WO2008044797A1

WO2008044797A1 - Osteopenia model animal

Info

Publication number: WO2008044797A1
Application number: PCT/JP2007/070309
Authority: WO
Inventors: Yoshiya Tomimori; Hisataka Yasuda
Original assignee: Oriental Yeast Co., Ltd.
Priority date: 2006-10-11
Filing date: 2007-10-11
Publication date: 2008-04-17

Abstract

Disclosed are: a method for producing an osteopenia model animal by administering RANKL; and an osteopenia model animal. Specifically disclosed are: a method for producing an osteopenia model animal, which comprises administering soluble RANKL or a fusion protein of soluble RANKL and an epitope tag to a non-human animal to stimulate the differentiation and activation of an osteoclast in the non-human animal; and an osteopenia model animal produced by the method.

Description

Description Bone loss model animal Technical field

The present invention relates to an osteopenia animal model in which the bone mass is reduced as compared to a normal animal. Background art

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. In fact, 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.

On the other hand, ovariectomy (see non-patent documents 3 to 6), low calcium diet (see non-patent documents 7 and 8), and nerve resection (see non-patent document 9) as bone loss models. ), Immobilization by hind limb suspension (see Non-Patent Document 10), etc., but all took about 1 to 4 weeks to develop bone loss. For this reason, it took time to evaluate drugs such as bone resorption inhibitors (such as bisphosphonates and Cathepsin K inhibitors) and bone formation promoters (such as parathyroid hormone (PTH)). In addition, the above animal model indirectly activates osteoclasts by estrogen loss, increased PTH, etc., so how effective is it in vivo when evaluating drugs? It was not easy to prove.

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. As a result, 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) It was found that a model animal for osteopenia can be produced. 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.

That is, the present invention is as follows.

[1] 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.

[2] The method for producing an osteopenia animal model according to [1], wherein the epitag is glutathione-S-transferase. [3] 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.

[4] The method for producing an osteopenia model animal according to [3], wherein an osteopenia animal model can be produced within 50 hours.

[5] The method for producing an osteopenia model animal according to [4], wherein an osteopenia animal model can be produced within 24 hours.

[6] The bone loss model animal according to any one of [1] to [5], wherein the non-human animal is an animal belonging to rodents.

[7] The method for producing a bone loss model animal according to [6], wherein the non-human animal is a mouse.

[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.

[9] Furthermore, 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.

[10] 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].

[1 2] The bone loss model animal according to [1 1], wherein the bone resorption marker level in the body is increased compared to normal individuals.

[1 3] The bone loss model animal according to [1 1], wherein bone density and / or unit bone mass have decreased compared to normal individuals.

[1 4] Furthermore, the osteopenia animal model according to [1 3], wherein the number of osteoclasts and / or the number of trabeculae has decreased compared to normal individuals.

[1 5] 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.

[1 6] 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 7] Increased bone mass, increased bone resorption marker level, increased bone density, increased unit bone mass, increased trabecular number, decreased osteoclast number in osteopenia model animals, Judgment is made using at least one selected from the group consisting of a decrease in osteoblastic surface and an increase in bone mass observed by CT as an index. Method.

[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.

[1 9] 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, Judgment is made using at least one selected from the group consisting of an increase in osteoblastic surface and an increase in bone mass observed by CT as an index. how to.

[20] 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.

[2 1] 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.

This specification includes the contents of the Japanese patent applications 2006-278029, 2007-095017 and international patent application PCT / JP2007 / 063871 which are the basis of the priority of this application and the Z or drawings. To do. Brief Description of Drawings

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 | concentration.

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.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

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-κ B l igand) is a ligand of RANK (NF-κ 受容 receptor activator) that is a member of TNF Suno ヽ⁰ — Family, and is an intracellular domain (from the Ν end of RANK). A domain consisting of amino acids 1 to 48), a type 2 transmembrane protein with a transmembrane domain and an extracellular domain

(Special Table 2002-509430 Publication, International Publication No. W098 / 46644 Pamphlet (Patent No.

No. 3523650))). 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. Also included are 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. Further, 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 A protein having Here, 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. Usually, an epitope tag is used for purification of a fusion protein. In the present invention, 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 acid sequence DDDDK; SEQ ID NO: 1 1), S tag (amino acid sequence KETAAAKFERQHIDSC; SEQ ID NO: 12), CruzTag09 (amino acid sequence MKAEFRRQESDR; SEQ ID NO: 1 3), CruzTag22 (amino acid sequence MRDALDRLDRLA; SEQ ID NO: 14), CruzTag41 (amino acid sequence MKDGEEYSRAFR; SEQ ID NO: 15), Glu-Glu tag (amino acid sequence EEEEYMPME; SEQ ID NO: 16), Ha. 11 tag (Amino acid sequence CTPTDVPDYASL; SEQ ID NO: 17), KT3 tag (amino acid sequence PPEPET; SEQ ID NO: 18), thioredoxin, maltose-binding protein (MBP), immune globulin Fc region, and 3 galactosidase It is not limited to those listed here. Of these, dartathione-S-transferase is preferred.

In order to obtain a fusion protein of soluble RANKL and epitope tag, the genes that code for both are linked and expressed. Fusion of 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.

