WO2015072527A1

WO2015072527A1 - Osteoclastogenesis inhibitor comprising netrin-1

Info

Publication number: WO2015072527A1
Application number: PCT/JP2014/080118
Authority: WO
Inventors: 健太丸山; 審良　静男
Original assignee: 国立大学法人大阪大学
Priority date: 2013-11-13
Filing date: 2014-11-13
Publication date: 2015-05-21
Also published as: JPWO2015072527A1

Abstract

The present invention addresses the problem of providing a drug capable of inhibiting fusion of osteoclasts so as to provide a prophylactic or therapeutic agent that is useful for bone destructive diseases. To achieve this purpose, provided is an osteoclastogenesis inhibitor that comprises netrin-1.

Description

Netrin-1 containing osteoclast formation inhibitor

The present invention relates to an osteoclast formation inhibitor, and in particular, to an osteoclast multinucleation inhibitor containing netrin-1.

Netrin-1 is a protein found as an axon guidance molecule that induces neuronal axons (Ishii et al, Neuron 1992: Non-patent document 1), and research is ongoing. It has gradually become clear that it has various roles.

Patent Document 1 suggests that netrin 1 has an inhibitory effect on migration of synovial fibroblasts and can be used for prevention or treatment of cartilage defects. However, it is described that the actual effect of netrin 1 is not remarkable (the same patent document [0133]), and it is also described that the osteoporosis is not included in the cartilage defect and the degenerative disease (the same patent document). [0047]). Bisphosphonates and anti-RANKL monoclonal antibodies are known as drugs that act on osteoclasts. Bisphosphonates prevent osteoclast recruitment, differentiation, adhesion, and survival. The anti-RANKL monoclonal antibody prevents osteoclast differentiation and proliferation by RANK / RANKL signals. These drugs are used as therapeutic agents for osteoporosis, but have been reported to have side effects such as jaw osteonecrosis.

Special table 2010-539123 gazette

An object of the present invention is to provide a drug that suppresses osteoclast cell fusion and to provide a prophylactic or therapeutic agent useful for bone destruction diseases.

Osteoclasts are large, dendritic motile cells. Bone marrow-derived monocytic cells are known to differentiate and fuse to become osteoclasts. For this reason, osteoclasts are multinucleated giant cells having several to several tens of nuclei.

The present inventors have surprisingly found that netrin 1 is specifically expressed in osteoblasts and synovial cells in the netrin family and suppresses fusion to become multinucleated cells. Thus, the present inventors have found that the formation of osteoclasts is inhibited, and have made further improvements to complete the present invention. In addition, the above-mentioned Patent Document 1 does not suggest that Netrin 1 in the Netrin family specifically suppresses fusion in the osteoclast formation process and suppresses multinucleation.

That is, the present invention includes, for example, the subject matters described in the following sections.
Item 1.
An osteoclast formation inhibitor containing netrin-1.
Item 2.
An osteoclast multinucleation inhibitor containing netrin-1.
Item 3.
The inhibitor according to

claim

1 or 2, which is used for prevention and / or treatment of a bone destruction disease caused by increased osteoclasts.
Item 4.
The inhibitor according to claim 3, which is used for prevention and / or treatment of osteoporosis.
Item 5.
The inhibitor according to any one of claims 1 to 4, which is a transvascular agent.

Furthermore, the present invention includes the subject matter described in the following section, for example.
Item 1A.
A method for inhibiting osteoclast formation, comprising administering netrin 1 to a subject in need thereof.
Item 2A.
A method for inhibiting osteoclast multinucleation, comprising administering Netrin-1 to a subject in need thereof.
Item 3A.
Item 2. The method according to Item 1A or 2A, which is a method for preventing and / or treating a bone destruction disease caused by increased osteoclasts.
Item 4A.
Item 3. The method according to Item 3A, wherein the bone destruction disease caused by increased osteoclasts is osteoporosis.
Item 5A.
Item 4. The method according to any one of Items 1A to 4A, wherein Netrin 1 is transvascularly administered.

