WO2008044797A1 - Animal modèle d'ostéopénie - Google Patents

Animal modèle d'ostéopénie Download PDF

Info

Publication number
WO2008044797A1
WO2008044797A1 PCT/JP2007/070309 JP2007070309W WO2008044797A1 WO 2008044797 A1 WO2008044797 A1 WO 2008044797A1 JP 2007070309 W JP2007070309 W JP 2007070309W WO 2008044797 A1 WO2008044797 A1 WO 2008044797A1
Authority
WO
WIPO (PCT)
Prior art keywords
bone
rankl
animal
osteopenia
gst
Prior art date
Application number
PCT/JP2007/070309
Other languages
English (en)
Japanese (ja)
Inventor
Yoshiya Tomimori
Hisataka Yasuda
Original Assignee
Oriental Yeast Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/JP2007/063871 external-priority patent/WO2008044379A1/fr
Application filed by Oriental Yeast Co., Ltd. filed Critical Oriental Yeast Co., Ltd.
Priority to US12/445,050 priority Critical patent/US8334261B2/en
Priority to EP11169483.2A priority patent/EP2389803B1/fr
Priority to CA2666443A priority patent/CA2666443C/fr
Priority to EP07830043A priority patent/EP2084961A4/fr
Priority to JP2008538785A priority patent/JP5219823B2/ja
Priority to AU2007307504A priority patent/AU2007307504B2/en
Publication of WO2008044797A1 publication Critical patent/WO2008044797A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the present invention relates to an osteopenia animal model in which the bone mass is reduced as compared to a normal animal.
  • Osteoclasts which control bone destruction, are large multinucleated cells derived from monocyte / macrophage hematopoietic cells.
  • the progenitor cells are differentiated and matured into osteoclasts by being regulated by osteoblasts / stromal cells on the bone surface.
  • the osteoclast differentiation factor (RANKL) belongs to the tumor necrosis factor (TNF) family that is induced on osteoblasts / stromal cells by bone resorption factor. It is a membrane-bound protein and is an essential factor for osteoclast differentiation 'maturation (see Non-Patent Documents 1 and 2). It is known that a part of RANKL is cleaved by meta-oral protease in the extracellular region and becomes soluble.
  • soluble RANKL induces osteoclasts from bone marrow cells, splenocytes, progenitor cells in peripheral blood, or macrophage progenitor cells such as macrophage cell lines in the presence of M-CSF in vitro. It is known to do.
  • Non-patent literature 1 Yasuda et al., Proc Natl Acad Sci USA 95: 3597, 1998
  • Non-patent literature 2 Lacey et al., Cell 93: 165, 1998
  • Non-Patent Document 3 Thompson et al., Bone 17 (Suppl.): S125, 1995
  • Non-Patent Document 4 Wronski et al., Calcif Tissue Int 42: 179, 1988
  • Non-Patent Document 5 Wronski et al., Endocrinology 123: 681, 1988
  • Non-Patent Document 6 Wronski et al., Calcif Tissue Int 45: 360, 1989
  • Non-Patent Document 7 de Winter et al., Calcif Tissue Res 17: 303, 1975
  • Non-Patent Document 8 Geusens et al., J Bone Miner Res 6: 791, 1991
  • Non-Patent Document 9 Wakley et al., Calcif Tissue Int 43, 383, 1988
  • Non-Patent Document 1 0 Globus et al., J Bone Miner Res 1: 191, 1986 Disclosure of Invention
  • An object of the present invention is to provide a method for producing an osteopenia animal model by administering RANKL and an osteopenia animal model.
  • the present inventors have intensively studied a method for producing a bone loss model animal that can solve the problems of conventional bone loss model animals.
  • administration of soluble RANKL or fusion protein of soluble RANKL and Epitope tag directly to non-human animals directly activates osteoclasts and causes bone loss rapidly (within a few days)
  • This osteopenia animal model is effective for rapid drug evaluation.
  • the mechanism of bone loss in the osteopenia model animal of the present invention is extremely simple: RANKL promotes osteoclast differentiation and activation. It is a pure osteoclast inhibitor (bisphosphonate, Cathepsin). It is extremely effective as an evaluation system when developing K inhibitors. It can also be used to evaluate drugs that increase bone mass (such as PTH) by comparing the speed of bone loss once it returns to its original level.
  • the present invention is as follows.
  • Bone mass comprising administering soluble RANKL or a fusion protein of soluble RANKL and an epitope tag to a non-human animal, and promoting osteoclast differentiation and activation in the non-human animal body
  • a method for producing a reduction model animal comprising administering soluble RANKL or a fusion protein of soluble RANKL and an epitope tag to a non-human animal, and promoting osteoclast differentiation and activation in the non-human animal body
  • a bone loss model of [1] or [2] can be produced within one week after administration of soluble RANKL or a fusion protein of soluble RANKL and an epitope tag to a non-human animal.
  • a method for producing a reduction model animal is
  • [8] [1 :! A method for producing an osteopenia animal model according to any one of [7], wherein bone loss of different severity is achieved by changing the dose of soluble RANKL or a fusion protein of soluble RANKL and an epitope tag A method for creating a bone loss model animal that produces a disease model animal.
  • the ovary is removed from an animal to which soluble RANKL or a fusion protein of soluble RANKL and an epitope tag is administered, and the osteopenia model animal of any of [1] to [: 8] Production method.
  • Osteopenia model animal can be created within 72 hours after administration of soluble RANKL or fusion protein of soluble RANKL and epotope tag to non-human animals [9] How to make animals.
  • [1 1] A bone loss model animal produced by any one of the methods [1] to [1 0].
  • At least one of blood estrogen concentration, blood PTH concentration and blood 0PG concentration is not changed compared to normal individuals [1 1] to [1 4] Le animals.
  • Bone resorption inhibitor or a bone resorption inhibitor candidate substance was administered to any bone loss model animal of any one of [1 1] to [1 5] and decreased in the bone loss model animal It is a method for evaluating the effect of the bone resorption inhibitor or the bone resorption inhibitor candidate substance by using whether or not the bone mass increases as an index, and when the bone mass increases, A method for evaluating a bone resorption inhibitor or a bone resorption inhibitor candidate substance to be determined.
  • [1 8] [1 1;] to any one of the osteopenia model animals of [1 5] is administered with an osteogenesis promoter or an osteogenesis promoter candidate substance, and decreases in the osteopenia model animal
  • This is a method for evaluating the effect of the osteogenesis promoting agent or the osteogenesis promoting agent candidate substance by using whether or not the increased bone mass is an index, and is effective in promoting osteogenesis when the bone mass increases.
  • a method for evaluating an osteogenesis promoter or an osteogenesis promoter candidate substance is a method for evaluating the effect of the osteogenesis promoting agent or the osteogenesis promoting agent candidate substance by using whether or not the increased bone mass is an index, and is effective in promoting osteogenesis when the bone mass increases.
  • a hormone or hormone receptor in a bone loss model animal produced by administering a soluble RANKL or a fusion protein of soluble RANKL and an epitope tag by the method according to claim 8 and further extracting the ovary
  • a method for evaluating the effect of the hormone or hormone receptor modulator using the modulator as an indicator of whether or not the decreased bone mass increases in the osteopenia model animal, wherein the bone mass increased A method for evaluating a hormone or a hormone receptor modulator, which is judged to be effective in inhibiting bone resorption.
  • Hormone or hormone receptor modulator is selective estrogen receptor A method for evaluating a [20] honolemon or honolemon receptor modulator, which is a body modulator.
  • [2 2] Increased bone mass, increased bone formation marker level in body, increased bone density, increased bone mass, increased trabecular number, decreased osteoclast number in osteopenia model animals, Evaluate [2 0] or [2 1] hormone or hormone receptor modulators based on at least one selected from the group consisting of an increase in osteoblastic surface and an increase in bone mass observed by CT how to.
  • FIG. 1 is a graph showing the blood Ca concentration in mice administered and not administered with GST-RANKL.
  • FIG. 2 is a graph showing blood CTx concentrations in mice administered and not administered with GST-RANKL.
  • FIG. 3 is a graph showing blood TRAP-5b concentrations in mice administered with and without GST-RANKL.
  • FIG. 4 is a graph showing blood osteocalcin concentrations in mice administered with and without GST-RANKL.
  • FIG. 5 is a graph showing blood ALP concentrations in mice administered with and without GST-RANKL.
  • FIG. 6 is a graph showing femur bone density in mice administered and not administered with GST-RANKL.
  • FIG. 7 is a graph showing the unit bone mass of the tibia in mice administered GST-RANKL and mice not administered.
  • FIG. 8 is a graph showing the number of tibia osteoclasts in mice administered with and without GST-RANKL.
  • Figure 9 shows tibia bones in mice administered and not treated with GST-RANKL. It is a figure which shows the number of powers.
  • FIG. 10 is a photograph showing bone morphology of mice treated with GST-RANKL and measured with a mouth-mouth CT of the femurs of mice not administered with GST-RANKL.
  • FIG. 11 is a diagram showing the osteoblastic surface of the tibia in mice administered with and without GST-RANKL.
  • FIG. 12 is a graph showing blood Ca concentration in mice administered with 213 nmol to 852 nmol of GST-RANKL and in mice not administered with GST-RANKL.
  • Fig. 13 is a graph showing blood CTx concentrations in mice administered with 213mnol to 852nmol of GST-RANKL and in mice not administered with GST-RANKL.
  • FIG. 14 is a graph showing blood TRAP-5b concentrations in mice administered with 213 nmol to 852 nmol of GST-RANKL and mice not administered with GST-RANKL.
  • FIG. 15 is a graph showing femoral bone density in mice administered with 213 nmol to 852 nmol of GST-RANKL and mice not administered with GST-RANKL.