The nucleotide sequence of DNA encoding GST fused protein to the protein consisting of the 127th amino acid sequence from the 127th amino acid sequence to the 317th amino acid sequence in the amino acid sequence of RANKL in SEQ ID NOS: 19 and 20 and the protein The amino acid sequence of is shown. 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. In addition, 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. In addition, a vector in which a gene encoding an epitope tag such as glutathione-S-transferase is previously incorporated can also be used.

Introduction of DNA into a vector can be performed by a known method. 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.

Examples of 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. In this case, when the expression product is 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. For example, 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. In addition, when expressed as a fusion protein with a histidine tag, the protein having the histidine tag binds to the chelate column and can be purified using the chelate column. Furthermore, when any epitope tag is 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.

In addition, 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. When continuous administration is performed, the administration interval is not limited, and for example, administration may be performed for several days every day. Also, by adjusting the total dose, The degree of bone loss in the model animal can be adjusted, for example, when a single dose is given,

By increasing the dose at one time, a model animal with a large degree of bone loss can be produced.

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. In the following description, 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. As a control, animals that have been administered PBS or the like can be used as normal animals when creating a model animal for dose reduction.

(1) 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., and urinary bone resorption markers include urinary CTx or NTx, urinary hydroxyproline And urinary pyridinoline, deoxypyridinoline and the like. In addition, 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.

(2) Serum bone formation marker concentration (level) 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.

(3) 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. Depending on the dosage of soluble RANKL or fusion protein of soluble RANKL and Epitope tag, in the osteopenia animal model of the present invention, when the bone density of the femur or tibia is measured by pQCT, the growth plate For example, 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.

In addition, 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.For example, 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. When 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.

In addition, the amount of cortical bone salt decreases compared to normal animals. Bone mineral content is the amount of bone mineral (hydroxyapatite) and reflects bone density. Bone mineral content can be measured by pQCT. When 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.

Furthermore, cortical bone thickness is reduced compared to normal animals. Cortical bone thickness can be measured by pQCT. When 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.

(4) In bone morphometry, bone volume / total tissue volume (BV / TV), trabecular number (Tb. N), trabecular width (Tb. Th; trabecular thickness) Compared to decrease. 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. In the osteopenia animal model of the present invention, 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.

(5) 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.

(6) 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.

In addition, when 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.

(7) Normal bone thickness (0. Th; osteoid thickness, ES / BS; eroded surface / bone surface) and osteoclast tatsuki wrapping surface (Oc. S / Bs; osteoclast surface / bone surface) normal Increased compared to other animals. Osteoid refers to the state prior to calcification to become a bone matrix, and 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.

(8) When bone morphology is observed, bone loss is observed compared to normal animals. The bone shape can be measured by micro CT or the like.

(9) In the osteopenia animal model of the present invention, since estrogen is synthesized in the body, the blood estrogen concentration does not vary compared to normal animals. This is different from the model animal produced by extracting the conventional ovary. In addition, blood PTH levels do not increase compared to normal animals. This is different from the model animals produced by feeding animals with a low calcium diet.

Furthermore, in the model animals produced by removing the conventional ovaries and in the model animals produced by feeding animals with a low calcium diet, the blood OPG (osteoprotegerin) concentration decreases. It does not change in the osteopenia animal model of the invention. Further, 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. In this case, the ovaries are preferably removed after administration of RANKL. By combining ovariectomy and soluble RANKL, or administration of a soluble RANKL and an epitope protein, a bone loss model animal similar to a physiological menopausal state can be produced. 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.

Among the above features, 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. In addition, a decrease in cortical bone mineral content is also observed.

In addition, 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. . In other words, 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. Here, a severe bone loss model animal is a model animal in which the above-mentioned characteristics are strongly manifested. For example, bone resorption marker concentration (level) in body fluid force Normal animal of the same type (normal individual) 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 . In addition, it is a model animal whose bone density is relatively low compared to normal animals. When 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. Furthermore, in bone morphometry, animals with normal bone volume / total tissue volume (BV / TV), trabecular number (Tb. N) and trabecular width (Tb. Th; trabecular thickness) In Compared to normal animals, for example, it is 10% or more, preferably 20% or more, more preferably 30% or more, more preferably 40% or more, and particularly preferably 50% or more. Model animal. Furthermore, in bone morphometry, osteoid thickness (0. Th; osteoid thickness), resorption surface (ES / BS; eroded surface / bone surf ace) and osteoclast surface (0c. 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.

Therefore, as a typical example of the osteopenia animal model of the present invention, 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.

After administration of soluble RANKL and fusion protein of soluble RANKL and epotope tag to animals, the above characteristics gradually appeared over time, and after the characteristics appeared most prominently, this time, bone formation The above features are gradually lost and eventually return to normal. That is, the osteopenia animal model obtained by administering soluble RANKL and a fusion protein of soluble RANKL and an epitope tag is reversible with respect to the feature of bone loss.

In particular, among the above features, 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.

As will be described later, 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. In this case, 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. Can evaluate the effect of candidate substances. In addition, 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. Specifically, 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.

