separate experiments and then obtaining a mean value of the three experimental results and a standard deviation thereof. In FIG. 5, C denotes a control group, Mt denotes a peptide having an amino acid sequence set forth in SEQ D NO: 14, and WTdenotes a having an amino acid sequence set forth in SEQ D NO: 8.
[49] FIG. 6 are images illustrating a cell permeable RANK inhibiting peptide inhibiting an actin ring formation in osteoclasts. In FIG. 6, Mtand WT denote peptides having an amino acid sequence of SEQ D NO: 14 and SEQ D NO: 8, respectively; and
[50] FIG. 7 are images illustrating RANKL or LPS injection-induced bone destruction inhibition effect of a cell permeable RANK inhibiting peptide in a mouse (A) and a graph showing degree of bone destruction inhibition in quantification form (B). Specifically, a ratio of an absorbed area to the entire area in a sectional view of the calvaria was measured to quantify the degree of bone destruction inhibition. The absorbed area refers to a bone marrow cavity in calvaria, and an increase or decrease in
the absorbed area was compared to the control group to quantify the degree of bone destruction inhibition. An average value was obtained from six kinds of samples and a standard deviation thereof was obtained. The relative absored area of control group was set to 1. When bone marrow cavity destruction caused by RANKL or LPS injection was increased, the relative absored area of the bone marrow cavity was greater than the control group 1. However, when a RANKL inhibiting peptide treatment was performed, the relative absored area of the bone marrow cavity was decreased to the level of the control group. Best Mode
[51] The present invention will be described in further detail with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
[52] Example
[53] Example 1: Preparation of Fusion Polypeptide Including Cell Permeable Sequence and RANK Cytoplasmic Tail Domain Sequence
[54] Herein, a fusion polypeptide composed of 11 amino acids in which a human transcription factor Hph-1 that has a cell permeable sequence YAR VRRRGPRR (SEQ D NO: 7) is connected to an N-terminal of a human RANK cytoplasmic tail domain- derived polypeptide. The human RANK cytoplasmic tail domain-derivd polypeptide is a polypeptide fragment having a 5-12 contiguous amino acid sequence selected from an amino acid sequence set forth in SEQ D NO: 1 that has an amino acid sequence of KGDIIVVYVSQT corresponding to an amino acid sequence 543-554 of a human RANK (corresponding to an amino acid sequence 531-542 of a mouse RANK) and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1. Polypeptide fragments used in the current example have amino acid sequences set forth in SEQ D NOS: 1-6, and fusion polypeptides (hereinafter referred to as test group fusion peptides 1-6) in which a cell permeable sequence set forth in SEQ D NO: 7 is connected to an N-terminal of each of the polypeptide fragments, have amino acid sequences set forth in SEQ D NOS: 8-13. As for a control group, an IV sequence of the amino acid sequences set forth in SEQ D NOS: 8-13 was substituted with a LA sequence to prepare polypeptides having amino acid sequences set forth in SEQ D NOS: 14-19 (hereinafter referred to as control group fusion peptides 1-6). The amino acid sequences of the test group and control group polypeptide are shown in Table 1.
[55] Table 1.
[Table 1] [Table ]
[56] The polypeptides shown in Table 1 were synthesized by Peptron, Inc. (Korea).
[57] FIG. 1 illustrates domains showing RANK inhibiting activity in RANK cytoplasmic tail regions of humans and mouse (A) and fusion peptides connected to a cell permeable sequence (B).