  • FIG. 16 is a photograph showing bone morphology of mice treated with 213 nmol to 852 nmol of GST-RANKL and of the femurs of mice not administered with microscopic CT.
  • FIG. 17 is a graph showing the blood TRAP-5b concentration when risedronate was administered to osteopenia model mice produced by administering GST-RANKL.
  • FIG. 18 is a graph showing the blood CTx concentration when risedronate was administered to osteopenia model mice produced by administering GST-RANKL.
  • FIG. 19 is a graph showing the ALP concentration in blood when risedronate was administered to an osteopenia model mouse produced by administering GST-RANKL.
  • FIG. 20 is a graph showing blood osteocalcin concentration when risedronate was administered to a mouse model for osteopenia produced by administration of GST-RANKL.
  • FIG. 21 is a graph showing the bone density of the femur when risedronate is administered to an osteopenia model mouse produced by administering GST-RANKL.
  • Fig. 22 is a photograph showing the bone morphology measured by micro-CT of the femur of a mouse administered with risedronate to an osteopenia model mouse produced by administering GST-RANKL.
  • Figure 23 shows blood levels in mice administered and not treated with soluble RANKL. It is a figure which shows Ca density
  • FIG. 24 is a graph showing blood CTx concentrations in mice administered and not administered soluble RANKL.
  • FIG. 25 is a graph showing blood TRAP-5b concentrations in mice with and without soluble RANKL.
  • FIG. 26 is a graph showing the blood osteocalcin concentration in mice with and without soluble RANKL.
  • FIG. 27 is a diagram showing blood ALP concentrations in mice with and without soluble RANKL.
  • FIG. 28 is a graph showing femur bone density in mice administered and not administered soluble RANKL.
  • FIG. 29 is a diagram showing unit bone mass in mice administered with GST-RANKL and mice administered with PBS.
  • FIG. 30 is a diagram showing trabecular width in mice administered with GST-RANKL and mice administered with PBS.
  • FIG. 31 shows the number of trabeculae in mice administered with GST-RANKL and mice administered with PBS.
  • FIG. 32 shows the osteoid thickness in mice administered with GST-RANKL and mice administered with PBS.
  • FIG. 33 is a diagram showing the absorption surface in mice administered with GST-RANKL and mice administered with PBS.
  • FIG. 34 is a graph showing the number of osteoclasts in mice administered with GST-RANKL and mice administered with PBS.
  • FIG. 35 is a diagram showing the osteoclast surface in mice administered with GST-RANKL and mice administered with PBS.
  • FIG. 36 is a photograph showing a TRAP-stained image showing an increase in osteoclasts in a mouse administered with GST-RANKL and a mouse administered with PBS.
  • FIG. 37 shows the blood Ca, TRAP-5b and ALP concentrations when risedronate was administered to mice administered with GST-RANKL.
  • FIG. 38 shows the bone density when risedronate was administered to mice administered with GST-RANKL.
  • Fig. 39 is a photograph showing the results of image analysis using a mouth-to-mouth CT when risedronate was administered to mice administered with GST-RANKL.
  • FIG. 40 shows blood Ca, ALP and TRAP-5b concentrations when mice treated with GST-RANKL were treated with etidronate, and mouth-mouth, or risedronate.
  • FIG. 41 is a graph showing bone density when etidronate, and-andoate or risedronate was administered to mice administered with GST-RANKL.
  • Fig. 42 is a photograph showing the results of image analysis using micro-CT when etidronate, and oral mouthnate, or risedronate was administered to mice administered with GST-RANKL.
  • FIG. 43 shows blood Ca, TRAP-5b, and ALP concentrations in mice that had been ovariectomized after administration of GST-RANKL.
  • FIG. 44 is a graph showing the bone density of a mouse administered with GST-RANKL and excised from the ovaries.
  • Fig. 45 is a photograph showing the results of image analysis using micro-CT of mice that had been treated with GST-RANKL and removed their ovaries.
  • FIG. 46 is a graph showing blood Ca, ALP and TRAP-5b concentrations when CSTBL / 6 mice were administered GST-RANKL and PBS.
  • FIG. 47 shows the bone density when GST-RANKL and PBS were administered to C57BL / 6 mice.
  • Fig. 48 is a photograph showing the results of image analysis using micro CT when GST-RANKL and PBS were administered to C57BL / 6 mice.
  • FIG. 49 is a diagram showing blood Ca, ALP and TRAP-5b concentrations when PTH was administered to mice that had been GST-RANKL-administered and ovariectomized.
  • FIG. 50 is a graph showing the bone density when PTH is administered to mice in which ovary has been removed by administration of GST-RANKL.
  • FIG. 51 is a photograph showing the results of image analysis using micro-CT when GTH-RANKL is administered and PTH is administered to a mouse whose ovaries have been removed.
  • FIG. 52 shows the Ca, ALP and TRAP-5b concentrations in male mice administered with GST-RANKL and PBS.
  • FIG. 53 shows the bone density of male mice administered with GST-RANKL and PBS.
  • FIG. 54 is a photograph showing the results of image analysis using micro CT of male mice administered with GST-RANKL and PBS.
  • FIG. 55 shows the blood Ca, ALP and TRAP-5b concentrations in ICR mice administered with GST-RANKL and PBS.
  • FIG. 56 shows the bone density of ICR mice administered with GST-RANKL and PBS.
  • FIG. 57 is a photograph showing the result of image analysis using micro CT of ICR mice administered with GST-RANKL and PBS.
  • FIG. 58 shows the blood Ca, ALP and TRAP_5b concentrations in fisher rats administered with GST-RANKL and PBS.
  • FIG. 59 shows the bone density of fisher rats administered with GST-RANKL and PBS.
  • FIG. 60 is a photograph showing the results of image analysis using micro CT of fisher rats administered with GST-RANKL and PBS.
  • FIG. 61 shows the RANKL concentration in the serum of mice that received GST-RANKL once.
  • FIG. 62 shows the concentrations of Ca, ALP and TRAP-5b in the serum of mice that received GST-RANKL and PBS once.
  • FIG. 63 shows the bone density of mice that received GST-RANKL and PBS once.
  • FIG. 64 is a photograph showing the result of image analysis using micro CT of a mouse administered with GST-RANKL and PBS once.
  • FIG. 65 shows the serum Ca, ALP and TRAP-5b concentrations in mice administered with GST-RANKL and PBS for 7 consecutive days.
  • FIG. 66 shows the bone density of mice administered with GST-RANKL and PBS for 7 consecutive days.
  • FIG. 67 is a photograph showing the results of image analysis using micro CT of mice administered with GST-RANKL and PBS for 7 consecutive days.
  • FIG. 68 shows the bone density of mice administered GST-RANKL and PBS to the calvaria.
  • Figure 69 shows a photograph showing bone mass when LFM-A13 is administered to GST-RANKL-treated mice.
  • FIG. 70 shows the unit bone mass (Bone volume / tissue volumej) when LFM-A13 is administered to GST-RANKL-treated mice.
  • FIG. 71 shows the trabecular width (Trabecular / thickness) when LFM-A13 is administered to GST-RANKL-treated mice.
  • FIG. 72 is a graph showing the number of trabecular bone (Trabecular / Aberration) when LFM-A13 is administered to GST-RANKL-treated mice.
  • Fig. 73 shows the number of osteoclasts (Osteoclast number / bone perimeter) when LFM-A13 is administered to GST-RANKL-treated mice.
  • Fig. 74 shows the absorption surface (Eroded surface / bone surf ace) when LFM-A13 is administered to GST-RANKL-treated mice [S3.
  • FIG. 75 shows the blood Ca concentration when LFM-A13 was administered to GST-RANKL-treated mice.
  • FIG. 76 shows the serum Ca, ALP and TRAP-5b concentrations when raloxifone was administered to GST-RANKL-treated mice from which the ovaries had been removed.
  • FIG. 77 shows the total bone density when raloxifene was administered to GST-RANKL-treated mice from which the ovaries had been removed.
  • FIG. 78 shows the cortical bone mineral density when raloxifene is administered to GST-RANKL-treated mice from which the ovaries have been removed.
  • FIG. 79 shows the cortical bone thickness when raloxifene was administered to GST-RANKL-treated mice from which the ovaries had been removed.
  • FIG. 80 is a photograph showing the results of image analysis using micro CT when raloxifene was administered to GST-RANKL-treated mice from which the ovaries had been removed.
  • FIG. 81 shows the blood Ca, ALP and TRAP_5b concentrations when raloxifene was administered to GST-RANKL-treated mice from which the ovaries had been removed.
  • Figure 8-2 shows that raloxifene was administered to GST-RANKL-treated mice from which the ovaries were removed. It is a figure which shows cancellous bone density in the case.
  • FIG. 83 shows the total bone density when PTH was administered to GST-RANKL-treated mice.
  • FIG. 84 shows the cortical bone density when PTH was administered to GST-RANKL-treated mice.
  • FIG. 85 is a graph showing the amount of cortical bone mineral when GTH-RANKL-administered mice are administered PTH.
  • FIG. 86 shows the cortical bone thickness when PTH was administered to GST-RANKL-treated mice.
  • FIG. 87 is a graph showing cortical bone density in the diaphysis when PTH is administered to GST-RANKL-treated mice.
  • FIG. 88 is a photograph showing the results of image analysis using micro CT when PTH was administered to GST-RANKL-treated mice.
  • FIG. 89 shows the serum Ca, ALP, and TRAP_5b concentrations when GST-RANKL was administered to 12-week-old mice.
  • FIG. 90 shows the total bone density when GST-RANKL was administered to 12-week-old mice.
  • FIG. 91 is a photograph showing the results of image analysis using micro CT when GST-RANKL was administered to 12-week-old mice.
  • the osteopenia animal model of the present invention can be produced by administering a soluble RANKL or a fusion protein of soluble RANKL and an epitope tag to a non-human animal,
  • RANKL Receptor activator of NF- ⁇ B l igand
  • RANK NF- ⁇ ⁇ ⁇ receptor activator
  • a domain consisting of amino acids 1 to 48), a type 2 transmembrane protein with a transmembrane domain and an extracellular domain
