JP2001086982A - Bone resorption regulator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine for regulating the bone resorption caused by osteoclast, and further to provide a method for screening the medicine. SOLUTION: It is discovered that a vascular endothelial growth factor(VEGF) has activities for promoting formation and existence of the osteoclast. The activities is mediated by a vascular endothelial growth factor receptor type 1 (VEGFR-1). The decrease of the osteoclast is successfully carried out by inhibiting the activation of the VEGFR-1 of M-CSF-deficient mouse. The formation and existence of the osteoclast is regulated by using the medicine for regulating the activation of the VEDFR-1, and thereby the therapy of the disease accompanying the abnormality of the bone resorption become possible.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a differentiation induction system for osteoclasts targeting vascular endothelial growth factor receptor type 1 and differentiation, survival and / or bone formation of osteoclasts using the differentiation induction system. The present invention relates to a method for screening a compound that regulates resorption, and an agent for regulating differentiation, survival, and / or bone resorption of osteoclasts targeting vascular endothelial growth factor receptor type 1.

[0002]

BACKGROUND OF THE INVENTION Recessive mutations in the osteopetrosis; op gene on chromosome 3 cause a marked reduction in osteoclasts, monocytes, and macrophages in various organs (Marks, SC , Jr., and
PW Lane. 1976. J. Hered. 67:11; Marks, SC, Jr.
1982. Am. J. Anat. 163: 157; Wiktor-Jedrzejczak,
W. et al. 1982. J. Exp. Med. 156: 1516). This reduction was due to the insertion of a single base pair present in the coding region of the macrophage colony stimulating factor (M-CSF) gene, resulting in a functional macrophage colony stimulating factor (M-CSF or CSF-
1) is lost (Yoshida, H. et al.
1990. Nature (Lond.). 345: 442), recombinant human M-CSF
(RhM-CSF) can be supplemented (Felix, R.
et al. 1990. Endocrinology. 127: 2592; Kodama, H.
et al. 1991. J. Exp. Med. 173: 269; Wiktor-Jedrzejc
zak, W. et al. 1991. Exp. Hematol. 19: 1049; Sundqu
ist, KTet al. 1995. Bone. 16:39). The fact that M-CSF acts directly on cells of the osteoclast lineage allows c-Fms, a receptor for M-CSF, to be expressed in vitro (Kodama, H. et al. 1991. J. E.
xp.Med. 173: 1291) and in vivo (Hofstetter, W. e.
Natl. Acad. Sci. USA. 89: 9637). These results indicate that M-CSF plays an essential role in osteoclast and macrophage differentiation in certain organs under physiological conditions.

[0003] In op / op mice, however, serious symptoms of osteopetrosis are clearly apparent only when young, and the symptoms gradually improve as osteoclasts increase (Marks, SC, Jr. , and PW Lane. 1976. J.
Hered. 67:11; Marks, SC, Jr. 1982. Am. J. Anat. 1
63: 157; Begg, SK et al. 1993. J. Exp. Med. 177: 2
37). The present inventors have previously shown that administration of rhM-CSF at a high dose (5 μg / mouse or more) induces recruitment of osteoclasts of op / op mice even with a single administration, and allows the osteoclasts to survive. Have been found to maintain long-term active bone resorption (Kodama, H. et al. 1993. J. Bone Mi
ner. Res. 8:45; Niida, S. et al. 1994. J. Bone Min.
er. Res. 9: 873). This suggests the presence of other factors involved in osteoclast bone resorption under M-CSF deficiency.
Some data has been reported on this, but it remains controversial. For example, op / op
Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to be effective in ameliorating bone marble disease in mice, but it has been suggested that GM-CSF is involved in osteoclast differentiation in vitro. (MacDonald, BR et al. 1
986. J. Bone Miner. Res. 1: 227; Kurihara, N. et al.
1989. Blood. 74: 1295; Takahashi, N. et al. 1991.
J. Bone Miner. Res. 6: 977). Also, Wiktor-Jedrzejc
zak et al. (Wiktor-Jedrzejczak, W. et al. 1994. Endocri
nology. 134: 1932) and Nilsson et al. (Nilsson, SK e)
t al. 1995. Blood. 86:66) report that GM-CSF can ameliorate macrophage deficiency but not osteoporosis. Most recently Myint et al. (Myint, YY et
al. 1999. Am. J. Pathol. 154.553), GM-CSF and
Interleukin 3 was reported to induce osteoclast development in op / op mice at low doses.