[58] Example 2: Osteoclast Differentiation Inhibition Effect of Cell Permeable RANK Inhibiting Peptide
[59] A bone marrow cell collected from a five- week old mouse (C57BL/6J) was loaded onto a 48-well plate at a density of 4xlO4 cells/well, and then cultured for three days using 30 ng/ml of hM-CSF (R&D system Inc.) that is a macrophage differentiation factor-containing differentiation medium (α-MEM (Invitrogen. co.,), 10%(v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 100 βg /ml streptomycin) at 370C under 95%(v/v) air and 5% (v/v) CO2. As a result, a macrophage derived from a bone marrow cell was obtained. Then, in addition to 30 ng/ml of hM-CSF, 100 ng/ml of a mouse RANKL extracellular domain fragment (position 150 to 316) that is an active osteoclast differentiation factor comparable to a full length mouse RANKL (SEQ D NO: 22) (Lacey et al. Cell, 93: 165, 1998) which had been expressed in an E.coli and then purified was added to the obtained macrophage cell and the resultant macrophage cell was cultured in the same conditions as described above to cause osteoclast differ-
entiation. The 158aa mouse RANKL extracellular domain fragment was cloned into Sail-Not I site of pET28b(+) vector (Novagen), which is a commonly used bacterial expression vector and containing a His tag sequence for the purification of the expressed protein.
[60] At this time, the test group fusion peptide 1 according to the present invention and the control group fusion peptide 1 were respectively added thereto while the concentration of the test group fusion peptide 1 and the control group fusion peptide 1 differed at lμM, 2.5μM, and 5μM and the culturing was performed. During experiments, the differentiation medium was exchanged every other day. As a result, osteoclast differentiation occurred when the control group fusion peptide 1 was used, and osteoclast differentiation did not occur when the test group fusion peptide 1 was used.
[61] FIG. 2 are images illustrating osteoclast stained with tartrate resistant acid phosphatase (TRAP) showing an effect of a RANK inhibiting peptide according to a concentration of the RANK inhibiting peptide when an osteoclast precursor separated from a mouse bone marrow cell is treated with RANKL to cause osteoclast differentiation (A) and a graph showing degree of differentiation (B). FIG. 2 shows results of such an experiment that bone marrow -derived macrophage/monocyte cells (osteoclast precursors) were cultured using the test group fusion peptide 1 and the control group fusion peptide 1 for three days, respectively, and then on the fourth day, the cultured product was stained with a TRAP and identified using an optical microscope. Specifically, the TRAP staining was performed such that the cultured cell was fixed using 3.7% formaldehyde for 15 minutes at room temperature, washed twice, and then a staining solution prepared by mixing acetate, a fast gargnet GBC base, a naphthol AS-BI phosphate, sodium nitrite, and tartrate in a mixture ratio described in the specification of an acid phosphatase, Leukocyte (TRAP) kit™ (Sigma Co.) was added to a 200 μJl /well and then reacted together at 370C for 20 minutes.
[62] Example 3: Osteoclast Differentiation Inhibition Effect of Shortened Cell Permeable
RANK Inhibiting Peptide
[63] The current experiment was performed to identify a minimum active domain of a
RANK inhibiting peptide. Specifically, an osteoclast differentiation inhibition effect was identified using test group fusion peptides 1-6 having amino acid sequences set forth in SEQ D NO: 8 to 13 in which some amino acids were removed. At this time, control group fusion peptides 1-6 having amino acid sequences set forth in SEQ D NO: 14-19 were used as a control group.
[64] Osteoclast differentiation inhibition experiments were performed in the same manner
as in Example 2, except that the concentration of each of the test group fusion peptides 1-6 and control group fusion peptides 1-6 was 5μM.
[65] FIG. 3 is a view illustrating test group fusion peptides 1-6 and control group fusion peptides 1-6.
[66] FIG. 4 are images of osteoclasts stained with TRAP showing an osteoclast differentiation inhibiting effect of the test group fusion peptides 1-6 and the control group fusion peptides 1-6 (A) and a graph showing degree of differentiation (B).
[67] Referring to FIG. 4, it was identified that a fragment having a 6-12 contiguous amino acid sequence and an amino acid sequence of HVVYV (SEQ D NO: 4) is as small a fragment of RANK inhibiting motif having an amino acid sequence set forth in SEQ D NO: 1 as possible to have an osteoclast differentiation inhibition activity. On the other hand, polypeptides having amino acid sequences set forth in SEQ D NOS: 5 and 6 prepared by respectively removing I at an N-terminal and V at a C-terminal of the IIVVYV (SEQ D NO: 4) sequence did not show an osteoclast differentiation inhibition activity.