  • RANKL From the 152th amino acid from the N-terminal in the extracellular domain The domain is a homology domain of the TNF ligand family. Soluble RANKL does not contain an intracellular domain. RANKL has functions such as osteoclast differentiation activation, lymphocyte differentiation, dendritic cell activation, mammary epithelial cell differentiation and lymph node formation.
  • Soluble RANKL includes soluble RANKL derivatives and soluble RANKL analogs.
  • the animal species from which soluble RANKL is derived is not limited, and RANKL derived from any animal species such as human-derived RANKL, mouse-derived RANKL, rat-derived RANKL and the like can be used.
  • the full-length base sequence and amino acid sequence of RANKL derived from human are shown in SEQ ID NOs: 1 and 2, respectively.
  • the soluble RANKL derivative or the soluble RANKL analog includes a protein having a partial RANKL amino acid sequence, such as a RANKL tranchete, and having a RANKL activity.
  • the soluble RANKL derivative preferably comprises a TNF ligand family homology domain starting from amino acid 152 in the amino acid sequence of SEQ ID NO: 2.
  • Examples of the soluble RANKL derivative include a protein consisting of the 127th amino acid to the 317th amino acid sequence, a protein consisting of the 140th amino acid to the 317th amino acid sequence, or the 159th amino acid to the Examples include a protein comprising the 317th amino acid sequence.
  • RAMLs derived from animal species other than human RANKL which are composed of amino acid sequences corresponding to the partial amino acid sequences of human RANKL.
  • the soluble RANKL derivative or the soluble RANKL analog includes an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and A protein having activity, or an amino acid sequence of a protein consisting of a partial sequence of the amino acid sequence of RANKL, including an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and the activity of RANKL
  • 1 or several is 1 to 9, preferably 1 to 5, and more preferably 1 or 2.
  • Examples of the epitope tag that forms a fusion protein with soluble RANKL include a protein or peptide having a sequence that can bind to a specific compound such as an antibody.
  • an epitope tag is used for purification of a fusion protein.
  • an epitope tag has a function of enhancing the activity of soluble RANKL.
  • Epitope tags include glutathione-S-transferase (GST), 2-12, preferably 4 or more, more preferably 4-7, more preferably 5 or 6 histidine polyhistidine, FLAG Tag (amino acid sequence DYKDDDDK; SEQ ID NO: 3), Myc tag (amino acid sequence EQKLISEEDL; SEQ ID NO: 4), V5 tag (amino acid sequence GKPIPNPLLGLDST; SEQ ID NO: 5), Xpress tag, HQ tag (amino acid sequence HQHQHQ; SEQ ID NO: 6), HA tag (amino acid sequence YPYDVPDYA; SEQ ID NO: 7), AU1 tag (amino acid sequence DTYRYI; SEQ ID NO: 8), T7 tag (amino acid sequence MASMTGGQQMG; SEQ ID NO: 9), VSV-G tag (amino) Acid sequence YTDIEMNRLGK; SEQ ID NO: 10), DDDDK tag (amino
  • a gene encoding RANKL and a gene encoding an epitope tag can be performed by conventional gene recombination techniques. In this case, it can be carried out by introducing an appropriate restriction site. Make sure that no stop codon appears between the genes to be fused.
  • the distance between the genes to be fused is not limited, and a linker may be included between them. Also, match the open reading frames of the two genes.
  • the above epitope tag may be fused to the N-terminal side of the amino acid sequence of RANKL, or may be fused to the C-terminal side.
  • SEQ ID NO: 2 1 And 22 shows the base sequence of DNA encoding the protein in which GST is fused to the protein comprising the amino acid sequence from the 140th amino acid to the 317th amino acid sequence in the amino acid sequence of RANKL, and the amino acid sequence of the protein.
  • SEQ ID NOS: 23 and 24 include a nucleotide sequence of DNA encoding a protein in which GST is fused to a protein comprising the 159th amino acid sequence to the 317th amino acid sequence in the RANKL amino acid sequence, and The amino acid sequence of the protein is shown.
  • the fusion gene thus prepared can be incorporated into an appropriate expression vector available for expression, and the target fusion protein can be recovered and purified. In this case, it can also be expressed in a cell-free system (cell-free system).
  • Any vector can be used as long as it can replicate in a host cell such as a plasmid, phage, or virus.
  • the vector contains a replication origin, a selectable marker, and a promoter, and may contain an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyaduration signal, and the like as necessary.
  • a vector in which a gene encoding an epitope tag such as glutathione-S-transferase is previously incorporated can also be used.
  • the vector preferably contains a polylinker with various restriction sites within it, or preferably contains a single restriction site.
  • a specific uniform site in a vector can be cleaved with a specific restriction enzyme, and DNA can be inserted into the cleaved site.
  • An expression vector containing the fusion gene can be used for transformation of an appropriate host cell to cause the host cell to express and produce a fusion protein encoding the fusion gene.
  • host cells include bacterial cells such as E. coli, Streptomyces, Bacillus subtilis, fungal cells, baker's yeast, yeast cells, insect cells, and mammalian cells.
  • the transformation can be performed by a known method such as calcium chloride, calcium phosphate, DEAE-dextran mediated transformation, electroporation, ribophatation and the like.
  • the obtained recombinant fusion protein can be separated and purified by various separation and purification methods. For example, ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, etc. alone or as appropriate Can be used in combination.
  • the expression product when expressed as a fusion protein with GST or the like, it can be purified using the properties of the protein or peptide fused with the target protein.
  • GST when expressed as a fusion protein with GST, GST has affinity for dartathione, so it must be efficiently purified by affinity chromatography using a column in which daltathione is bound to a carrier. Can do.
  • the protein having the histidine tag binds to the chelate column and can be purified using the chelate column.
  • any epitope tag when used, it can be purified by affinity chromatography using an antibody that recognizes the epitope associated with the epitope tag.
  • Animal species to which soluble RANKL or fusion protein of soluble RANKL and Epitope tag is administered are not limited, and mice, rats, guinea pigs, hamsters, horses, horses, hidges, pigs, monkeys, inu, cats, etc. All mammals other than humans are targeted, and osteopenia model animals can be produced in these animals. Both males and females can be used to create osteopenia model animals. Further, the age of the animal used for producing the osteopenia model animal is not limited, and the osteopenia model animal of the present invention can be produced using an elderly animal. For example, when a mouse is used as an animal, a model animal for osteopenia can be produced using a mouse 1 to 5 weeks old, preferably 4 to 12 weeks old.
  • the dose of the soluble RANKL or the fusion protein of soluble RANKL and epotope tag to the animal is not limited and can be appropriately determined depending on the animal species. For example, 10 nmol to 5000 nmol per animal, preferably 50 nmol to 1000 nmol, or 100 ug / kg to 50 mg / kg, preferably 500 g / kg to 5 mg / kg per body weight of the animal to be administered.
  • the route of administration is not limited, and may be administered by intravenous injection, intraperitoneal injection, subcutaneous injection, intramuscular injection, suppository, eye drops, calvarial administration, and the like.
  • the number of administrations is not limited, and it may be a single administration or a plurality of continuous administrations of 2 to h several times.
  • the administration interval is not limited, and for example, administration may be performed for several days every day.
  • the degree of bone loss in the model animal can be adjusted, for example, when a single dose is given,
  • a soluble RANKL or a fusion protein of soluble RANKL and an epitope tag When a soluble RANKL or a fusion protein of soluble RANKL and an epitope tag is administered to an animal, it directly differentiates and activates osteoclasts in the animal body. This makes it possible to produce bone loss model animals more quickly.
  • the osteopenia model animal of the present invention has the following characteristics.
  • a normal animal refers to an animal that does not suffer from a bone metabolic disease and has not been administered a soluble RANKL or a fusion protein of soluble RANKL and an epitope tag.
  • animals that have been administered PBS or the like can be used as normal animals when creating a model animal for dose reduction.
  • Bone resorption marker concentration in body fluid (level) Force S temporarily increased compared to normal animals of the same type (normal individuals). Although it depends on the dose of soluble RANKL or fusion protein of soluble RANKL and epitopog, it is, for example, 1.1 times or more, preferably 1.2 times or more, more preferably 1.3 times that of normal animals. More than double, particularly preferably 1.4 or more.
  • Serum bone resorption markers include serum calcium, serum collagen degradation products (CTx ⁇ type 1 collagen cross-linked C-telopeptide ⁇ , NTx ⁇ type 1 collagen bridge N-telopeptide ⁇ , PICP ⁇ type I procollagen- C-propeptide ⁇ , or PINP
  • ⁇ Type I procollagen-N-peptide ⁇ serum tartrate-resistant acid phosphatase (TRAP-5 b), etc.
  • urinary bone resorption markers include urinary CTx or NTx, urinary hydroxyproline And urinary pyridinoline, deoxypyridinoline and the like.
  • hydroxylysine glycosides (in serum and urine), bone shaloprotein (in serum) and the like can be mentioned. These markers are elevated when osteoclasts proliferate and promote activity. These markers are usually used as bone metabolism markers in osteoporotic bone metabolic diseases. These markers can be measured by a colorimetric method or an immunoassay using a specific antibody.
  • Serum bone formation marker concentration does not vary from normal animals.