Furthermore, 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. Can do. 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. . It takes several weeks or more for a model animal that has just had its ovaries removed before the drug effect is recognized and the drug can be evaluated.However, when the bone loss model animal of the present invention with the ovaries removed is used. The evaluation can be performed within a period of less than half, for example, within 2 weeks, preferably within 1 week, more preferably within a few days. Known selective estrogen receptor modulators include raloxifene and the like. It can also be used to evaluate hormone-like compounds of unknown action and hormone receptor modulators. Evaluation of hormones or hormone receptor modulators etc. is based on whether hormones or hormone receptor modulators are administered to osteopenia model animals and whether or not the decreased bone mass increases in osteopenia model animals. Can be evaluated.

In addition, the 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

After administration of the RANKL and Epitope tag fusion protein, it is desirable to evaluate from the time when the bone mass continues to decrease and the bone loss declines to the time when the bone loss once increases again. In this case, 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. In this case, 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. Specifically, 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. Examples of 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.

Furthermore, 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.

Furthermore, in the 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. In addition, after administration of soluble RANKL or a fusion protein of soluble RANKL and an epitope tag, 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 present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Production of osteopenia model mice (1) Preparation of GST-RANKL

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. 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. Subsequently, it was washed with a washing buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM DTT, 0.1% (v / v) Triton X-100). Thereafter, elution was performed with a Glutathione solution (20 mM reduced glutathione, 50 mM Tris-HC1, pH 8.0). The molecular weight and purity of GST-RANKL purified by SDS-PAGE were confirmed and filtered. The molecular weight was 47.0 kDa and the purity was 95% or more. In addition, the endotoxin concentration was measured by a limulus amebocyte lysate assay and confirmed to be less than lEU / V g.

GST-RANKL administration study

GST-RANKL, 57 nmol (low dose) and 426 nmol in 10 7-week-old female C57BL / 6N mice

(High dose) was administered intraperitoneally three times every 24 hours, and whole blood was collected 1.5 hours after the third dose. A group to which PBS was similarly administered was used as a comparison target.

Blood samples collected from whole blood were measured for serum bone resorption parameters (calcium, CTx, TRAP-5b) and bone formation parameters {osteocalcin, al force phosphatase (ALP)}. Calcium was measured by the 0CPC method (丽 0, 272-21801) and CTx (Nordic

Bioscience Diagnostics), TRAP-5b (IDS Ltd, SB-TR103)

(Biomedical Technologies Inc.) It ELISA method trowel was measured and measured by ALP i Bessey-Lowry method (WAK0, 274-04401).

After blood collection, 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.

For the femur, using pQCT, 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

By administration of GST-RANKL, the Ca concentration in the blood increased significantly by approximately 1.4 times (P-0.01) at high doses (Fig. 1). CTx, a collagen metabolite, was approximately 1.5 times more significant (p> 0.01) compared to the control group at high doses (Fig. 2). As for TRAP-5b, the GST-RANKL high dose significantly increased by about 1.5 times (p 0.01) (Fig. 3). Regarding osteocalcin and ALP in blood, there was no change in GST-RANKL administration at both high and low doses (Figures 4 and 5). Bone density and bone morphometry

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).

As a result of bone morphometry, 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).

When the bone morphology of the femur was measured by micro CT, significant bone loss was observed in the high-dose GST-RANKL administration group (Fig. 10).

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.

High dose of GST-RANKL increased bone resorption parameters, decreased bone density, unit bone mass, decreased number of trabeculae, and increased number of osteoclasts. Use as a model mouse for osteopenia. Therefore, it is simpler than the conventional method, and a bone loss model mouse can be produced in a shorter time. W

Osteoblast surface

High doses of GST-RANKL resulted in increased osteoclast numbers, decreased bone mass, and bone resorption. Further examination of the osteoblast surface revealed that it was significantly elevated (Figure 11). This suggests that this is a model in which bone formation is promoted by an increase in the number of osteoclasts and increased bone resorption due to the activation of osteoclasts, that is, the bone resorption and bone formation coupling are recognized. ing. However, there is no change in the osteocalcin concentration in serum, which is a marker for osteoblasts, or the activity of phosphatase activity, suggesting that osteoblast activation has just begun. Example 2 Production of osteopenia model mice (2)

GST-RANKL was prepared in the same manner as in Example 1.

RANKL administration study

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.

Blood samples collected from whole blood were measured for serum bone resorption parameters (calcium, CTx, TRAP-5b) and bone formation parameters (osteocalcin, al force phosphatase (ALP). Calcium was measured by the 0CPC method (WAK0, 272-21801), CTx (Nordic Bioscience Diagnostics) TRAP—5b (IDS Ltd, SB-TR103) and osteocalcin (Biomedical Technologies Inc.) ί ELISA method ί The measurement was performed by the ALP It Bessey-Lowry method (WAK0, 274-04401).

After collecting the whole blood, 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.

Each measured value was tested by comparison with the control group by Dunnett method.

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. Regarding 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.