[68] Example 4: Osteoclast Bone Absorption Inhibition Effect of Cell Permeable RANK
Inhibiting Peptide
[69] The osteoclast bone absorption inhibition effect was identified using OAAS ™
(Oscotec Inc.: Korea) that is a cell culture container coated with calcium phosphate nanocrystal for measuring a degree of bone absorption.
[70] A bone marrow cell collected from a five- week old mouse (C57BL/6J) was loaded onto a OAAS™ 48-well plate at a density of 4xlO4 cells/well, and then cultured for three days using 30 ng/ml of hM-CSF (R&D system Inc.) that is a macrophage differentiation factor-containing differentiation medium (α-MEM (Invitrogen. co.), 10%(v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 100 βg /ml streptomycin) at 370C under 95%(v/v) air and 5% (v/v) CO2. As a result, a macrophage derived from a bone marrow cell was obtained. Then, in aάϊtion to 30 ng/ml of hM- CSF, 100 ng/ml of a mouse RANKL that is an osteoclast differentiation factor which had been expressed in an E.coli and then purified was added to the obtained macrophage cell and the resultant macrophage cell was cultured in the same conditions as described above to cause osteoclast differentiation.
[71] After osteoclast differentiation was caused in the 48-well plate, the medium used was altered to a new differentiation medium. At this time, the osteoclast cell was treated with each of polypeptides having amino acid sequences set forth in SEQ D NOS: 8 and 14 having a concentration of 5 μM and then bone absorption was identified for 48
hours. Then, the OAAS™ plate was washed using distilled water to remove cells attached to the OAAS™ plate after the cell culturing and then a 4-5% sodium hypochlorite solution was added thereto and the resultant solution was left to sit for 5 minutes. Then, the resultant OAAS™ plate was washed and dried, and bone absorption was identified. The osteoclast absorption and tissue measurement were identified on the basis of images obtained using Axiovert 200 (Carl Zeiss, Gottingen, Germany), that is an optical microscope including a CCD camera therein. The obtained images were analyzed using Image-pro Plus4.5 (Media Cybernetics, Inc.) that is a surface measuring program.
[72] FIG. 5 are images illustrating that a bone absorption activity of an activated osteoclast is inhibited by a cell permeable RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NOS: 8 and 14 (A) and a graph showing degree of differentiation in quantification for the peptide having an amino acid sequence set for th in SEQ D NO: 8 and 14(B).
[73] Referring to FIG. 5, when the osteoclast cell was treated with a RANK inhibiting peptide, bone absorption activity of osteoclast was decreased by 88% compared to when the osteoclast cell was treated with a control group. Such results show that the RANK inhibiting peptide inhibits, in addition to osteoclast differentiation, sustaining and functioning of a complete osteoclast.
[74] Example 5: Actin Ring -Formation Inhibition Effect of Cell Permeable RANK inhibiting peptide
[75] In order to identify an actin ring-formation inhibition effect of a complete osteoclast, osteoclast differentiation was caused in the 48-well plate as described in Example 2 and then, RANKL was not supplied for 6 hours to stop a cell signal delivery due to RANKL.
[76] Then, the RANKL, the RNAK inhibiting peptide (SEQ D NO: 8), and the control group peptide (SEQ D NO: 14) were used and then the culturing was performed for 36 hours. After the culturing, cells were fixed using 3.7% (v/v) formaldehyde. A per- meabilization was made in the fixed cells using 0.1% (v/v) TritonX-lOQ and then the resultant cells were treated with 2μg/ml of Phalloidin-FITC (Sigma Chem. co., U.S.A) at room temperature for 1.5 hours. Then, actin ring formation was identified using a fluorescence microscope.