  • Serum osteocalcin, as serum bone formation marker Examples include alkaline phosphatase (especially bone-specific alkaline phosphatase). These markers are osteoblast-derived proteins that increase in concentration when osteoblasts proliferate. These markers are usually used as bone metabolic markers in osteoporotic bone metabolic diseases. These markers can be measured by an immunoassay using a colorimetric method or a specific antibody. Soluble RANKL or fusion protein of soluble RANKL and Epitope tag do not act directly on osteoblasts, but indirectly due to the force-pulling phenomenon of bone resorption and osteogenesis. One concentration may vary. The presence or absence of variation varies depending on the dose of soluble RANKL or the fusion protein of soluble RANKL and an epitope tag, the number of doses, and the time course after administration.
  • the bone resorption marker (1) and the bone formation marker (2) are collectively referred to as a bone metabolism marker.
  • Bone density decreases compared to normal animals.
  • Bone density is a numerical representation of the density of mineral components such as calcium in bone.
  • Bone density includes cancellous bone density, total bone density, and cortical bone density. In the present invention, when simply referred to as bone density, it can be referred to as cancellous bone density. It can be measured by ' ⁇ density, pQCT (peripheral quantitative computerized tomography), DXA (Dual Energy X-Ray Absorptiometry), etc.
  • the growth plate when the bone density of the femur or tibia is measured by pQCT, the growth plate
  • the bone density is 1% or more, 2% or more, 3% or more, 5% or more, preferably 7.5% or more, more preferably 10% or more, and most preferably 20%. More than that.
  • the total bone density is lower than that of normal animals, and when the bone density of the femur or tibia is measured by pQCT, it varies depending on the distance from the growth plate.
  • the total bone density is 1% or more, 2% or more, 3% or more, 5% or more, preferably 7.5% or more, more preferably 10% or more, and most preferably 20% or more.
  • Cortical bone density is also lower than that of normal animals.
  • the bone density of the femur or tibia is measured by PQCT, it varies depending on the distance from the growth plate. 2% or more, 3% or more, 5% or more, preferably 7.5% or more, more preferably 10% or more, and most preferably 20% or more.
  • Bone mineral content is the amount of bone mineral (hydroxyapatite) and reflects bone density. Bone mineral content can be measured by pQCT.
  • the cortical bone mineral content of the femur or tibia is measured by pQCT, depending on the distance from the growth plate, for example, the cortical bone mineral content is 5% or more, preferably 7.5% or more, more preferably 10%. In addition, it is preferably reduced by 15% or more, and most preferably by 20% or more.
  • cortical bone thickness is reduced compared to normal animals.
  • Cortical bone thickness can be measured by pQCT.
  • the cortical bone thickness of the femur or tibia is measured by pQCT, depending on the distance from the growth plate, for example, the cortical bone thickness is 5% or more, preferably 7.5% or more, more preferably 10% or more, More preferably, it decreases by 15% or more, and most preferably by 20% or more.
  • Unit bone mass refers to the area of the entire trabecular bone that occupies the entire tissue area in the pathological section.
  • the trabecular bone is the part of the epiphysial cancellous bone that appears to be finely loosened.
  • the unit bone mass and the number of trabecular bone depend on the dose of soluble RANKL or fusion protein of soluble RANKL and Epitope tag, but compared to normal animals, For example, it is reduced by 10% or more, preferably 20% or more, more preferably 30% or more, further preferably 40% or more, and particularly preferably 50% or more.
  • the number of osteoclasts is increased compared to normal animals.
  • the number of osteoclasts can be measured by bone morphometry according to a conventional method.
  • the number of osteoclasts depends on the dose of soluble RANKL or the fusion protein of soluble RANKL and epitope tag, but for example 20% or more, preferably 30% or more, more preferably 40% compared to normal animals. % Or more, more preferably 50% or more, particularly preferably 60% or more.
  • Osteoblast surface / bone surface (0b. S / BS) is significantly increased compared to normal animals.
  • the osteoblast surface is the attachment of osteoblasts across the trabecular surface. This is the percentage (%) of the surface.
  • the osteoblast surface depends on the dose of soluble RANKL or fusion protein of soluble RANKL and epitope tag, for example, 20% or more, preferably 30% or more, more preferably 40% compared to normal animals. % Or more, more preferably 50% or more, particularly preferably 60% or more.
  • the dose of the fusion protein of soluble RANKL and Epitope tag is increased, for example, when administered to mice at 2 mg / kg, it takes time until the coupling phenomenon between bone resorption and bone formation is observed. The appearance of changes in the blast surface may be delayed. Therefore, depending on the time of observation of the osteoblast surface, an increase in the osteoblast surface may not be observed.
  • Osteoid refers to the state prior to calcification to become a bone matrix
  • resorption surface refers to the proportion of the surface that has been eroded by the erosion of the entire trabecular surface.
  • the osteoclast surface refers to the ratio of the surface to which osteoclasts adhere on the entire trabecular surface.
  • Osteoid thickness, resorption surface, and osteoclast cell surface are, for example, 20% or more, preferably 30 compared to normal animals, depending on the dose of soluble RANKL or fusion protein of soluble RANKL and Epitope tag. % Or more, more preferably 40% or more, further preferably 50% or more, particularly preferably 60% or more.
  • the bone shape can be measured by micro CT or the like.
  • a model animal for osteopenia can be produced by administering soluble RANKL or a fusion protein of soluble RANKL and an epitope tag to the animal, and further removing the ovary.
  • the ovaries are preferably removed after administration of RANKL.
  • the present invention includes a model animal for osteopenia that can be produced by administering soluble RANKL or a fusion protein of soluble RANKL and an epitope tag, and further extracting the ovaries.
  • the osteopenia model animal can be used as a human disease model animal having abnormalities in bone metabolism after menopause.
  • bone loss, bone density and unit bone mass observed by micro-CT directly reflecting bone loss are particularly characteristic, followed by osteoclast number / An increase in bone perimeter) and a decrease in the number of trabeculae are characteristic.
  • a decrease in cortical bone mineral content is also observed.
  • the above characteristics become more prominent as the amount of GST-RANKL administered increases, and the degree of characteristics depends on the dosage.
  • the degree of the above characteristics indicates the severity of osteopenia, and the osteopenia animal model of the present invention can control the severity by changing the amount of GST-RANKL to be administered. .
  • a small amount of GST-RANKL should be administered when trying to obtain a mild bone loss model animal, and a large amount of GST when trying to obtain a severe bone loss model animal.
  • -RANKL should be administered.
  • a severe bone loss model animal is a model animal in which the above-mentioned characteristics are strongly manifested.
  • bone resorption marker concentration (level) in body fluid force Normal animal of the same type Is a model animal that has risen relatively relatively temporarily compared to normal animals, for example, a model animal that has increased by a factor of 1.2 or more, preferably 1.3 or more times, more preferably 1.4 or more times that of normal animals .
  • it is a model animal whose bone density is relatively low compared to normal animals.
  • the bone density of the femur or tibia is measured by pQCT, it varies depending on the distance from the growth plate. %, Preferably 10% or more, more preferably 20% or more.
  • S / Bs; osteoclast surface / Done surface is a model animal with a relatively large increase compared to normal animals, for example, 20% or more, preferably 30% or more, more preferably 40% or more, more preferably 50% or more compared to normal animals. Particularly preferred is a model animal that is elevated by 60% or more.
  • an animal in which bone density and / or unit bone mass is decreased as compared with a normal animal, and an increase in the number of osteoclasts and Z or Examples include animals in which the number of trabeculae is reduced compared to normal animals. Furthermore, there are animals in which a decrease in trabecular width is observed compared to normal animals, and animals in which increases in osteoid thickness, resorption surface, osteoclast surface, etc. are observed compared to normal animals.
  • the bone resorption marker (1) varies after administration of soluble RANKL or soluble RANKL and epitope-tagged fusion protein, but gradually increases after the creation of osteopenia model animals Eventually, it will return to the normal value.
  • osteopenia animal model of the present invention when evaluating or screening a drug related to bone metabolism using the osteopenia animal model of the present invention, when the bone mass is decreasing according to the purpose, when the bone mass is most reduced, Alternatively, use a model animal at an appropriate time when the bone mass once decreased is increasing again. Therefore, an animal whose bone mass is decreasing after administration of soluble RANKL or a fusion protein of soluble RANKL and an epitope tag, regardless of the strength of the above-described characteristics, The characteristics of Are included in the osteopenia animal model of the present invention.