[0004] c-Fms is a member of the eight platelet derived growth factor receptor (PDGFR) family (Kondo, K. et al.
1998. Gene. 208: 297). Vascular endothelial growth factor (Vascular
Receptors specific to Endothelial Growth Factor (VEGF) include two receptor tyrosine kinases belonging to PDGFR, VEGFR-1 / Flt-1 and VEGFR-2 / KDR / Flk-1
And neuropilin-1 have been identified (Neufeld, G. et.
al. 1999. FASEB J.13: 9). Unlike all VEGFR-expressing endothelial cells, cells of the monocyte / macrophage lineage mainly express VEGFR-1 (Neufeld, G. et al. 1999. FASE
B J. 13: 9; Berleon, B. et al. 1996. Blood. 87: 333.
6; Clauss, M. et al. 1996. J. Biol. Chem. 271: 1762
9). VEGFR-1 mediates the chemotactic response to VEGF and placental growth factor type 1 (PlGF-1) (Neufeld, G. et al. 1999.
FASEB J. 13: 9; Berleon, B. et al. 1996. Blood. 87:
3336; Clauss, M. et al. 1996. J. Biol. Chem. 271: 1
7629; Park, JE et al. 1994. J. Biol. Chem. 169: 2
5646; Sawano, A. et al. 1996. Cell Growth Differ.
7: 213; Hiratsuka, S. et al. 1998. Proc. Natl. Aca
d. Sci. USA. 95: 9349). PlGF-1 has high homology to VEGF and is expressed in endothelial cells of umbilical vein and placenta. Despite such findings, the relationship between VEGF and PlGF-1, and their receptor VEGFR-1 and bone resorption by osteoclasts was not known.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a novel system for inducing osteoclast differentiation targeting vascular endothelial growth factor receptor type 1, differentiation and survival of osteoclasts using the system, and It is an object of the present invention to provide a method for screening a compound that regulates bone resorption, and an agent for regulating differentiation, survival, and / or bone resorption of osteoclasts targeting vascular endothelial growth factor receptor type 1. And

[0006]

Means for Solving the Problems The present inventors have found that macrophages and osteoclasts have a close relationship in the cell lineage. Therefore, vascular endothelial growth factor (VEGF), which is a cytokine that acts on macrophages, has been developed. It was thought that it might have an effect on osteoclast formation. Therefore, an experiment was performed in which VEGF was administered to a bone osteopetrosis model mouse (op / op mouse) in which the M-CSF gene was deleted and osteoclast formation was impaired. As a result, they found that VEGF could completely complement M / CSF deficiency in op / op mice in bone resorption by osteoclasts.

[0007] One administration of recombinant human VEGF (rhVEGF) to op / op mice produced osteoclasts.
The formed osteoclasts are mainly VEGF receptor type 1 (VEGFR-
1) was expressed. Administration of recombinant human placental growth factor type 1 (rhPlGF-1) in place of VEGF
The formation of osteoclasts in the same manner as described above indicated that the VEGF signal was mediated by VEGFR-1. Osteoclasts induced by recombinant human M-CSF died when VEGF was neutralized by administration of the VEGFR-1 / Fc chimeric protein, but could be survived by subsequent administration of recombinant human M-CSF. did it. There was little difference in bone resorption activity between osteoclasts supported by recombinant human M-CSF and osteoclasts supported by endogenous VEGF. In the op / op mice, the osteoclasts increase with aging, and the bone marble disease is improved.
The administration of anti-VEGF antibody to this mouse resulted in the loss of most osteoclasts, so the improvement of age-related osteopetrosis of op / op mice was due to endogenously produced VEGF, It has been shown that the appearance of osteoclasts in mice requires endogenously produced VEGF. Furthermore, experiments examining the support for osteoclast differentiation in vitro (in vitro) also revealed that VEGF could replace the activity of M-CSF.

[0008] Observation of the fine morphology of osteoclasts formed by the administration of VEGF revealed a ruffled boeder and a clear zone, and the Golgi apparatus and many mitochondria were observed. The characteristics of various osteoclasts were shown. When mature osteoclasts were isolated and examined for the in vitro activity effect of M-CSF or VEGF, extension of osteoclasts was observed with the addition of VGEF.

The above results demonstrate that M-CSF and VEGF have overlapping functions in bone resorption by osteoclasts. The presence of either cytokine is sufficient to support the entire process of bone resorption by osteoclasts, namely osteoclast differentiation, osteoclast survival, and active bone resorption. VEGF is mediated through a different receptor than M-CSF, revealing a unique type of cytokine signal redundancy that uses different ligand-receptor combinations.