[77] FIG. 6 are images illustrating that a cell permeable RANK inhibiting peptide inhibits an actin ring formation in osteoclast. Referring to FIG. 6, when treated with a control group, complete actin rings were formed and sustained. On the other hand, when
treated with a RANK inhibiting peptide (SEQ D NO: 8 , incomplete and abnormal actin rings were formed.
[78] Example 6: RANKL or LPS-induced Bone Destruction Inhibition Effect of RANK
Inhibiting Peptide
[79] A RANKL or LPS-induced bone destruction inhibition effect was identified using a mouse. Specifically, the calvaria of a 7 week-old mouse was treated only with 10 mg/ kg of a control group peptide, the calvaria of a 7 week-old mouse was treated with 12.5 mg/kg of LPS and thenlθmg/kg of a control group peptide, and the calvaria of a 7 week-old mouse was treated with 2mg/kg of RANKL and then 10 mg/kg of a control group peptide (3 control group experiments). Separately, the calvaria of a 7 week-old mouse was treated only with 10mg/kg of a RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NO: 8, the calvaria of a 7 week-old mouse was treated with 12.5 mg/kg of LPS and then 10mg/kg of a RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NO: 8, and the calvaria of a 7 week-old mouse was treated with 2mg/kg of RANKL and then 10 mg/kg of a RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NO: 8 (3 experiment groups). The treatments according each of the six separate experiment group were conducted every day for four days. On the fifth day, the calvarias of the mice were collected and sections thereof were stained with H&E. A degree of bone destruction inhibition was identified using an image-pro Plus4.5 (Media Cybernetics, Inc.).
[80] FIG. 7 are images illustrating RANKL or LPS injection-induced bone destruction inhibition effect of a cell permeable RANK inhibiting peptide in a mouse (A) and a graph showing degree of bone destruction inhibition in a quantification form (B).
[81] Referring to FIG. 7, when the cell permeable RANK inhibiting peptide having an amino acid sequence set forth in SEQ D NO: 8 was injected together, bone destruction due to LPS injection was decreased by 52% on average, and bone destruction due to RANKL injection was decreased by 60% on average.
[82] While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims. Industrial Applicability
[83] A polypeptide having a RANK inhibition activity according to the present invention
has a RANK inhibition activity, so that osteoclast differentiation due to RANKL(RANK-ligand) can be inhibited, bone destruction due to osteoclast activated by RANKL can be inhibited, cell fusion of osteoclast precursors due to RANKL can be inhibited, actin protein ring-formation in osteoclast due to RANKL can be inhibited, bone destruction due to RANKL injected to a mouse can be inhibited, and bone destruction due to lipopolysaccharide (LPS) injection can be inhibited.
[84] A pharmaceutical composition according to the present invention can be used to treat or prevent a disease caused by osteoclast differentiation and osteoclast activation.
[85] A method of inhibiting osteoclast differentiation of an individual according to the present invention can inhibit osteoclast differentiation of an individual.
[86] A disease caused by osteoclast differentiation and osteoclast activation occurring in an individual can be treated or prevented using a method of treating or preventing a disease according to the present invention .
Claims
[I] A polypeptide having a RANK inhibiting activity, comprising a 6-12 contiguous amino acid sequence selected from an amino acid sequence set forth in SEQ D NO: 1 and 4-9 positions of the amino acid sequence set forth in SEQ D NO: 1.
[2] The polypeptide of claim 1, wherein the RANK inhibiting activity is selected from the group consisting of polypeptides having amino acid sequences set forth in SEQ D NOS: 1-4.
[3] The polypeptide of claim 1, wherein the polypeptide is connected to a cell permeable polypeptide.
[4] The polypeptide of claim 3, wherein the cell permeable polypeptide is connected to an N-terminal or C-terminal of the polypeptide having a RANK inhibiting activity of claim 1.
[5] The polypeptide of claim 3, wherein the cell permeable polypeptide is a leader signal sequence.