Furthermore, the present invention includes the subject matter described in the following section, for example.
Item 1B.
Use of netrin 1 for inhibiting osteoclast formation.
Item 2B.
Use of netrin 1 for inhibiting osteoclast multinucleation.
Item 3B.
The use according to Item 1B or 2B, which is for prevention and / or treatment of a bone destruction disease caused by increased osteoclasts.
Item 4B.
Item 3. The use according to Item 3B, wherein the bone destruction disease caused by increased osteoclasts is osteoporosis.
Item 5B.
Item 4. The use according to any one of Items 1B to 4B, wherein Netrin 1 is administered transvascularly.

Furthermore, the present invention includes the subject matter described in the following section, for example.
Item 1C.
Use of netrin 1 in the manufacture of an osteoclast formation inhibitor.
Item 2C.
Use of netrin 1 in the production of an osteoclast multinucleation inhibitor.
Item 3C.
The use according to claim 1C or 2C, wherein the osteoclast formation inhibitor or the osteoclast multinucleation inhibitor is a drug for prevention and / or treatment of a bone destruction disease caused by osteoclast enhancement.
Item 4C.
The use according to claim 3C, wherein the bone destruction disease caused by increased osteoclasts is osteoporosis.
Item 5C.
The inhibitor according to any one of claims 1 to 4C, wherein the agent or the medicament is a transvascular agent.

The inhibitor of the present invention can suppress the fusion of osteoclasts and suppress the formation of osteoclasts. Therefore, the inhibitor of the present invention can prevent and / or treat a bone destruction disease caused by osteoclast enhancement. In particular, it is useful for prevention and / or treatment of osteoporosis and the like. In addition, the inhibitor of the present invention can specifically suppress only fusion without affecting osteoclast differentiation (ie, the activity of transcription factors involved in differentiation and the expression state of their target genes). Therefore, it is expected to have almost no side effects.

Expression of Netrin family genes by real-time PCR using RNA prepared from mouse osteoblasts, synovial fibroblasts, macrophages, or osteoclasts It is the graph which showed the relative expression ratio when the level was investigated and the expression level of netrin 1 in osteoblasts was set to 1. Monocyte-derived macrophages (MDM) were cultured in a medium supplemented with each concentration of

netrin

1 and 50 ng / ml of RANKL (receptor activator of nuclear factor-κBandligand), and the number of TRAP + MNCs (TRAP positive multinucleated cells) after 3 days It is a graph which shows the result of having measured. It can be seen that the formation of osteoclasts (TRAP positive multinucleated cells) is suppressed depending on the concentration of netrin-1. The results of confirming the formation of TRAP staining and resorption pits after culturing monocyte-derived macrophages (MDM) in a medium supplemented with various concentrations of

netrin

1 and 50 ng / ml RANKL are shown. It is a photograph. It is a figure which shows the culture schedule of MDM. It is a graph which shows the result of having measured the number of TRAP + MNCs after culture | cultivating MDM by the schedule shown in FIG. Human monocytes were cultured for 5 days in a medium containing M-CSF (100 ng / ml), and then cultured for 4 days in a medium containing human netrin 1 (400 ng / ml) and human RANKL (100 ng / ml), and then TRAP + MNCs It is a graph which shows the result of having measured the number of. It can be seen that the formation of osteoclasts is suppressed by netrin-1. Human monocytes were cultured for 5 days in a medium containing M-CSF (100 ng / ml), and then cultured for 4 days in a medium containing human netrin 1 (400 ng / ml) and human RANKL (100 ng / ml). It is a photograph which shows the result of having dyed. It can be seen that the formation of osteoclasts is suppressed by netrin-1. It is a graph which shows the result of having measured the number of TRAP + MNCs similarly to the case where the density | concentration of netrin 1 is 400 ng / ml, stimulating simultaneously with RANKL, observing with time, and acquiring the data of FIG. It can be seen that the formation of osteoclasts is suppressed by netrin-1. It is a graph which shows the result of having culture | cultivated MDM with the culture medium which added netrin 1 (400 ng / ml) and RANKL of various density | concentrations, and measured the nucleus number of the TRAP positive cell 4 days afterward. It can be seen that netrin 1 suppresses multinucleation of osteoclasts. MDM was cultured in a medium supplemented with netrin 1 (400 ng / ml) and RANKL (60 ng / ml), and the expression level (relative ratio) of molecules already known as osteoclast differentiation markers was measured by real-time PCR. It is a graph which shows a result. An ovariectomy or sham operation was performed on a 12-week-old mouse, a postmenopausal osteoporosis model mouse and its control mouse were prepared, and Netrin 1 was administered. It is a figure which shows a netrin 1 administration schedule. It is a photograph which shows the result of having analyzed the femur of each mouse | mouth prepared according to the schedule of FIG. 11 with the micro CT scanner. It can be seen that netrin 1 has improved osteoporosis. It is a graph which shows the result of having analyzed the bone mass / tissue volume (BV / TV) and uterine weight (Uterine における weight) in the femur of each mouse prepared according to the schedule of FIG.