When bone density was measured using PQCT, at the position of 0.6 thigh proximal to the growth plate, GST-RANKL213 nmol administration was 11%, 426 nmol administration was 19% (P-0.01), 852 A 30% (P 0.01) decrease in bone density was observed with nmol administration. Each of 0.8 mm is 23 ° /. (P-0.01), 29% (p-0.01), 42% (p-0.01), 1.0 mm, 23 ° / each. (P <0.01), 31% (p <0.01), 44% (p <0.01) decreased bone density (Fig. 15). Such a dose-dependent decrease in bone density was also confirmed by image analysis using micro CT (Fig. 16). Based on the above results, the conventional method required several weeks for preparation of model mice and required special techniques such as ovariectomy (0VX). However, in this method, a simple method of intraperitoneal administration was used. Model mice can be made in time. In addition, 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. Example 3 Evaluation of therapeutic agents for osteoporosis using mice administered with GST-RANKL

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) and bone formation parameters

(Osteocalcin, ALP) was measured. The femur was measured for bone density using pQCT and micro CT. Each measured value was tested by comparison with the control group by Dunnett's method.

TRAP-5b, a bone resorption marker, showed a significant increase of approximately 1.5 times in the GST-RANKL-treated group compared to the control group (P 0.01), and CTx was 1.4-fold. A significant increase was observed (P 0. 05). However, 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. In addition, 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. These results were also confirmed in the image analysis using micro CT. Fig. 21 shows the bone density of the femur, and 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 (manufactured by Peprotech) 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. Although it was not the difference, it showed an upward trend of 1.25 times (Figure 25). There was no change in osteocalcin and al force rephosphatase, which are bone formation markers (Figs. 26 and 27). The bone density of the femur decreased significantly by about 20% (Fig. 28). From the above, 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. Example 5 Evaluation of osteopenia model mouse

Morphological measurement

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), and osteoclast surface (Oc. S / BS) increased approximately L 8 times (p> 0.001) (Fig. 3 2-3 5). When this section was stained with TRAP, more osteoclasts were observed compared to the control mouth group (Fig. 36). From this result, it was found that not only serum and bone density but also bone loss can be evaluated by measuring bone morphology such as the number of osteoclasts and osteoclast surface. In addition, 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. However, depending on the dose of GST-RANKL, 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. Examination of drug evaluation using bisphosphonate (risedronate)

7-week-old C57BL / 6 female mice were dosed with risedronate at 3, 10, and 30 μg / kg.

It was administered subcutaneously every 24 hours from the day before GST-RANKL administration until the end of the experiment. 3rd time

After 5 hours of GST-RANKL administration, 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.

Although there was no significant change in Ca, 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. In addition, 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. 01), 35% (p <0. 01), 35% (p <0. 01). This decrease in bone density is 12% (ρ 0. 05) and 18% (ρ at doses of 3 μg / kg with risedronate at 0.6, 0.8, and 1.0 mm, respectively. 0.05), 24%, 10 g / kg at 13% (p 0.05), 28%, 30%, 30 μg / kg at 10% (p 0.05), 18% (p 0. 05) and 20% (p <0. 05) (Fig. 3 8). These results were also confirmed in image analysis using micro CT (Fig. 39). Examination of drug evaluation comparing multiple bisphosphonates

7-week-old female C57BL / 6N mice were dosed with etidronate at 3, 30 mg / kg, alendronate at 3, 30, 300 μg / kg, risedronate at 1, 10, 100 μg / 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.

Ca significantly decreased compared to RANKL administration when alendronate was administered at 300 IX g / kg and risedronate at 1 or 100 g / kg, respectively (p 0.05, p 0.01, p < (O. 01) was seen (Fig. 40).

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). In addition, risedronate administration was 1, 10, and 100 β, respectively. W was significantly suppressed at a dose of 200 g / kg (p 0.05, p 0.01, p <0.01) (Figure 40). In ALP, 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. There was a decrease of p (0.01), 13% (p 0.01), and 18% (p 0.01). This decrease in bone density was reduced by 2 mg to 4% (p 0.05) at all positions at 30 mg / kg after administration of etido mouthnate. Similarly, alendronate decreased to 1% (p <0.01) and 5% (p0.05) at 0.6 and 0.8 mm, respectively, at a dose of 300 μg / kg. Was suppressed. With risedronate administration, the decrease was suppressed to 1% at 0.6 mm at a dose of 100 μg / kg (P 0.01) (Fig. 41). These results were also confirmed in image analysis using micro CT (Fig. 42).

Based on the above results, it was possible to evaluate the pharmacological effects of pre-administration of bisphosphonates from the 1st generation to the 3rd generation one day before RANKL administration. The doses that showed this effect were 30 mg / kg for etidronate, 300 μg / kg for alendronate, and 100 wg / kg for risedronate, reflecting the strength of their respective pharmacological effects. . From these results, it was found that the bone loss model with RANKL administration can be applied to screening and evaluation of new drugs with stronger pharmacological effects. In addition, the time from bisphosphonate pre-administration to mouse dissection was only 73.5 hours, and pharmacological effects could be evaluated in just 4 days, including measurement of bone metabolism markers and bone density. It was also found that it can be applied as a rapid bone resorption inhibitor evaluation system. Short-term 0VX mouse production method

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. As for 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.In the GST-RANKL-treated Sham (day 27) and OVX (day O) groups, Compared to PBS control Sham (day 27) group and Sham (day O) group of each control group, approximately 0.6% at 0.6 mm, approximately 4%, approximately 14% at 0.8 mm (p 0.01), At about 9% and 1.0 mm, 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).