[6] The polypeptide of claim 5, wherein the leader signal sequence is selected from the group consisting of Kaposi fibroblast growth factor signal sequence, HIV-I tat (48-60), D-amino acid-substituted HIV-I tat (48-60), arginine-substituted HIV-I tat (48-60), Drosophila Antennapaedia(43-58), 7 or more amino acid- containing virus RNA binding peptide, 7 or more arginine-containing DNA binding peptide, and 6-8 arginines-containing polyarginine polypeptide.
[7] The polypeptide of claim 6, wherein the virus RNA binding peptide is selected from the group consisting of HIV-I rev (34-50), HTLV-II rev (4-16), a brome mosaic virus Gag (7-25) and a pollok house virus coat protein (35-49).
[8] The polypeptide of claim 6, wherein the DNA binding peptide is selected from the group consisting of human c-R>s (139-164), human c-Jun (252-279), and a yeast transcription factor GCN4 (231-252).
[9] The polypeptide of claim 3, wherein the cell permeable polypeptide is a human transcription factor Hph-1 set forth in SEQ D NO: 7.
[10] The polypeptide of claim 3, wherein the polypeptide is selected from the group consisting of polypeptides having amino acid sequences set forth in SEQ D NOS: 8-11.
[I I] A pharmaceutical composition for treating or preventing a disease caused by osteoclast differentiation and osteoclast activation, comprising the polypeptide having a RANK inhibiting activity of any one of claims 1 through 10 and a phar-
maceutically acceptable carrier.
[12] The composition of claim 11, wherein the disease is selected from the group consisting of osteoporosis, arthritis, bone destruction caused by cancers, metabolic bone diseases, leukemia, and multiple myeloma.
[13] The composition of claim 11, wherein the polypeptide is encapsulated by a liposome or exists in a form of a virus or vector comprising a gene encoding the polypeptide.
[14] A method of inhibiting osteoclast differentiation of an individual, comprising administering the pharmaceutical composition of claim 11 to an individual so as to inhibit a RANK-meditated signal delivery.
[15] A method of treating or preventing a disease caused by osteoclast differentiation and osteoclast activation, comprising administering the pharmaceutical composition of claim 11 to an individual so as to inhibit a RANK-meditated signal delivery.
[16] The method of claim 15, wherein the disease is selected from the group consisting of osteoporosis, arthritis, bone destruction caused by cancers, metabolic bone diseases, leukemia, and multiple myeloma.
[17] A nucleic acid encoding the polypeptide having a RANK inhibiting activity of any one of claims 1 through 10.
[18] A vector comprising the nucleic acid of claim 17.
[19] A host cell transformed with the vector of claim 18.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020070037155A KR100885545B1 (en) | 2007-04-16 | 2007-04-16 | A polypeptide inhibiting RANK, pharmaceutical composition comprising the same, method of inhibiting osteoclast differentiation, a method of treating or preventing a disease caused by differentiation or activation of osteoclast, nucleic acid encoding the polypeptide, vector comprising the nucleic acid and a host cell transformed with the vector |
| KR10-2007-0037155 | 2007-04-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2008127066A1 true WO2008127066A1 (en) | 2008-10-23 |
Family
ID=39864115
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2008/002133 WO2008127066A1 (en) | 2007-04-16 | 2008-04-16 | Peptide inhibitors of receptor activator of nf-kb, and pharmaceutical composition including the same |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR100885545B1 (en) |