  • the osteopenia animal model of the present invention is produced by a simple mechanism not involving other substances as described above. Therefore, within 1 week, preferably within 3 days (72 hours or less), more preferably within 2 days after administration of soluble RA dish L or soluble RANKL and epitopic tag fusion protein to normal animals ( Within 50 hours), more preferably within 24 hours, it will have the above characteristics and a bone loss model animal will be created.
  • Osteoporosis hypercalcemia, Paget's disease, renal osteodystrophy, kuru disease / osteomalacia, rheumatoid arthritis, etc. It can be used as a disease model animal. Specifically, it can be used as follows.
  • the bone loss model animal of the present invention can be used for evaluation of a bone resorption inhibitor or screening of a new bone resorption inhibitor (bone resorption inhibitor). In the present invention, these are sometimes referred to as evaluation of a bone resorption inhibitor or a bone resorption inhibitor candidate substance.
  • Known bone resorption inhibitors include risedronate, etidronate, bisphosphonates such as alendronate, canorecitonin, Cathepsin K inhibitor, and proton pump inhibitor. Bone resorption inhibitors work when bone mass is decreasing. Therefore, it is desirable to evaluate at the time when bone mass is decreased after administration of soluble RANKL or fusion protein of soluble RANKL and epotope tag.
  • the bone resorption inhibitor or the bone resorption inhibitor candidate substance is administered to the osteopenia model animal of the present invention, and the bone resorption inhibitor or the bone resorption inhibitor depends on whether the bone loss is suppressed or not.
  • the candidate substance may be administered before administration of soluble RANKL or a fusion protein of soluble RANKL and an epitope tag, for example, 1 to 3 days, preferably 1 day before. In other words, it is only necessary to use an index as to whether the decrease in bone mass or the increase in bone mass in the osteopenia model animal is reduced.
  • the increase in bone mass was achieved by reducing the bone resorption marker level in the bone loss model animal, increasing bone density, increasing unit bone mass, increasing trabecular number, trabecular width, osteoid Judgment can be made using at least one selected from the group consisting of thickness, increase in resorption surface, decrease in the number of osteoclasts, increase in osteoblast surface, and increase in bone mass observed by CT.
  • the model animal of the present invention By using it, the drug can be evaluated within a few days, for example, about 3 to 4 days.
  • the osteopenia model animal of the present invention from which the ovaries have been removed can be used for drug evaluation of hormones such as estrogen androgen or hormone receptor modulators or screening of new hormone receptor modulators.
  • hormones such as estrogen androgen or hormone receptor modulators or screening of new hormone receptor modulators.
  • a selective estrogen receptor modulator which is a hormone receptor modulator, is one of the bone formation and absorption inhibitors, and has the effect of suppressing bone resorption through the action of the hormone estrogen. Evaluation with normal wild animals with endogenous estrogen is difficult.
  • By extracting the ovary (0VX) of the osteopenia animal model of the present invention it is possible to perform drug evaluation of a selective estrogen receptor modulator much faster than the evaluation using a model animal that simply removes the ovary. .
  • osteopenia animal model of the present invention can be used for evaluation of osteogenesis promoters or screening for new osteogenesis promoters. In the present invention, these may be referred to as evaluation of osteogenesis promoter or osteogenesis promoter candidate substance. Bone formation promoters are effective when bone mass is once reduced and restored. Therefore, soluble type RANKL or soluble type
  • the osteogenesis promoter or osteogenesis promoter candidate substance is administered to the osteopenia animal model of the present invention, and the osteogenesis promoter or The effect of the osteogenesis promoter candidate substance can be evaluated.
  • the candidate substance is preferably administered after administration of soluble RANKL or a fusion protein of soluble RANKL and an epitope tag to an animal and a decrease in bone mass is observed. The evaluation may be based on whether or not the decreased bone mass increases in the osteopenia model animal.
  • the increase in bone mass can be achieved by increasing the bone formation marker level in the body, increasing bone density, increasing unit bone mass, trabecular number, trabecular width, Judgment can be made using at least one selected from the group consisting of an increase in thickness, a decrease in the number of osteoclasts and resorption surface, an increase in the osteoblast surface, and an increase in bone mass observed by CT.
  • osteogenesis promoters include PTH (parathyroid hormone).
  • the above bone resorption inhibitor and osteogenesis promoter can be used as a therapeutic agent for osteoporosis and osteopenia.
  • the osteopenia animal model of the present invention can be used as an experimental animal for bone metabolism research. That is, in the osteopenia animal model of the present invention, bone formation (coupling) accompanying bone resorption occurs and can be used for basic research such as elucidation of the mechanism for regulating bone remodeling. It can also be used to search for coupling factors that couple bone resorption and bone formation. It can also be used to evaluate drugs that inhibit the RANKL signal and to study mechanism including osteoclast differentiation. Examples of drugs that suppress the RANKL signal include LFM-A13, which is an inhibitor of the Tec kinase family.
  • osteopenia animal model of the present invention since bone loss is observed in only about 1 to 2 days after administration of soluble RANKL or a fusion protein of soluble RANKL and an epitope tag, universities, etc. Can be used to practice bone loss in vivo.
  • a bone loss model animal is sent to a research institution such as a university or a pharmaceutical manufacturer to provide the boner of the present invention to the researcher. It is possible to provide a model animal for dose reduction.
  • the cDNA encoding human RANKL residues 140-317 was ligated with Sal I and Not I sites by PCR, and pGEX-4T-2 (GE healthcare) was used with these endonucleases.
  • Genclo Accession Number U13854 was cloned downstream of Glutathione S-transferase.
  • the cells After induction of protein expression by IPTG (final concentration: 0.5 mM) in BL21 (DE3) Escherischia coli (invitrogen), the cells are extracted into buffer — (50 mM Tris-HCl, pH 8.0, lOO mM NaCl, The suspension was suspended in 1 mM EDTA, 1 mM DTT, l% (v / v) TritonX-100) and crushed using a sonicator at 4 ° C. After centrifugation at 18000 X g for 15 min, the supernatant was recovered and applied to a Glutathione Sepharose column.
  • buffer — 50 mM Tris-HCl, pH 8.0, lOO mM NaCl
  • the suspension was suspended in 1 mM EDTA, 1 mM DTT, l% (v / v) TritonX-100) and crushed using a sonicator at 4 ° C. After centrifugation at 18000
  • the femur, tibia, cerebrum, lung, heart, liver, thymus, spleen, kidney, and skin were collected, and the cerebrum, lung, heart, liver, thymus, spleen, kidney, and skin were stained with HE. Thus, spontaneous lesions were observed.
  • the proximal end of the distal growth plate is 0.6 jobs, 0.8 mm, 1.
  • Bone density was measured at the position of 0 mm cancellous bone, and a section was prepared for the tibia and bone morphology was measured. Each measured value was compared with the control mouth group by Dunnett's method and tested. Bone resorption parameters and bone formation parameters
  • GST-RANKL As a result of measuring bone mineral density of the femur by PQCT, at a position 0.6 mm proximal to the growth plate, GST-RANKL was administered at a high dose of 10%, 0.8 at 23%, and 20 at 20% decrease. It was observed. Anova; Dunnett's method was used to test for significant differences. When GST-RANKL was administered at a high dose, p was 0.01 at all positions measured. In addition, no significant difference was obtained when GST RANKL was administered at a low dose (Fig. 6).
  • the unit bone mass and trabecular number decreased to about 50% by the high dose of GST-RANKL, and the number of osteoclasts increased. In addition, no decrease was observed at low doses (Figs. 78 and 9).
  • the collected cerebrum, lung, heart, liver, thymus, spleen, kidney and skin were stained with HE, and no abnormal findings or spontaneous lesions were observed in all groups.
  • GST-RANKL was prepared in the same manner as in Example 1.
  • GST-RANKL213 nmol, 426 nmol, and 852 nmol were intraperitoneally administered every 24 hours three times to 10 7-week-old female C57BL / 6N mice, and whole blood was collected 1.5 hours after the third administration. PBS was administered in the same manner as a comparison target.
  • GST-RANKL 213 nmol, 426 nmol and 852 nmol correspond to 10 20 g and 40 g, respectively.
  • the femur and tibia were collected from the mouse, and for the femur, the bone density of the cancellous bone of 0.6, 0.8, and 1.0 mm was measured proximally from the growth plate using pQCT. went.
  • GST-RANKL administration increased serum Ca, CTx, and TRAP-5b in a dose-dependent manner (Figs. 12, 13, and 14 respectively).
  • the increase in calcium was significantly increased to p 0.05 at 213 nmol administration, and more pronounced at 0.01 at 426 nmol and 852 nmol administration. An increase was seen.
  • CTx and TRAP-5b no significant increase was observed at 213 nmol, but a marked increase was observed at 0.01 and 426 nmol and 852 nmol, respectively.
  • the conventional method required several weeks for preparation of model mice and required special techniques such as ovariectomy (0VX).
  • ovariectomy a simple method of intraperitoneal administration was used.
  • Model mice can be made in time.
  • there was no technology for creating model mice that could easily change the degree of osteoporosis and bone loss but by changing the dose of GST-RANKL, it was possible to increase bone resorption parameters and decrease bone mass.
  • a model mouse can be created that can be freely adjusted to match the degree of osteoporosis and osteopenia.
  • GST-RANKL was prepared in the same manner as in Example 1.