Hereinafter, the present invention will be described in more detail.

The present invention relates to an osteoclast formation inhibitor containing netrin-1 and more particularly to an osteoclast multinucleation inhibitor containing netrin-1.

“Netrin 1” as used herein refers to netrin 1 of various organisms (preferably vertebrate netrin 1, more preferably mammalian netrin 1), and in particular has substantially the same biological activity as human netrin 1. Including natural sources and those obtained by genetic recombination methods. Naturally derived netrin 1 can be extracted and purified from various organisms by various methods. The netrin 1 produced by the gene recombination method can be produced in bacteria such as E. coli, yeast cells, Chinese hamster ovary (CHO) cells, C127 cells, COS cells, HEK293 cells and other cultured cells derived from animals. Extracted, separated and purified can be used. The netrin 1 obtained by the genetic recombination method includes a mutant or fragment of the netrin 1 as long as it has an osteoclast multinucleation inhibitory action. For example, netrin-1 isoforms, splicing variants, mutants (including point mutations, insertion mutations, deletion mutations), derivatives, proteins cleaved from netrin-1 precursor protein, netrin-1 gene and other genes Proteins expressed by fusion are also included. Specific examples of preferred netrin 1 include the following proteins (a) and (b).
(A) a protein comprising the full-length amino acid sequence of netrin 1 of various organisms (ie, full-length netrin 1 protein of various organisms)
(B) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the full-length amino acid sequence of netrin 1 of various organisms, and having an inhibitory effect on osteoclast multinucleation of netrin 1 of various organisms The full-length amino acid sequence is not particularly limited as long as it is a known full-length amino acid sequence of netrin 1. Preferable examples include the full-length amino acid sequence of netrin 1 such as human, mouse, rat, monkey, dog and rabbit. An example of the full-length amino acid sequence of human netrin 1 is shown as SEQ ID NO: 1, and an example of the full-length amino acid sequence of mouse netrin 1 is shown as SEQ ID NO: 2 in the sequence listing.

The number of one or more in (b) is preferably 1 to 100, more preferably 1 to 75, still more preferably 1 to 50, still more preferably 1 to 30, and more preferably 1 to 20 More preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 is particularly preferable.

Specific preferred examples of the protein (b) include the following (b-1) to (b-6).
(B-1): protein consisting of the amino acid sequence of SEQ ID NO: 3 (positions 28 to 604 of SEQ ID NO: 1) (b-2): 80% or more (preferably 85% or more, more preferably) with the amino acid sequence of SEQ ID NO: 3 Is composed of amino acid sequences having homology of 90% or more, more preferably 95% or more, even more preferably 97% or more, particularly preferably 98% or more, and particularly preferably 99% or more. Protein (b-3) having inhibitory action: 80% or more (preferably 85% or more, more preferably 90% or more, more preferably 95% or more, even more preferably 97% or more) with the amino acid sequence of SEQ ID NO: 3. A tamper which has an amino acid sequence having an identity of 98% or more, more preferably 99% or more, and an osteoclast multinucleation inhibitory activity. Quality (b-4): Protein consisting of the amino acid sequence of SEQ ID NO: 4 (positions 22 to 604 of SEQ ID NO: 2) (b-5): 80% or more (preferably 85% or more, more than the amino acid sequence of SEQ ID NO: 4) Preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 98% or more, more particularly preferably 99% or more) Protein (b-6) having oxidization inhibitory activity: 80% or more (preferably 85% or more, more preferably 90% or more, more preferably 95% or more, still more preferably 97% or more) with the amino acid sequence of SEQ ID NO: 4. , Particularly preferably 98% or more, more particularly preferably 99% or more) and has an activity of inhibiting osteoclast multinucleation. The present invention is based on the novel finding that only netrin 1 is expressed in osteoblasts and synovial fibroblasts in the netrin family, and the fusion of osteoclasts is suppressed by netrin 1. It is a thing. Since the osteoclast formation inhibitor of the present invention contains netrin 1, it can suppress osteoclasts from fusing and becoming multinucleated, thereby suppressing the formation of osteoclasts. For this reason, it is useful for the prevention and / or treatment of bone destruction diseases caused by the increase of osteoclasts.