Based on the above results, 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.

As a result of bone density measurement by pQCT, compared with PBS group, it was 5, 7, 9% at 0.6 mm at 0, 1, 4 weeks, 10, 11, 15%, 1.0 mm at 0.8 mm, respectively. Showed a decrease in bone density of 16, 13, and 11%. At all positions of 0.6 mm, 0.8 ram, and 1.0 mm, bone density decreased as compared to the PBS group until 4 weeks after RANKL administration, and then PBS administration at 6 weeks. It recovered to the same level as the group (Fig. 47). These results were also confirmed by image analysis using micro CT (Fig. 48).

From the above results, it was found that the 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.

Evaluation of bone mass increase by PTH

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.

Serum ALP decreased about 24% in all GST-RANKL administration groups, and Ca was not changed. In TRAP-5b, 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). Compared with the PBS-administered + Sham group, there was no difference between the PBS-administered 0VX group and the RANKL-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. When PTH was administered to the PBS + Sham group, a significant increase in bone density was observed only at the 1.0 mm position (P 0.05). Even when PTH was administered to the 0VX group, an increase in bone density was observed but not significant. On the other hand, when PTH was administered to the GST-RANKL + Sham group, the decrease in bone density was significantly suppressed (P 0.05), which was higher than that in the PBS + Sham group (Fig. 50). These results were also confirmed by image analysis using micro CT (Fig. 51). Based on the above results, model mice with osteopenia due to GST-RANKL administration can promote not only bone resorption inhibitors such as bisphosphonate but also bone formation like PTH by changing the evaluation period and period. It was found that it can also be used for evaluation of agents. In addition, it was found that administration of GST-RANKL activated bone formation, enabling PTH to be evaluated in a shorter period of time and with higher sensitivity. Evaluation in Oss

C57BL / 6N mice GST-RANKL was intraperitoneally administered 3 times every 24 hours at 1 mg / kg in 7-week-old males, and femur and serum were collected 1.5 hours after the third administration and administered PBS Compared to the 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.

Image analysis was performed using CT.

Ca increased by 7% compared to the PBS-administered group, but there was no significant difference, and TRAP-5b There was a significant increase of about 65% (p <0.003). There was no change in ALP (Fig. 52). The femur bone density was measured at 0.6, 0.8, and 1.0 mm proximal to the growth plate.It was about 6% at 0.6 mra and about 10% at 0.8 mm. At 1.0 mm, a decrease of about 15% (p 0.03) was observed (Fig. 53). These results were also confirmed by image analysis using micro CT (Fig. 54).

From these results, it was found that the osteopenia model mouse by GST-RANKL administration can be used regardless of sex, unlike the conventional ovariectomized model mouse. In addition, hind limb suspension, low calcium diet, nerve resection, etc. are known as models that can be used in males, but the GST-RANKL administered bone loss model is superior in that it can be created in a shorter time. Evaluation with other strains of mice

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. .

Although there was no change in Ca in the blood compared with the control group, TRAP-5b increased 1.8 times compared with the PBS-treated group (p 0.01). There was no change in ALP (Figure 5 5). The bone density of the femur was measured at 1.0, 1.2, 1.4 orchid position proximal to the growth plate. About 1.0% at 1.0 mm compared to the control group, 1.2 mm Then, about 20% and 1.4 mm, a decrease of about 22% (p 0.05) was observed (Fig. 56). These results were also confirmed by image analysis using micro CT (Fig. 57). From these results, it was found that bone loss model mice by RANKL administration can be produced not only in C57BL / 6 mice but also in other strains of mice. Study using Fisher rats.

Fischer rats 7 week old females received GST-RANKL at 1 mg / kg three times by intraperitoneal route every 24 hours. Serum and femur were collected 1.5 hours after the third administration and PBS was administered. Compared to the group. Serum is used to measure bone resorption markers Ca, TRAP-5b (using IDS Ltd, SB-TR102 kit) and bone formation marker ALP. Femur is measured by bone density and micro CT using pQCT. Image analysis was performed.

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 く 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.

Single dose of GST-RANKL

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.

Serum human RANKL concentration rose rapidly after administration and peaked after 4 hours. After that, it decreased rapidly and became undetectable after 24 hours (Fig. 61). There was no change in Ca in serum, and TRAP-5b significantly increased about 1.7 times (p 0.01) 12 hours after administration. In addition, ALP showed a significant decrease of about 30% (p <0.01) in the GST-RANKL administration group (Fig. 62). 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. About 4%, about 5% for mm, about 9% for about 0.8 mm, about 9%, about 12% for 1.0 mm (p <0.05), about 15% (p <0.01) Decrease was observed (Fig. 63). These results were also confirmed by image analysis using micro CT (Fig. 64).