| WO (1) | WO2008127066A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10030050B2 (en) | 2014-07-31 | 2018-07-24 | Caregen Co., Ltd. | Peptide having osteoclast differentiation and activation inhibition, and use of same |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101510743B1 (en) | 2013-07-23 | 2015-04-10 | (주)케어젠 | Peptides Having an Inhibitory Activity on Osteoclast Differentiation and Uses Thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007016606A2 (en) * | 2005-08-02 | 2007-02-08 | The Uab Research Foundation | Compositions and methods for modulating rank activities |
-
2007
- 2007-04-16 KR KR1020070037155A patent/KR100885545B1/en not_active IP Right Cessation
-
2008
- 2008-04-16 WO PCT/KR2008/002133 patent/WO2008127066A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007016606A2 (en) * | 2005-08-02 | 2007-02-08 | The Uab Research Foundation | Compositions and methods for modulating rank activities |
Non-Patent Citations (2)
| Title |
|---|
| KANAZAWA K. AND KUDO A.: "Self-Assembled RANK Induces Osteoclastogenesis Ligand-Independently", J. BONE & MIN. RES., vol. 20, no. 11, 2005, pages 2053 - 2060 * |
| XU D. ET AL.: "A Novel Receptor Activator of NF-kappaB(RANK) Cytoplasmic Motif Plays an Essential Role in Osteoclastogenesis by Commiting Macrophages to the Osteoclast Lineage", J. BIOL. CHEM., vol. 281, no. 8, 2006, pages 4678 - 4690 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10030050B2 (en) | 2014-07-31 | 2018-07-24 | Caregen Co., Ltd. | Peptide having osteoclast differentiation and activation inhibition, and use of same |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20080093334A (en) | 2008-10-21 |
| KR100885545B1 (en) | 2009-02-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DE60131146T2 (en) | MATERIALS AND METHODS CONTAINING HYBRID VASCULAR ENDOTHELIC GROWTH FACTORS DNA AND PROTEINS AND SCREENING METHOD FOR MODULATORS | |
| US7611711B2 (en) | VEGFR-3 inhibitor materials and methods | |
| US20030125537A1 (en) | Vascular endothelial growth factor D(VEGF-D) antibodies and vectors, and methods of use | |
| JP5854283B2 (en) | Cell growth promoting peptide and use thereof | |
| PT975754E (en) | Osteoprotegerin binding proteins and receptors | |
| AU2002225265A1 (en) | VEGFR-3 Binding peptides and their use for inhibiting angiogenesis | |
| JP6311935B2 (en) | Synthetic peptide that suppresses expression of type 2 TNF receptor and use thereof | |
| JP2015128431A (en) | Cell growth promoting peptide and use thereof | |
| JPWO2007072980A1 (en) | New use of apelin | |
| WO2008127066A1 (en) | Peptide inhibitors of receptor activator of nf-kb, and pharmaceutical composition including the same | |
| Boosani et al. | Validation of Different Systems for Tumstatin Expression and its in-vitro and iv-vivo Activities | |
| KR101637497B1 (en) | Peptide for promoting bone formation and use thereof | |
| JP6655011B2 (en) | Peptide that induces multinucleation of cells and its use | |
| JP7405361B2 (en) | Antitumor peptides and their uses | |
| EP2706113B1 (en) | Synthetic peptide capable of inducing expression of type-2 tnf receptor and use thereof | |
| KR20010043090A (en) | NOVEL POLYPEPTIDE, cDNA ENCODING THE SAME AND UTILIZATION THEREOF | |
| CA3133147A1 (en) | Core master regulators of glioblastoma stem cells | |
| EP2083073B1 (en) | Reagent containing fused protein of soluble rankl with epitope tag | |
| JP4696543B2 (en) | Novel protein, its pharmaceutical use, promoter of gene of the protein and its use | |
| EP2812015B1 (en) | Peptides targeting receptor activator of nuclear factor-kappa b (rank) and their applications | |
| US20050222074A1 (en) | Modulators of bone homeostasis identified in a high-throughput screen | |
| WO2003076467A1 (en) | Compositions and methods for inducing and regulating bone formation | |
| WO2008014410A2 (en) | Zimlig2 polynucleotides and polypeptides and methods of use | |
| KR20010030984A (en) | POLYPEPTIDE, cDNA ENCODING THE SAME, AND USE OF THEM | |
| JP2000186045A (en) | Bone resorption inhibitor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 08741378 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 08741378 Country of ref document: EP Kind code of ref document: A1 |