The ligand of VEGFR-1 such as VEGF or PlGF-1 is V
Compounds that activate VEGFR-1, such as VEGFR-1 ligands, have been shown to activate EGFR-1, differentiate and form osteoclasts, and promote bone resorption. Can be used as a therapeutic agent for diseases including osteopetrosis, in which osteoporosis is reduced. Conversely, a drug that suppresses VEGFR-1 activation can be used as a drug that inhibits osteoclast formation and suppresses bone resorption. It is considered that such an agent exerts a higher effect when used in combination with an agent that suppresses signal transduction of the M-CSF / c-Fms system, inhibits osteoclast formation, and suppresses bone resorption. Thereby, prevention and treatment of osteoporosis and the like can be performed.

The screening system using the differentiation, survival, and / or bone resorption of osteoclasts due to the activation of VEGFR-1 as an index provides not only screening of drugs for regulating bone resorption but also VEGFR-1. It may also be used to screen for agents that control other functions via. For example, VEGFR-1 plays an important role in promoting angiogenesis during development, promoting angiogenesis in adults, and enhancing vascular endothelial permeability. Therefore, a compound that promotes the activity of VEGFR-1 is useful as an agent for regenerating blood vessels lost due to a disease injury such as injury or infarction. In tumor cells, the expression of VEGFR-1 ligand such as VEGF is enhanced, which induces angiogenesis and enlarges the tumor. Therefore, an agent that suppresses VEGFR-1 activation is useful as an anticancer agent because it suppresses vascular induction. In order to isolate such a compound that promotes or suppresses vascular induction, the screening system of the present invention using bone cell formation and bone resorption as an index can be used. In addition to the above, the above-mentioned screening system can be applied to obtain compounds that regulate various signalings via VEGFR-1. That is, osteoclast differentiation, survival,
The method of the present invention for screening for a compound that promotes or suppresses bone resorption is useful for isolating a compound for promoting or suppressing VEGFR-1 mediated signal transduction. VEGFR-1 is known to mediate the signal of endothelial cell proliferation response, and is known to regulate cell functions in various tissues in vivo, and compounds that can be isolated by the screening method of the present invention include: VEGF
It is useful to control these functions via R-1.

The present invention provides a system for inducing osteoclast differentiation by VEGFR-1 mediated signals, screening for compounds that regulate osteoclast differentiation and bone resorption using the differentiation inducing system, and targeting VEGFR-1. For drugs to regulate the differentiation and bone resorption of osteoclasts, more specifically,
(1) A method for inducing osteoclast differentiation, wherein cells expressing vascular endothelial growth factor receptor type 1 are cultured in the presence of a compound that activates vascular endothelial growth factor receptor type 1. (2) the method according to (1), wherein the compound that activates vascular endothelial growth factor receptor type 1 is a ligand for vascular endothelial growth factor receptor type 1; The method according to (2), wherein the ligand for endothelial growth factor receptor type 1 is vascular endothelial growth factor or placental growth factor type 1, (4) cells expressing vascular endothelial growth factor receptor type 1 are non-adherent Bone marrow cells,
(1) the method according to any one of (3), (5) a method of screening for a compound that promotes or inhibits osteoclast differentiation, survival, and / or bone resorption,
(A) a step of contacting vascular endothelial growth factor receptor type 1 with vascular endothelial growth factor receptor type 1 in the presence of a test compound; (b) the vascular endothelial growth factor receptor type 1 and the vascular endothelial growth factor Or a method comprising the steps of: detecting the binding to placental growth factor type 1; (c) selecting a compound that promotes or inhibits the binding; (6) osteoclast differentiation, survival, and / or bone resorption A method of screening for a compound that promotes the following: (a) contacting a test compound with a cell that expresses vascular endothelial growth factor receptor type 1;
(B) detecting differentiation, survival, and / or bone resorption of osteoclasts; (c) selecting a compound that promotes the differentiation, survival, and / or bone resorption;
(7) A method for screening a compound that inhibits osteoclast differentiation, survival, and / or bone resorption,
(A) contacting cells expressing vascular endothelial growth factor receptor type 1 with vascular endothelial growth factor or placental growth factor type 1 in the presence of a test compound; (b) differentiation and survival of osteoclasts;
And / or detecting bone resorption, (c) the differentiation,
Selecting a compound that inhibits survival and / or bone resorption, (8) vascular endothelial growth factor receptor 1
The method according to (6) or (7), wherein the cells expressing the type are non-adherent bone marrow cells, (9) a method for screening for a compound that promotes the induction of blood vessels, Contacting a test compound with a cell expressing factor receptor type 1, (b) detecting osteoclast differentiation, survival, and / or bone resorption; (c) the differentiation, survival,
And / or a step of selecting a compound that promotes bone resorption, (10) a method of screening for a compound that inhibits the induction of blood vessels, wherein (a) a vascular endothelial growth factor receptor in the presence of a test compound Contacting cells expressing type 1 with vascular endothelial growth factor or placental growth factor type 1; (b) detecting differentiation, survival and / or bone resorption of osteoclasts; (c) said differentiation and survival And / or selecting a compound that inhibits bone resorption. (11) The cells expressing vascular endothelial growth factor receptor type 1 are non-adherent bone marrow cells, (9) or (10). (12) differentiation of osteoclasts, comprising, as an active ingredient, a compound that activates vascular endothelial growth factor receptor type 1;
Agents to promote survival and / or bone resorption,
(13) the agent according to (12), wherein the compound that activates vascular endothelial growth factor receptor type 1 is a ligand for vascular endothelial growth factor receptor type 1; (14) vascular endothelial growth factor receptor type 1 The agent according to (13), wherein the ligand for is vascular endothelial growth factor or placental growth factor type 1, (15) the compound that activates vascular endothelial growth factor receptor type 1, (5) or (6). And a compound isolated by the screening method described in (12), (16) used for treating a disease or injury selected from the group consisting of marble disease, low-rotation osteoporosis, and fracture. The agent according to any one of (12) to (15), (17) differentiation and survival of osteoclasts comprising, as an active ingredient, a compound that inhibits activation of vascular endothelial growth factor receptor type 1; / Or an agent for inhibiting bone resorption, (18) a compound that inhibits activation of vascular endothelial growth factor receptor type 1 is a vascular endothelial growth factor receptor type 1 and a ligand for vascular endothelial growth factor receptor type 1 (17) the agent according to (17), which is a compound that inhibits the binding to vascular endothelial growth factor receptor 1; And (20) the compound that inhibits activation of vascular endothelial growth factor receptor type 1 is a compound isolated by the screening method according to (5) or (7).
(17) The agent according to (17), which is used for treating a disease selected from the group consisting of high-rotation osteoporosis, bone metastatic cancer, osteosarcoma, hypercalcemia, and bone destruction in rheumatoid arthritis. A drug according to any one of (17) to (20), (22) a blood vessel induction promoter comprising a compound isolated by the method according to (9) as an active ingredient,
(23) An anticancer agent comprising a compound isolated by the method according to (10) as an active ingredient.