This result shows that GST-RANKL can significantly reduce bone mass with a single dose of 1 mg / _kg . However, comparing the single dose and the double dose, the two doses are 0.6 mm, 0.8 mm, and 1.0 mm proximal to the growth plate. Although a significant decrease in bone density is observed, this is desirable. It shows that the density can be reduced. When GST-RANKL was administered twice, the bone loss reduction effect increased compared to the dose of 0.5 rag / kg and 1 mg / kg when the dose was 2 mg / kg. However, the degree of bone loss can be freely adjusted by increasing the dose. That is, even with a single dose, for example, by setting the dose to 2 mg / kg, a significant bone density can be achieved at all three points: 0.6 mm, 0.8 ram, and 1.0 mm on the proximal side of the growth plate. It can be expected that there will be a decrease.

Daily administration of GST-RANKL for 7 days

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.

Ca, a bone resorption marker, increased by about 20%, but no significant difference was obtained. TRAP-5b also showed an upward trend of about 30%, but no significant difference. ALP was also significantly increased by about 30% (p 0.02) (Fig. 6 5). The bone density of the femur was measured at a distance of 0.6, 0.8, 1.0 difficult to the proximal side of the growth plate. At 0. 05) and 0.8 mm, there was a decrease of about 32% (p <0. 05), and at 1.0 mm, a decrease of about 47% (p <0.01). (Figure 6 6). These results were also confirmed by image analysis using micro CT (Fig. 67).

Based on the above results, it was speculated that a bone pulling phenomenon between bone resorption and bone formation occurred due to an increase in the bone formation marker as well as the bone resorption marker after continuous administration for 7 days. Therefore, it was found that this model mouse can be used not only for bone loss and osteoporosis research but also for elucidating the coupling phenomenon.

GST-RANKL administered to mouse calvaria

GST-RANKL was administered to the calvaria for 3 days in 8-week old female C57BL / 6N mice. Experiment started

On day 5, femurs were collected and their bone density was evaluated by pQCT. As experimental groups, GST-RANKL was administered at 0.5 mg / kg twice a day, experimental group 1, GST-RANKL was administered at 1 mg / kg.

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.

As a result of bone density measurement by PQCT, bone density decreased at all measured positions in experimental group 1 compared to PBS group. In experimental group 2, bone density decreased at 0.8 mm and 1.0 mm proximal to the growth plate (Figure 6 8).

In addition, when comparing experimental group 1 and experimental group 2, the bone density decreased more in experimental group 1, so that the same dose of GST-RANKL per day was more than a single dose. It was found that the decrease in bone density was more remarkable with multiple administrations. Example 6 Evaluation of RANKL signal inhibitory compounds using osteopenia model mice

Seven-week-old C57BL / 6N female mice were administered GST-RANKL three times every 24 hours with 20 μg GST. LFM-A13 (20 mg / kg body weigh) or physiological saline was administered 1 hour before GST-RANKL administration. Blood Ca and bone morphology were measured 1.5 hours after the third GST-RANKL administration. In addition, 3D image analysis was performed by micro CT.

Tec and Btk, one of the Tec family kinases, have been suggested to play an important role in osteoclast differentiation from analyzes using K0 mice. 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 匪 des et al., J. Leukoc. Biol., 78, 524-532, 2005).

From the results of microscopic image analysis, administration of LFM-A13 suppressed bone loss due to GST-RANKL administration (Fig. 69). In bone morphometry, the decrease in unit bone mass (BV / TV) by GST-RANKL administration was significantly (p 0.01) suppressed by LFM-A13 administration (Fig. 70). Bone width (Tb. Th) and trabecular number (Tb. N) decrease with GST-RANKL administration, but significantly with LFM-A13 administration (p 0. 05 and p 0. 01), respectively. The decrease was suppressed (Figures 7 1 and 7 2). The number of osteoclasts (N. 0c / B. Pm) and resorption surface (ES / BS) are increased by GST-RANKL administration, but each LFM-A13 administration has a significant increase (p> 0.01). (Figures 7 3 and 7 4). The blood Ca concentration increased with GST-RANKL administration but was significantly (p 0.05) suppressed by LFM-A13 administration (Fig. 75). Based on the above results, GST-RANKL-administered osteopenia model can be applied to evaluation of drugs that inhibit RANKL signaling and mechanism research including osteoclast differentiation Example 7 Examination of drug evaluation using selective estrogen receptor modulator (raloxifene)

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.) And ultrapure water (P. O.) were used as comparison targets for GST-RANKL and raloxifene, respectively. 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 Ca and TRAP-5b levels in blood did not change significantly, and only a significant increase in Ca was observed in the PBS-treated 0VX group compared to the PBS-treated Sham group (Fig. 76). ALP

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). In bone density measurements using pQCT, there was no significant change in cancellous bone density between groups. On the other hand, 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. GST-RANKL administration In the 0VX group, 10% (p-0.05) and 11% at a position of 1.0 mm proximal to the growth plate, respectively, by administration of laguchi xifene l mg / kg and 10 mg / kg (p 0. 05) increased (Fig. 7 7). In addition, raloxifene 10 mg / kg in the GST-RANKL-administered 0VX group resulted in a total bone density of 10% (p-0.05) at 0.6 and 0.8 mm, respectively, closer to the growth plate. 11% (p <0. 05) increased (figure

7 7). For cortical bone mineral density by pQCT measurement, PBS administration 0VX group and GST-RANKL administration