  • GST-RANKL426 nmol was administered intraperitoneally three times every 24 hours to 5 to 6 females of 7-week-old C57BL / 6N mice.
  • the osteoporosis drug (risedronate) was administered 0.01 mg from 3 days before GST-RANKL administration.
  • the drug was administered subcutaneously at / kg and continued every 24 hours until the end of the experiment.
  • Serum and femur and tibia were collected 5 hours after the third administration of GST-RANKL.
  • Serum bone resorption parameters calcium, CTx, TRAP_5b
  • bone formation parameters
  • TRAP-5b a bone resorption marker
  • CTx was 1.4-fold.
  • a significant increase was observed (P 0. 05).
  • GST-RANKL and risedronate combined administration group TRAP-5b and CTx decreased by about 30% compared to the control mouth group. ( Figures 17 and 18).
  • the decrease in the GST_RANKL and risedronate combination group is considered to be due to the risedronate suppressing the osteoclasts originally present in the mouse.
  • Al force phosphatase and osteocalcin which are bone formation markers, were not changed in the GST-RANKL administration group, GST-RANKL, and risedronate combination administration group compared to the control mouth group ( Figures 19 and 20). .
  • the bone density was significantly decreased in the GST-RANKL-treated group compared to the control group, and 20% at any position of 0.6 thigh, 0.8 mm, and 1.0 mm proximal to the growth plate. A significant decrease was observed (p 0. Q1).
  • the GST-RANKL and risedronate combination group showed bone mineral density values similar to those of the control group.
  • Fig. 21 shows the bone density of the femur
  • Fig. 22 shows the bone morphology measured by micro CT. From the above results, it can be used for the evaluation of new therapeutic agents by administering osteoporosis and osteopenia treatment drug to this model mouse from the above results, and in the osteopenia model mouse using conventional 0VX etc. Compared to drug evaluation, it will be possible to shorten the time of several weeks and significantly reduce the amount of drug used.
  • Example 4 Production of osteopenia model mouse using soluble RANKL
  • 57 nmol and 426 nmol of soluble RANKL were intraperitoneally administered to 10 females of 7-week-old C57BL / 6N mice every 24 hours three times, and serum 1.5 hours after the third administration At the same time, the femur was collected. Serum was measured for bone resorption markers and bone formation markers, and femur was measured for bone density at 0.6, 0.8, and 1.0 mm proximal to the growth plate by PQCT. Bone resorption markers, blood calcium and CTx, were significantly increased by about 1.4 and 1.5 times, respectively, with high doses of 426 nmol ( Figures 2 and 2), and TRAP-5b was significant.
  • osteopenia model mouse can be produced specifically for GST-RANKL. This technology can be applied to all soluble RANKL and can be used for drug evaluation as well as GST-RANKL.
  • mice Seven-week-old female C57BL / 6N mice were administered GST-RANKL at 2 mg / kg three times every 24 hours, then dissected, and the tibia was stained with toluidine blue, and morphometry was performed.
  • the control group was similarly administered solvent (PBS).
  • GST-RANKL intraperitoneally administered tibia bone morphology was measured and compared to the control mouth group, the unit bone mass (BV / TV) was about 30% (P 0.03), trabecular width (Tb. Th) decreased by about 10% (P-0.03), and the number of trabeculae (Tb. N) decreased by about 20% (p-0.05) (Figs. 29-31).
  • the osteoid thickness (0.
  • Th is about 1.2 times (p ⁇ 0.04)
  • the resorption surface (ES / BS) is about 1.8 times (p ⁇ 0.001)
  • the number of osteoclasts (N Oc / B. Pm) increased approximately 1.9 times (p ⁇ 0. 001)
  • osteoclast surface (Oc. S / BS) increased approximately L 8 times (p> 0.001) (Fig. 3 2-3 5).
  • GST-RANKL administration significantly increased osteoid thickness, but no increase in osteoblast surface was observed.
  • Significant in the resorption system items such as resorption surface, number of osteoclasts, and osteoclast surface. Since the increase was observed, it was found that GST-RANKL did not act on osteoblasts but caused bone loss by directly differentiating and activating osteoclasts. That is, the mechanism of GST-RANKL-administered bone loss was not bone formation, but increased bone resorption.
  • the number of doses, and the time course after administration bone formation may be promoted by the coupling phenomenon, and an increase in the osteoblast surface may be observed.
  • femur and serum were collected, and TRAP-5b, calcium (Ca), Al force phosphatase (ALP) was measured, and the femur was analyzed for bone density by pQCT and image analysis using micro CT.
  • TRAP-5b significantly increased approximately 2-fold by administration of RANKL (p 0.01).
  • the elevated TRAP-5b concentration was about 30% (p. 01), 50% (p. 0.01), 70% (p. ⁇ 0. 01) and decreased in a dose-dependent manner.
  • ALP was significantly decreased (p ⁇ 0.01) compared to the RANKL single administration group by risedronate 30 w.g / kg administration (Fig. 37).
  • the femoral bone was measured by pQCT at a position of 0.6, 0.8, 1.0 mm proximal to the growth plate, and compared with the PBS-administered group, the RANKL-administered group showed 27% (p. ⁇ 0.
  • mice 7-week-old female C57BL / 6N mice were dosed with etidronate at 3, 30 mg / kg, alendronate at 3, 30, 300 ⁇ g / kg, risedronate at 1, 10, 100 ⁇ g / kg. It was administered subcutaneously every 24 hours from the day before RANKL administration until the end of the experiment. GST-RANKL was administered intraperitoneally 3 times every 24 hours at 1 mg / kg. After the third GST-RANKL administration, the femur, tibia, and serum were collected 5 hours later, and TRAP_5b, calcium (Ca), and alkaliphosphatase (ALP) in the serum were measured. The femur was examined by pQCT. Density measurement and image analysis were performed using micro CT.
  • TRAP-5b increased by about 40% with RANKL administration, but no significant difference was obtained.
  • Alendronate administration significantly suppressed the increase in TRAP-5b at a dose of 300 g / kg (p 0.01).
  • risedronate administration was 1, 10, and 100 ⁇ , respectively.
  • W was significantly suppressed at a dose of 200 g / kg (p 0.05, p 0.01, p ⁇ 0.01) ( Figure 40).
  • alendronate and risedronate decreased about 20 to 30% (p 0.01) compared to the RANKL group, and this decrease was dose-dependent (Figure 40).
  • the femur was measured by PQCT at a bone density of 0.6, 0.8 and 1.0 mm proximal to the growth plate.
  • mice Seven-week-old female C57BL / 6N mice were bred for 25 days, and GST-RANKL was intraperitoneally administered twice every 24 hours at 1 mg / kg. Ovariectomy was performed 24 hours after the second administration, and femur, tibia, and serum were collected 24 hours after ovariectomy. These samples were compared to samples from a group that was raised 4 weeks after ovariectomy. PBS was administered as a comparative control for GST-RANKL, and sham surgery was performed as a comparative control for ovariectomy. (Sham) was done.
  • Bone metabolism markers were TRAP-5b, calcium (Ca), and alkaline phosphatase (ALP) in blood. Femur was measured for bone density by pQCT and image analysis by micro CT. The day when 0VX or Sham operation was performed after the start of breeding was expressed as (day X).
  • TRAP-5b increased about 1.8 times in all GST-RANKL administration groups compared with PBS administration + Sham (day 27) group, but no significant difference was observed.
  • Ca is about 11 ° / in the OVX (dayO) group compared to Sham (dayO). However, there was no change in all the 0VX treatment groups.
  • ALP there was no change in the Sham (dayO) group and the OVX (dayO) group, compared with the PBS-treated Sham (day27) group, about 40% (p ⁇ 0.p.) In the GST-RANKL-treated OVX (day27) group. 01) was observed (Fig. 4 3).
  • the bone density of the femur was measured at 0.6, 0.8, and 1.0 mm proximal to the growth plate.
  • the decrease was about 16% (p ⁇ 0. 01) and about 12% (p ⁇ 0. 05).
  • the GST-RAML-administered 0VX (day 27) group had a significant decrease of about 4%, about 13%, and about 15% (p 0.01), compared with the PBS-treated Sham (day 27) group. There was no difference from the Sham (day 27) group administered with GST-RANKL (Fig. 44). These results were also confirmed by image analysis using micro CT (Fig. 45).
  • the osteopenia model mouse produced by GST-RANKL administration showed the same symptoms as the conventional ovariectomized mouse. Usually, it takes more than 4 weeks to produce a model mouse with osteopenia by ovariectomy, but ovariectomy after GST-RANKL administration can shorten the production period to only 72 hours. It was. Mice that have been ovariectomized after GST-RANKL administration have almost lost their estrogen one day after excision, and their hormone balance is similar to that of a model mouse model for osteopenia due to normal ovariectomy. Therefore, it was possible to easily and quickly produce a mouse having almost the same state as the model for osteopenia due to ovariectomy. this
  • the GST-RANKL / 0VX model is a bone loss model that is physiologically similar to that of postmenopausal women and can be created in a short period of 72 hours. Bone healing that can be applied to rapid drug evaluation
  • GST-RANKL was intraperitoneally administered at a dose of 1 mg / kg twice every 24 hours to 7-week-old female C57BL / 6N mice, and 24 hours after the second administration was defined as 0 week 0, 1, 4, 6, Eight-week serum and thigh bones were collected and compared with mice that received PBS in the same manner. Bone metabolism markers were measured for TRAP-5b, calcium (Ca), and alkaline phosphatase (ALP) in the blood, and the femur was analyzed for bone density by pQCT and image analysis using microphone-mouth CT. There was no significant difference in Ca in the serum, but TRAP-5b increased approximately 3 times compared to the PBS-administered group 24 hours after RANKL administration (week 0). Showed behavior. As for ALP, an increase of about 1.4-fold was observed at 0 and 1 week, and after that, behavior similar to that of the PBS-administered group was observed (Fig. 46).