[0013]

DETAILED DESCRIPTION OF THE INVENTION 1. To induce osteoclast differentiation
METHODS The present inventors utilize vascular endothelial growth factor (VEGF) or placental growth factor type 1 (PlGF-1) to activate vascular endothelial growth factor receptor type 1 (VEGFR-1) to achieve osteoclasts. It has been found that cells can be differentiated and bone resorption can be promoted. Therefore, the present invention provides a method for inducing differentiation of cells expressing the receptor into osteoclasts by targeting and activating VEGFR-1. The method of the present invention comprises contacting a cell that expresses vascular endothelial growth factor receptor type 1 (VEGFR-1) with a compound that activates vascular endothelial growth factor receptor type 1 (VEGFR-1). That is, it can be carried out by culturing cells expressing vascular endothelial growth factor receptor type 1 in the presence of a compound that activates vascular endothelial growth factor receptor type 1. The culturing can be performed, for example, in vitro or in vivo.

The compound that activates vascular endothelial growth factor receptor type 1 used in the method of the present invention is not particularly limited as long as it has the ability to activate vascular endothelial growth factor receptor type 1; Ligands for endothelial growth factor receptor type 1 are preferred. Vascular endothelial growth factor receptor 1
Ligands for the type include vascular endothelial growth factor (VEG
F), and placental growth factor type 1 (PlGF-1),
These ligands can be particularly suitably used in the method of the present invention. In addition, a fragment containing a receptor binding portion of these proteins, a synthetic peptide, a synthetic compound, or the like may be used.

Cells expressing vascular endothelial growth factor receptor type 1 are not particularly limited as long as they express vascular endothelial growth factor receptor type 1 and can differentiate into osteoclasts. Bone marrow cells, spleen cells and the like. Further, as shown in Example 7, cells already differentiated into osteoclasts can be activated by the method of the present invention. The cells may be cultured according to a known method. in vitro
May be performed, for example, by culturing the cells in a culture solution containing the compound. For example, for non-adherent bone marrow cells, the method described in Example 4 is used. 7 can be performed.