In the 0VX group, ³ 4% (p <0. 05) and 49% (p <0. 01) decrease at a position of 1.0 mm proximal to the growth plate, respectively, compared to the PBS-treated Sham group. It was seen (Figure 7 8). GST-RANKL administration (In the group, raloxifone was administered at 10 mg / _kg , and the cortical bone mineral content was 87% (p <0. 05), 118% (p <0. 01), 137%

(p く 0.01) increased (Fig. 7 8). In the GST-RANKL-administered 0VX group, raloxifene 1 mg / kg was administered, and cortical bone mineral density was 93% at a position of 0.8 mm and 1.0 mm proximal to the growth plate. 05), increased by 136% (p 0.01) (Fig. 7 8). Similarly, the cortical bone thickness measured by pQCT was ³⁵ % at a position of 1.0 mm proximal to the growth plate in the PBS-treated 0VX group and GST-RANKL-treated 0VX group compared to the PBS-treated Sham group. p (0.05), 49% (p 0.01) showed a significant decrease (Fig. 7 9). In the 0VX group treated with GST-RANKL, 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). In addition, when raloxifene l mg / kg was administered in the 0VX group treated with GST-RANKL, cortical bone thickness was 101% at a position of 0.8 and 1.0 mm proximal to the growth plate, respectively.

(p 0.05) and 137% (p 0.01) increased (Fig. 79). These results were also confirmed in image analysis using micro CT (Fig. 80). From these results, it was found that osteopenia model mice with RANKL administration can be used for drug evaluation of selective estrogen receptor modulators such as raloxifene. Example 8 Evaluation of bone mass increase using low dose PTH

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 μg / 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.

There was no significant change in Ca and TRAP-5b in the blood, and in the GST-RANKL-administered group, administration of 80 g / kg PTH resulted in a slight but significant decrease in Ca (p 0. 05) ( Fig 8 In the GST-RANKL-treated group, Do-method and ALP were significantly reduced by administration of 80; U g / kg of PTH (p 0.01), and also decreased by administration of 40 g / kg of PTH (Fig. 8 1). In the GST-RANKL administration group, the cancellous bone density was 25% (p 0.01) at the position of 0.6 and 0.8 mm proximal to the growth plate compared to PBS S administration with PTH administration of 40 // g / kg. , 28%

(p 0.05) increased (Fig. 82). In the GST-RANKL administration group, administration of 80 g / kg PTH increased the cancellous bone density by 20% (p <0.01) at a position 0.6 mm proximal to the growth plate (Fig. 82). On the other hand, the total bone density in the GST-RANKL administration group was 14 (%) at a position of 0.6, 0.8, and 1.0 mm proximal to the growth plate compared to PBS administration with 4 () g / kg PTH administration, respectively. (p 0.01), 17% (p 0.01), 19% (p <0.01) (Fig. 83). In addition, in the GST-RANKL administration group, 10% (p 0.01) and 13% were similarly administered by 80 / ^ g / kg PTH administration.

(p <0.01), increased by 13% (p <0.01) (Fig. 83). 40 in the GST-RANKL group

; zg / kg sputum administration compared to PBS administration, cortical bone density is 0.8 on the proximal side of the growth plate,

4 ° / at 1.0 mm position. (P <0.01), increased by 5% (p <0 01) ((Fig. 8 4).

In the 0VX. Group treated with GST-RANKL, administration of 80 g / kg PTH increased cortical bone density by 3% (p 0.05) at a position of 1.0 mm proximal to the growth plate (Fig. 84). In cortical bone mineral content

In the GST-RANKL administration group, 80% (p 0.01) and 123% (p 0.01),

Increased by 100% (p <0.01), and PTH of 80 μg / kg was ⁴⁶ % (p + 0 05),

It increased by 83% (p 0.01) and 77% (p 0.01) (Fig. 85). Similarly, in terms of cortical bone thickness

In the GST-RANKL administration group, compared to PBS administration with PTH of 40 g / kg, 86% (p 0.01) and 127% (p 0.01),

98% (p 0.01) ± 50%, 50% (p 0.05) by 80 // g / kg PTH administration,

They increased by 89% (p 0.01) and 79% (p 0.01) (Fig. 86). On the other hand, cortical bone density in the diaphysis was reduced by 3% (p <0.01) by GST-RANKL compared to the control group treated with PBS (Fig. 87). In the GST-RANKL-administered group, the 40 and 80 g / kg sputum doses increased by 3% (p 0.01) and 3% (p 0.01), respectively, compared to PBS (Fig. 87). These results were also confirmed in image analysis using micro CT (Fig. 88). From these results, it was found that osteopenia model mice with RANKL administration can detect the bone mass-increasing effect of low dose PTH in a short period of time. Example 9 Production of a model mouse with bone loss by administering a soluble protein of RANKL and GST to a 12-week-old mouse

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.