  • the bone density of the femur was measured at 0.6 mm, 0.8 mm, and 1.0 mm proximal from the growth plate by PQCT.
  • osteopenia model mouse by RANKL administration is useful for comparing the recovery of bone mass. That is, it was found that it is possible to evaluate a drug that promotes bone mass increase in the period up to 6 weeks after two administrations of RANKL.
  • GST-RANKL was administered to 7-week-old female C57BL / 6N mice at 1 mg / kg twice every 24 hours, and ovariectomy was performed 24 hours after the second administration. From 24 hours after ovariectomy, parathyroid hormone (PTH) was administered subcutaneously at 160 ⁇ ug / kg for 10 consecutive days. In addition, PBS administration and sham surgery (Sham) were performed as comparative controls, and serum markers and The bone density of the femur was measured.
  • PTH parathyroid hormone
  • Serum ALP decreased about 24% in all GST-RANKL administration groups, and Ca was not changed.
  • all PTH-treated groups showed a significant increase of about 1.5 times (p 0.05) compared to the non-PTH-treated groups (Fig. 49).
  • p 0.05 the PBS-administered + Sham group
  • Bone density was measured at 0.6 mm, 0.8 thigh, and 1.0 mm proximal to the growth plate.
  • the GST-RANKL + Sham group was approximately 17% of the PBS + Sham group, respectively. There was a decrease of (p ⁇ 0. 05), 15% (p ⁇ 0. 05), 18% (p ⁇ 0.05). In the 0VX group, decreases of about 5%, 4%, and 8% were observed, respectively. However, the bone density was not sufficiently decreased 10 days after 0VX. In the GST-RANKL + 0VX group, there was no decrease in bone density compared to the PBS + Sham group.
  • Bone metabolism markers are TRAP-5b, calcium (Ca), and alkaline phosphatase (ALP) in blood. Femur is measured by bone density using pQCT and microphone mouth.
  • GCR-RANKL was intraperitoneally administered 3 times every 24 hours at 1 mg / kg to 7-week-old female ICR mice. Femur and serum were collected 1.5 hours after the third administration and compared with the PBS group. did. Bone metabolism markers were TRAP-5b, calcium (Ca), and alkaline phosphatase (ALP) in blood. Femur was measured by bone density using pQCT and image analysis using micro CT. .
  • Serum TRAP-5b, Ca, and ALP did not change compared to the control group (Fig. 58), but the bone density of the femur was measured at a distance of 3 mm proximal to the growth plate. There was a decrease in% (p ⁇ 0.02) (Fig. 59). These results were also confirmed by image analysis using micro CT (Fig. 60). From this result, it was found that the bone loss model by RANKL administration can be used not only for mice but also for other animals such as rats.
  • GST-RANKL was intraperitoneally administered to 7-week-old female C57BL / 6N mice at 1 mg / kg, and serum bone resorption and bone formation markers were measured 12, 24, and 48 hours later. Serum human RANKL concentrations were collected before and after GST-RANKL administration, 2, 4, 8, 12 2, 24, 48, 72 hours, and measured using ELISA. The bone density of the femur 24 and 48 hours after administration was also measured by pQCT.
  • the bone density of the femur was measured at a position of 0.6, 0.8, 1.0 mm proximal to the growth plate. After 24 hours, the bone density after 48 hours was 0.6.
  • GST-RANKL was administered intraperitoneally for 7 days to 7-week-old female C57BL / 6 mice at 2 rag / kg / day, and serum and femur were collected 1.5 hours after the seventh administration. Serum and femur obtained were measured for serum markers and bone density by pQCT.
  • the experiment was conducted in a total of 3 groups: 2 experimental groups administered once a day and PBS groups administered with PBS as a comparative control.
  • the collected femurs were 0.6 mm, 0.8 mm proximal to the growth plate by pQCT, 1. Bone mineral density of cancellous bone was measured at 0 mm.
  • LFM-A13 is a drug that inhibits the activity of Tec kinase by binding specifically to the ATP binding region of Btk belonging to Tec family kinase (Mahajan et al., J. Biol. Chem., 274, 9587-9599, 1999; Fer ⁇ des et al., J. Leukoc. Biol., 78, 524-532, 2005).
  • GST-RANKL was intraperitoneally administered at a dose of 1 mg / kg every 7 hours to 7-week-old C57BL / 6N female mice, and ovariectomy (0VX) or sham surgery (Sham) 24 hours after the second administration After 24 hours, raloxifene 1 mg / kg and 10 mg / kg were orally administered every 24 hours for 14 days.
  • PBS i. P.
  • ultrapure water P. O.
  • Serum opal femurs were collected 24 hours after the administration of raloxifene on day 14.
  • the collected serum was measured for bone resorption markers (Ca, TRAP-5b) and osteogenesis markers (ALP) in the blood.
  • the femur was measured proximal to the growth plate by pQCT.
  • the bone density, bone mineral content, and cortical bone thickness were measured at a distance of 1.0 mm. Image analysis was performed by micro CT.
  • the GST-RANKL-treated 0VX group showed an increasing trend compared to the PBS-treated Sham group, but decreased significantly with raloxifene l mg / kg and 10 mg / kg respectively (p 0. 05, p 0 01) (Fig. 7 6).
  • raloxifene l mg / kg and 10 mg / kg respectively p 0. 05, p 0 01
  • Fig. 7 6 bone density measurements using pQCT
  • the total bone density was significantly decreased in the PBS-administered 0VX group compared to the PBS-administered Sham group, but the GST-RANKL-administered 0VX group showed a decrease but no significant difference.
  • raloxifene was administered at 10 mg / kg, and the cortical bone thickness was 92% (p.0. 01), 123% (p ⁇ 0.01), 133% (p ⁇ 0.01) increased (Fig. 7 9).
  • cortical bone thickness was 101% at a position of 0.8 and 1.0 mm proximal to the growth plate, respectively.
  • GST-RANKL was intraperitoneally administered at a dose of 1 mg / kg twice every 24 hours to 7-week-old female C57BL / 6N mice. After 24 hours from the second administration, wrinkles were continued for 10 days every 24 hours. Subcutaneous administration was performed at 40 and 80 ⁇ g / kg, respectively. PBS was administered as a comparative control of GST-RANKL and PTH administration. Serum and femur were collected from the mice 24 hours after the administration.
  • the collected serum was measured for bone resorption markers (Ca, TRAP-5b) and osteogenesis markers (ALP) in the blood, and femur was measured at 0.6, 0.8 on the proximal side of the growth plate by pQCT.
  • the total bone density, cortical bone density, cortical bone thickness, cortical bone mineral density, and cortical bone density of the diaphysis were measured at 1.0 mm. Image analysis was performed by micro CT.
  • cortical bone density is 0.8 on the proximal side of the growth plate
  • GST-RANKL was administered intraperitoneally at 12 mg / kg every 12 hours to male and female 12-week-old C57BL / 6N mice three times every 24 hours, and the serum and femur were evaluated 1.5 hours after the third dose. Collected. The collected serum is measured for bone resorption markers (Ca, TRAP-5b) and bone formation markers (ALP) in the blood, and the femur is checked for bone density by dual energy X-ray absorptiometry (DEXA). Measurement was performed and image analysis was performed by micro CT.
  • Ca, TRAP-5b bone resorption markers
  • ALP bone formation markers
  • DEXA dual energy X-ray absorptiometry
  • Serum TRAP-5b was significantly increased in males compared to PBS following GST-RANKL administration (p 0. 05). Although Ca and ALP showed an upward trend, no significant changes were observed (Fig. 89). On the other hand, Ca and TRAP-5b in blood were significantly increased in females by GST-RANKL administration compared to PBS administration (p 0. 05, p 0. 01). However, although ALP showed an upward trend, there was no significant change ( Figure 89). As a result of bone density measurement by DEXA, the total bone density decreased by 8% (p ⁇ 0.05) and 6% (p ⁇ 0. 05) in both males and females in the GST-RANKL administration group compared to the control group. (Fig. 90).
  • a model animal for osteopenia can be rapidly produced by a simple mechanism of direct differentiation / activation promotion of osteoclasts by administration of soluble RANKL or a fusion protein of soluble RANKL and an epitope tag. Can be created.
  • drug evaluation can be performed quickly.
  • osteopenia animal model of the present invention since osteoclast differentiation / activation occurs directly by RANKL, the effect of the bone resorption inhibitor on bone destruction caused by pure osteoclast action can be evaluated.
  • a model animal for bone loss If attention is paid to the speed with which the bone mass is restored after the production, it is possible to evaluate a bone mass increasing drug.
  • the ovary which normally takes several weeks to produce, with a physiologically similar hormone balance to menopause
  • a bone loss model animal can be created more rapidly than an excised model.
  • the osteopenia model animal of the present invention can be used for screening drugs used for the treatment of bone metabolic diseases and the like, and can also be used as a research animal for bone metabolic diseases.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention porte sur un procédé de production d'un animal modèle d'ostéopénie par l'administration de RANKL, et sur un animal modèle d'ostéopénie. L'invention porte spécifiquement sur un procédé de production d'un animal modèle d'ostéopénie, lequel procédé comprend l'administration de RANKL soluble ou d'une protéine de fusion de RANKL soluble et d'un marqueur d'épitope à un animal non humain pour stimuler la différenciation et l'activation d'un ostéoclaste dans l'animal non humain. L'invention porte également sur un animal modèle d'ostéopénie obtenu par le procédé.
PCT/JP2007/070309 2006-10-11 2007-10-11 Animal modèle d'ostéopénie WO2008044797A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US12/445,050 US8334261B2 (en) 2006-10-11 2007-10-11 Osteopenia animal model
EP11169483.2A EP2389803B1 (fr) 2006-10-11 2007-10-11 Méthodes utilisées d'une modèle animal pour l'ostéopénie
CA2666443A CA2666443C (fr) 2006-10-11 2007-10-11 Animal modele d'osteopenie
EP07830043A EP2084961A4 (fr) 2006-10-11 2007-10-11 Animal modéle d'ostéopénie
JP2008538785A JP5219823B2 (ja) 2006-10-11 2007-10-11 骨量減少症モデル動物
AU2007307504A AU2007307504B2 (en) 2006-10-11 2007-10-11 Osteopenia animal model