The bone destruction disease is not particularly limited as long as it is a bone destruction disease known to be caused by an increase in osteoclasts. For example, osteoporosis (such as age-related osteoporosis, postmenopausal osteoporosis), diabetic bone Reduction, renal osteodystrophy, rheumatoid arthritis bone destruction, bone loss associated with disuse syndrome, bone destruction associated with osteomyelitis, bone destruction and bone loss due to periodontal disease, bone destruction associated with gouty arthritis Examples thereof include bone destruction caused by cervism, joint destruction caused by infectious arthritis, and bone destruction caused by cholesteatoma otitis media. Of these, osteoporosis is preferable.

The osteoclast formation inhibitor or osteoclast multinucleation inhibitor (hereinafter, also referred to as “inhibitor of the present invention”) containing netrin 1 of the present invention is limited in its form as long as the effect of the present invention is exhibited. Although not, it is preferably a transvascular agent. That is, the dosage form is preferably administered through blood vessels. For example, injections, infusions, infusions can be exemplified.

Since Netrin 1 is a protein, the inhibitor of the present invention preferably includes the form of a protein preparation, which can be prepared in the same manner as known protein preparations. In particular, the preparation method is not limited.

For example, the inhibitor of the present invention may contain a pharmacologically acceptable carrier in addition to netrin 1, and further, isotonic agent, amino acid, surfactant, sulfur-containing reducing agent, antioxidant, pH adjustment Agents, diluents, solubilizers, excipients, soothing agents, buffering agents, or combinations thereof may be included. The inhibitor of this invention can be prepared by mixing these.

As a pharmacologically acceptable carrier, water (particularly injection water) can be preferably exemplified. Injectable water may be added at the time of use, in which case the inhibitor of the present invention may be a dry preparation.

Examples of the isotonic agent include saccharides such as polyethylene glycol, dextran, mannitol, sorbitol, inositol, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, and raffinose. These tonicity agents may be used alone or in combination of two or more.

Amino acids include alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, leucine, isoleucine, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, aspartic acid, glycine, serine, valine. . These amino acids may be used alone or in combination of two or more.

Surfactants include nonionic surfactants such as sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate, and sorbitan monopalmitate; glycerin such as glycerin monocaprylate, glycerin monomylate, and glycerin monostearate. Fatty acid ester; polyglycerin fatty acid ester such as decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate; polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate , Polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate, etc. Oxyethylene sorbitan fatty acid ester; polyoxyethylene sorbite fatty acid ester such as polyoxyethylene sorbite tetrastearate and polyoxyethylene sorbite tetraoleate; polyoxyethylene glycerin fatty acid ester such as polyoxyethylene glyceryl monostearate; polyethylene glycol distea Polyethylene glycol fatty acid esters such as rate; polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether and other poly Oxyethylene polyoxypropylene alkyl ether; polyoxyethylene Polyoxyethylene alkyl phenyl ethers such as rennonyl phenyl ether; polyoxyethylene hydrogenated castor oils such as polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogen castor oil); polyoxyethylene sorbite beeswax and other poly Oxyethylene beeswax derivatives; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; those having HLB 6-18 such as polyoxyethylene fatty acid amides such as polyoxyethylene stearamide; anionic surfactants such as sodium cetyl sulfate, Alkyl sulfates having an alkyl group of 10 to 18 carbon atoms such as sodium lauryl sulfate and sodium oleyl sulfate; average addition of ethylene oxide such as sodium polyoxyethylene lauryl sulfate Polyoxyethylene alkyl ether sulfates having 2 to 4 moles added and 10 to 18 carbon atoms in the alkyl group; alkylsulfosuccinic acids having 8 to 18 carbon atoms in the alkyl group, such as sodium lauryl sulfosuccinate Ester salts; natural surfactants such as lecithin, glycerophospholipid; fingophospholipids such as sphingomyelin; and sucrose fatty acid esters of fatty acids having 12 to 18 carbon atoms. These surfactants may be used alone or in combination of two or more.

Sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and carbon Examples thereof include those having a sulfhydryl group such as thioalkanoic acid having 1 to 7 atoms. These sulfur-containing reducing agents may be used alone or in combination of two or more.

Antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite Chelating agents such as sodium, triamyl gallate, propyl gallate or disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate and the like can be mentioned. In addition, inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate, which are usually added as antioxidants; organic salts such as sodium citrate, potassium citrate, and sodium acetate Also mentioned. These antioxidants may be used alone or in combination of two or more.

PH can be adjusted by adding a pH adjuster. The pH of the inhibitor of the present invention is preferably 4 to 8, and more preferably 5 to 7.5. However, the pH is not limited to these.

Such components are dissolved in an aqueous buffer known in the field of solution formulation or a mixture thereof, and a solution formulation is prepared using the aqueous buffer. Examples of aqueous buffers include phosphate buffer (preferably sodium monohydrogen phosphate-sodium dihydrogen phosphate system); citrate buffer (preferably sodium citrate buffer); and acetate buffer; Can be mentioned. Although there is no particular limitation, the concentration of the buffer is generally 1 to 500 mM, preferably 5 to 100 mM, and more preferably 10 to 50 mM.

The content ratio of netrin 1 in the inhibitor of the present invention is not particularly limited, and examples thereof include 0.1 to 99.9% by mass, and more preferably about 1 to 50% by mass.

The dose of the inhibitor of the present invention can be appropriately set according to the serious injury degree, sex, age, etc. of the patient so that the effects of the present invention are exhibited.

In addition, this invention also includes the method of preventing and / or treating the bone destruction disease resulting from an osteoclast enhancement by administering the inhibitor of this invention.

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the following examples.

The test materials used in the experiment are described below. Synovial fibroblasts are obtained from DBA / 1J female mice (purchased from Japan SLC Co., Ltd.) according to the description of Gao, B., et. Al., Arthritis Res Ther 8, R172. (2006). It was collected from the prepared collagen-induced arthritis (CIA) mouse. Monocyte-derived macrophages (MDM) were prepared as described in Maruyama, K., et al. J Immunol 177, 3799-3805 (2006). The following were used for each protein. Mouse RANKL® (462-TR, R & D Systems), mouse M-CSF (315-02, PeproTech), mouse netrin 1 (1109-N1, R & D Systems), human netrin 1 (ALX-522-100, Enzo Life Sciences). The mouse netrin 1 consists of the amino acid sequence of SEQ ID NO: 4. The human netrin 1 consists of the amino acid sequence of SEQ ID NO: 3. Unless otherwise specified below, the test material derived from the living body is derived from a mouse.

Examination of Netrin Family Expression Sites Real-time PCR using RNA prepared from mouse osteoblasts (Osteoblast), synovial fibroblasts, macrophages (Macrophage) or osteoclasts (Osteoclast), The expression level of each gene of the netrin family was examined. The relative expression ratio when the expression level of netrin 1 in osteoblasts is 1 is shown in FIG. Only netrin 1 was confirmed to be expressed in osteoblasts and synovial fibroblasts. It was also found that netrin 1 was not expressed in macrophages and osteoclasts.

Inhibition of osteoclast formation by Netrin-1 <Study 1>
Monocyte-derived macrophages (MDM) are cultured in a medium (basic composition: α-MEM plus 10% FCS) supplemented with each concentration of

netrin

1 and 50 ng / ml RANKL (receptor activator of nuclear factor-κB ligand). (Bone resorption assay plates (Iwai Chemicals Co., Ltd.) installed under the medium) The number of TRAP + MNCs (tartrate-resistant acid phosphatase-positive multinucleated cells) was measured 3 days later. TRAP (tartrate-resistant acid phosphatase) is one of the markers of osteoclasts. The results are shown in FIG. 2 (* p <0.05, n = 4). TRAP staining was performed using a TRAP / ALP Stain kit (Wako Pure Chemical Industries, Ltd.), and TRAP + MNCs was measured by counting the number of TRAP-positive trinuclear cells or more under a microscope.