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). These results were also confirmed in the image analysis using micro CT (Fig. 9 1) _{0 From} the results of male and female mice at 12 weeks of age, the model for osteopenia due to RANKL administration was selected at the age of weeks. It was also found that it can be used for relatively older mice. It was also found that model mice with osteopenia due to RANKL administration can be produced at 12 weeks of age regardless of sex. Industrial applicability

According to the present invention, 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. By using the osteopenia animal model obtained in this way, drug evaluation can be performed quickly. Further, in the 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. In addition, 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. Furthermore, by combining ovariectomy and administration of soluble RANKL or soluble RANKL and epitopic tag fusion protein, 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.

All publications and patent applications cited in this specification are incorporated herein by reference in their entirety. Sequence listing free text

SEQ ID NOs: 3 to 1 8

Claims

The scope of the claims

1. Osteopenia, comprising administering soluble RANKL or a fusion protein of soluble RANKL and an epitope tag to a non-human animal, and promoting differentiation and activation of osteoclasts in the non-human animal. How to create a model animal.

2. The method for producing an osteopenia model animal according to claim 1, wherein the epitope tag is glutathione-S-transferase.

3. The bone mass according to claim 1 or 2, wherein a model animal for osteopenia 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.

4. The method for producing an osteopenia model animal according to claim 3, wherein the osteopenia animal model can be produced within 50 hours.

5. The method for producing an osteopenia model animal according to claim 3, wherein the osteopenia animal model can be produced within 4 hours.

6. The osteopenia animal model according to any one of claims 1 to 5, wherein the non-human animal is an animal belonging to rodents.

7. The method for producing an osteopenia animal model according to claim 6, wherein the non-human animal is a mouse or a rat.

8. A method for producing an osteopenia animal model according to any one of claims 1 to 7, wherein the dosage of the soluble RANKL or the fusion protein of soluble RANKL and an epitope tag is changed, A method for producing an osteopenia model animal, which produces an osteopenia model animal of different severity.

9. Furthermore, 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 animal model according to any one of claims 1 to 8, Production method.

10. A model animal for osteopenia can be produced within 72 hours after administration of soluble RANKL or a fusion protein of soluble RANKL and an epitope tag to a non-human animal.

9. A method for producing an osteopenia animal model according to 9.

1 1. Bone loss produced by the method according to any one of claims 1 to 10. A hypoxic model animal.

1 2. The bone loss model animal according to claim 1, wherein the bone resorption marker level in the body is increased as compared with a normal individual.

1 3. The osteopenia animal model according to claim 11, wherein bone density and Z or unit bone mass are reduced as compared to normal individuals. '

14. Furthermore, the osteopenia animal model according to claim 13, wherein the number of osteoclasts and / or the number of trabeculae has decreased compared to a normal individual.

15. The osteopenia model according to any one of claims 11 to 14, wherein at least one of blood estrogen concentration, blood PTH concentration, and blood 0PG concentration is not changed compared to a normal individual. animal.

1 6. A bone resorption inhibitor or a bone resorption inhibitor candidate substance is administered to the osteopenia animal model according to any one of claims 1 1 to 15 and the osteopenia animal model is used. This 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 decreased bone mass increases as an index, and is effective in suppressing bone resorption when the bone mass increases. A method for evaluating a bone resorption inhibitor or a bone resorption inhibitor candidate substance that is judged to be present.

1 7. Increased bone mass, increased bone resorption marker level in bone loss model animals, increased bone density, increased unit bone mass, increased trabecular number, decreased osteoclast number, The bone resorption inhibitor or the bone absorption inhibitor candidate substance according to claim 16, which is determined using as an index at least one selected from the group consisting of an increase in osteoblastic surface and an increase in bone mass recognized by CT. How to evaluate.

1 8. A bone hypoplasia or candidate osteogenesis agent is administered to the osteopenia animal model according to any one of claims 1 1 to 15 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 decreased bone mass increases as an index, and is effective in promoting osteogenesis when the bone mass increases. A method of evaluating a bone formation promoter or a bone formation promoter candidate substance that is judged to be present.

1 9. Increased bone mass, increased bone formation marker level in the animal model of osteopenia, increased bone density, increased unit bone mass, increased trabecular number, decreased osteoclast number 19. The osteogenesis promoter or osteogenesis promoter according to claim 18, which is judged using at least one selected from the group consisting of: an increase in osteoblastic cell surface, and an increase in bone mass as detected by CT. A method for evaluating trapped substances.

20. A hormone or hormone receptor in a bone loss model animal produced by administering soluble RANKL or a fusion protein of soluble RANKL and an epitope tag by the method according to claim 8 and extracting the ovary. A method for evaluating the effect of the hormone or hormone receptor modulator using a modulator as an index as to whether or not the decreased bone mass in a bone loss model animal is increased. A method of evaluating a hormone or hormone receptor modulator that determines that bone resorption is effective when increased.

21. The method for evaluating a hormone or hormone receptor modulator according to claim 20, wherein the hormone or hormone receptor modulator is a selective estrogen receptor modulator.

2 2. Increased bone mass with increased bone formation marker level in bone loss model animals, increased bone density, increased unit bone mass, increased trabecular number, decreased osteoclast number, The hormone or hormone receptor modulator according to claim 20 or 21, wherein the hormone or hormone receptor modulator is determined using at least one selected from the group consisting of an increase in osteoblastic surface and an increase in bone mass recognized by CT. How to evaluate.