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP2006278029 2006-10-11
JP2006-278029 2006-10-11
JP2007-095017 2007-03-30
JP2007095017 2007-03-30
JPPCT/JP2007/063871 2007-07-05
PCT/JP2007/063871 WO2008044379A1 (fr) 2006-10-11 2007-07-05 Modèle animal de perte osseuse

Publications (1)

Publication Number Publication Date
WO2008044797A1 true WO2008044797A1 (fr) 2008-04-17

Family

ID=39282981

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2007/070309 WO2008044797A1 (fr) 2006-10-11 2007-10-11 Animal modèle d'ostéopénie

Country Status (1)

Country Link
WO (1) WO2008044797A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046644A1 (fr) 1997-04-15 1998-10-22 Snow Brand Milk Products Co., Ltd. Nouvelle proteine et methode de production associee
JP2002509430A (ja) 1996-12-23 2002-03-26 イミュネックス・コーポレーション Ｔｎｆスーパーファミリーのメンバーであるｎｆ−ｋａｐｐａ ｂの受容体アクティベーターに対するリガンド
JP2004526748A (ja) * 2001-03-22 2004-09-02 バーンズ − ジューウィッシュ・ホスピタル Ｒａｎｋリガンド融合タンパク質を使用する骨形成の刺激

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002509430A (ja) 1996-12-23 2002-03-26 イミュネックス・コーポレーション Ｔｎｆスーパーファミリーのメンバーであるｎｆ−ｋａｐｐａ ｂの受容体アクティベーターに対するリガンド
WO1998046644A1 (fr) 1997-04-15 1998-10-22 Snow Brand Milk Products Co., Ltd. Nouvelle proteine et methode de production associee
JP3523650B2 (ja) 1997-04-15 2004-04-26 三共株式会社 新規蛋白質及びその製造方法
JP2004526748A (ja) * 2001-03-22 2004-09-02 バーンズ − ジューウィッシュ・ホスピタル Ｒａｎｋリガンド融合タンパク質を使用する骨形成の刺激

Non-Patent Citations (23)

* Cited by examiner, † Cited by third party
Title
DE WINTER ET AL., CALCIF TISSUE RES, vol. 17, 1975, pages 303
FERNANDES ET AL., J. LEUKOC. BIOL., vol. 78, 2005, pages 524 - 532
GEUSENS ET AL., J BONE MINER RES, vol. 6, 1991, pages 791
GLOBUS ET AL., J BONE MINER RES, vol. 1, 1986, pages 191
JUJI T. AND TANAKA S.: "RANKL no Vaccine Chiryo", THE BONE, vol. 17, no. 5, 1 September 2003 (2003-09-01), pages 81 - 83, XP008115667 *
KISHIMOTO Y. ET AL.: "GST ga Yugo suru Koto ni Yori RANKL no Kassei wa Zokyo Sareru", DAI 24 KAI THE JAPANESE SOCIETY FOR BONE AND MINERAL RESEARCH GAKUJUTSU SHUKAI PROGRAM SHOROKUSHU, 6 July 2006 (2006-07-06), pages 252, XP003018737 *
LACEY ET AL., CELL, vol. 93, 1998, pages 165
MAHAJAN ET AL., J. BIOL. CHEM., vol. 274, 1999, pages 9587 - 9599
MITLAK B.H. AND COHEN F.J.: "SELECTIVE ESTROGEN RECEPTORS MODULATORS A LOOK AHEAD", DRUGS, vol. 57, no. 5, May 1999 (1999-05-01), pages 653 - 663, XP002948164 *
MIZUNO A. ET AL.: "Transgenic mice overexpressing soluble osteoclast differentiation soluble osteoclast differentiation factor (s-ODF) exhibit severe osteoporosis", J. BONE MINER. METAB., vol. 20, 2002, pages 337 - 344, XP009065128 *
SATO K.: "Hakotsu Saibo Shigeki Inshi -Kayosei RANKL o Chushin ni-", JAPANESE JOURNAL OF CLINICAL MEDICINE, vol. 62, no. 860, 28 December 2004 (2004-12-28), pages 228 - 231, XP008110300 *
See also references of EP2084961A4 *
THOMPSON D.D. ET AL.: "FDA guidelines and animal models for osteoporosis", BONE, vol. 17, no. 4, October 1995 (1995-10-01), pages 125S - 133S, XP008109752 *
THOMPSON ET AL., BONE, vol. 17, 1995, pages 125
TOMIMORI Y. ET AL.: "GST-RANKL Toyo ni yoru Kotsugensho-sho Model no Sakusei", THE 8TH ANNUAL MEETING OF JAPAN OSTEOPOROSIS SOCIETY PROGRAM SHOROKUGO, vol. 14, no. 57, 18 September 2006 (2006-09-18), pages 139, XP003018740 *
WADA T. ET AL.: "The molecular scaffold Gab2 is a crucial component of RANK signaling and osteoclastogenesis", NATURE MED., vol. 11, no. 4, April 2005 (2005-04-01), pages 394 - 399, XP008109754 *
WAKLEY ET AL., CALCIF TISSUE INT, vol. 43, 1988, pages 383
WITTRANT Y. ET AL.: "RANKL/RANK/OPG: new therapeutic targets in bone tumours and associated osteolysis", BIOCHIM. BIOPHYS. ACTA, vol. 1704, 2004, pages 49 - 57, XP004567005 *
WRONSKI ET AL., CALCIF TISSUE INT, vol. 42, 1988, pages 179
WRONSKI ET AL., CALCIF TISSUE INT, vol. 45, 1989, pages 360
WRONSKI ET AL., ENDOCRINOLOGY, vol. 123, 1988, pages 681
YASUDA ET AL., PROC NATL ACAD SCI USA, vol. 95, 1998, pages 3597
YASUDA H.: "'RANKL-RANK-OPG' Kotsu Soshosho Chiryo", vol. 3, no. 4, 1 October 2004 (2004-10-01), pages 16 - 21, XP003025835 *

Similar Documents

Publication Publication Date Title
Siddiqui et al. Physiological bone remodeling: systemic regulation and growth factor involvement
Lacey et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation
Redlich et al. Repair of local bone erosions and reversal of systemic bone loss upon therapy with anti-tumor necrosis factor in combination with osteoprotegerin or parathyroid hormone in tumor necrosis factor-mediated arthritis
Kondo et al. Cyclic adenosine monophosphate/protein kinase A mediates parathyroid hormone/parathyroid hormone‐related protein receptor regulation of osteoclastogenesis and expression of RANKL and osteoprotegerin mRNAs by marrow stromal cells
Karaplis et al. PTH and PTHrP effects on the skeleton
CA2681317C (fr) Peptide synthetique qui contient un peptide osteoplastique 1 (bfp1) permettant de stimuler la differenciation osteoblastique, et composition pharmaceutique comprenant ledit peptide synthetique
JP5749659B2 (ja) Ｂｍｐアンタゴニスト感受性が低下した骨形成タンパク質２（ｂｍｐ２）変異体
Hollnagel et al. Parathyroid Hormone Enhances Early and Suppresses Late Stages of Osteogenic and Chondrogenic Development in a BMP‐Dependent Mesenchymal Differentiation System (C3H10T½)
JP5219823B2 (ja) 骨量減少症モデル動物
Ruchon et al. Cellular localization of neprilysin in mouse bone tissue and putative role in hydrolysis of osteogenic peptides
Mukherjee et al. Insulin-like growth factor binding protein-5 in osteogenesis: facilitator or inhibitor?
WO2008044797A1 (fr) Animal modèle d'ostéopénie
NZ310391A (en) Polypeptides that stimulate bone growth
US8066980B2 (en) Osteogenic synthetic peptides, pharmaceutical compositions comprising the same, and medium containing the same
EP2083073B1 (fr) Reactif contenant une proteine fusionnee de rankl soluble avec un marqueur d'epitope
KR102358345B1 (ko) 개선된 골형성 단백질
Bidwell et al. Nmp4/CIZ closes the parathyroid hormone anabolic window
JP6105623B2 (ja) 核内因子κＢ活性化受容体（ＲＡＮＫ）をターゲティングするペプチドおよびそれらの適用
Le Henaff et al. Parathyroid Hormones
López-Herradón et al. Comparison of the osteogenic actions of parathyroid hormone-related protein (PTHrP) in diabetic and insulin-like growth factor-I (IGF-I) deficient mouse models.
Ueland et al. Bone metabolism and growth hormone deficiency
EP2311482A1 (fr) Peptides synthétiques ostéogènes de BMP-7, compositions pharmaceutiques et milieux de culture les contenant
Ferrari et al. Clinical and basic research papers-May 2010
Hancock Ultrastructural organisation and molecular interactions in the hypertrophic cartilage extracellular matrix
Bruzzaniti et al. High Bone Mass in Mice Lacking Cx37 Due

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: 07830043

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2008538785

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2666443

Country of ref document: CA

Ref document number: 12445050

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2007307504

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2007830043

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 1020097009575

Country of ref document: KR

ENP Entry into the national phase

Ref document number: 2007307504

Country of ref document: AU

Date of ref document: 20071011

Kind code of ref document: A