<Examination 2>
After culturing under each condition in Study 1 above, TRAP staining and the formation of absorption pits were confirmed. TRAP staining was performed using a TRAP / ALP Stain kit (Wako Co., Ltd.). The results are shown in FIG. The black arrow at the bottom (Pit formation) in FIG.

<Examination 3>
MDM was cultured under different culture conditions from Study 1, and the number of TRAP + MNCs was measured after 6 days. Specifically, four types of culture conditions 1 to 4 are prepared. Under any condition, M-CSF (Macrophage colony-stimulating factor) (25 ng / ml) is always added to the medium, and RANKL (50 ng / ml) is added. ) Was added to the medium after the third day, and the number of days to which netrin 1 (400 ng / ml) was added was changed under the culture conditions 1 to 4 (see FIG. 4). The measurement result of the number of TRAP + MNCs is shown in FIG. 5 (* p <0.05, n = 4).

<Examination 4>
Human monocytes are cultured for 5 days in a medium containing M-CFS (100 ng / ml), and then cultured for 4 days in a medium containing human netrin 1 (400 ng / ml) and human RANKL (100 ng / ml), followed by TRAP + MNCs. The number of was measured. PBS (Phosphate buffered saline) was used as a control for Netrin-1. The measurement results are shown in FIG. 6 (* p <0.05, n = 4). Moreover, the result of having performed TRAP dyeing similarly to examination 2 is shown in FIG.

From the above examinations 1 to 4, it was found that the addition of netrin 1 suppresses the formation of osteoclasts.

Confirmation of Osteoclast Fusion Inhibition by Netrin 1 The concentration of Netrin 1 was 400 ng / ml, stimulation was performed simultaneously with RANKL, observation was performed over time, and the number of TRAP + MNCs was measured in the same manner as in Study 1 above. PBS was used as a control for Netrin-1. The results are shown in FIG. 8 (* p <0.05, n = 3).

Further, MDM was cultured in a medium supplemented with netrin 1 (400 ng / ml) and various concentrations of RANKL, and the number of TRAP-positive cells was counted 4 days later. The results are shown in FIG. 9 (* p <0.05, n = 4).

From these results, it was found that Netrin 1 suppresses multinucleation (ie fusion) of osteoclasts.

Furthermore, MDM was cultured in a medium supplemented with netrin 1 (400 ng / ml) and RANKL (60 ng / ml), and as an osteoclast differentiation marker at 0, 36 and 72 hours after addition of RANKL. The expression level (relative ratio) of already known molecules was measured by real-time PCR. As a control, an experiment in which PBS was added instead of netrin 1 was also performed. The results are shown in FIG. The name of the molecular marker is described above each graph shown in FIG.
From the results, it was found that the expression level of the molecular marker did not change depending on the presence or absence of addition of netrin 1, and therefore netrin 1 did not suppress differentiation into osteoclasts.

From the above results, it was found that Netrin 1 suppressed osteoclast formation by suppressing multinucleation (ie, fusion) without suppressing differentiation into osteoclasts.

Examination of recovery of bone destruction disease with netrin 1 Ovariectomy (OVX) or sham operation (Sham) was performed on 12-week-old mice to prepare postmenopausal osteoporosis model mice and their control mice. Three weeks after surgery (ie, up to 15 weeks of age), the animals were reared on the schedule shown in FIG. 11 and sacrificed. In addition, as shown in FIG. 11, postmenopausal osteoporosis model mice were intravenously injected with netrin 1 or PBS under the condition of 10 μg / head / 0.5 weeks.

After sacrifice, the femur and uterus were removed, and the femur was analyzed with a micro CT scanner. A scanned image is shown in FIG. In addition, bone mass / tissue volume (BV / TV) and uterine weight (Uterine weight) in the femur of each mouse were analyzed. The results are shown in FIG. 13 (* p <0.05, n = 4).

From the above results, it was shown that Netrin 1 has an effect of improving osteoporosis.

Claims

An osteoclast formation inhibitor containing netrin-1.
An osteoclast multinucleation inhibitor containing netrin-1.
The inhibitor according to claim 1 or 2, which is used for prevention and / or treatment of a bone destruction disease caused by increased osteoclasts.
The inhibitor according to claim 3, which is used for prevention and / or treatment of osteoporosis.
The inhibitor according to any one of claims 1 to 4, which is a transvascular agent.