The method of the present invention can also be performed in vivo. In vivo culture can be performed, for example, by administering the compound into a living body. That is, by administering a compound that activates vascular endothelial growth factor receptor type 1 to a living body, it is possible to differentiate and form osteoclasts in the living body. Or,
The ligand may be expressed in the body by administering a vector that expresses a ligand for vascular endothelial growth factor receptor type 1 such as VEGF or PlGF-1. Administration may be systemic or local. Also, ex vivo administration
Can also be performed. That is, cells such as bone marrow cells or spleen cells are taken out of a living body and treated with a compound that activates vascular endothelial growth factor receptor type 1, or vascular endothelial growth factor receptor type 1 such as VEGF or PlGF-1 By introducing a vector expressing a ligand and returning the cells into the body, the formation of osteoclasts can be promoted. When a gene for a ligand for vascular endothelial growth factor receptor type 1 is introduced ex vivo, as long as the expression product can reach a cell capable of differentiating into an osteoclast, the gene may be transferred to a cell other than the osteoclast. it can. The method of the present invention can also be applied in combination with known compounds that promote osteoclast formation. Examples of such a compound include M-CSF / CSF-1 and the like.

[0017] 2. Promoter or inhibitor of osteoclast differentiation
The present invention also provides a vascular endothelial growth factor receptor type 1 (VEGFR-
Provided is a method for screening for a compound that promotes or inhibits osteoclast differentiation, survival, and / or bone resorption, which targets 1). One embodiment of the screening method of the present invention includes a method using the binding between vascular endothelial growth factor receptor type 1 and its ligand as an index. This screening includes the steps of (a) contacting vascular endothelial growth factor receptor type 1 with vascular endothelial growth factor receptor type 1 in the presence of a test compound, and (b) vascular endothelial growth factor receptor type 1 It can be carried out by a method including a step of detecting binding to vascular endothelial growth factor or placental growth factor type 1, and (c) a step of selecting a compound that promotes or inhibits the binding.

Specifically, for example, purified vascular endothelial growth factor receptor type 1 (VEGFR-1) is converted to BIACORE (Pharmacia Bi
(Otech), a purified ligand (eg, VEGF or PlGF-1) and a test compound are mixed and poured into the thin film, and the response is measured. Test compounds used for screening are not limited to these, but include, for example, cell extracts, expression products of gene libraries, synthetic low molecular weight compounds, synthetic peptides, modified peptides,
Natural compounds and the like. The test compound used for screening is appropriately labeled and used as necessary.
Examples of the label include, but are not limited to, radiolabels and fluorescent labels. As a control, the same measurement is performed in the absence of the test compound. Specific operations may be performed according to known literature (supervised by Shigeo Ohno and Yoshifumi Nishimura, Cell Engineering Separate Volume Experimental Protocol Series, Protein Experimental Protocol 1 Function Analysis, Chapter 10, pp. 164-179, Shujunsha). By this screening, a compound that promotes or suppresses the binding between VEGFR-1 and the vascular endothelial growth factor or placental growth factor type 1 can be obtained. It is also possible to screen the purified ligand labeled with VEGFR-1 and the test compound as an RIA by competitively binding in the same solvent, and then precipitating with an anti-VEGFR-1 antibody, and measuring its radioactivity and the like. . Thus, for example, VE
Compounds that bind to GFR-1 and inhibit VEGFR-1 activation (VE
GFR-1 antagonist) can also be isolated.

Another embodiment of the screening method of the present invention is a method utilizing the above-described osteoclast differentiation-inducing system of the present invention. The screening method for a compound that promotes osteoclast differentiation using the differentiation-inducing system comprises: (a) contacting a test compound with a cell that expresses vascular endothelial growth factor receptor type 1; , And (c) selecting a compound that promotes the differentiation.

The test compounds used for screening are not limited to these, but include, for example, cell extracts, expression products of gene libraries, synthetic low molecular compounds, synthetic peptides, modified peptides, natural compounds and the like. When a gene is used as a test compound, the gene is introduced into a cell and expressed. The cells used for the screening are not particularly limited as long as they express vascular endothelial growth factor receptor type 1 and can differentiate into osteoclasts. Examples include non-adherent bone marrow cells and spleen cells.

Specifically, for example, a test compound is added to a mouse bone marrow cell culture system, cultured for 7 to 10 days, and the number of generated osteoclasts is measured. A compound is selected such that the addition of the test compound significantly increases the number of osteoclasts. When a test compound is added, VEGF and / or PlGF-1 may be allowed to coexist, and a compound that further increases the number of osteoclasts formed may be selected.

As compounds obtained by the above screening, compounds having various points of action can be considered.
For example, those which directly act on vascular endothelial growth factor receptor type 1 to promote their function, those which act on molecules binding to vascular endothelial growth factor receptor type 1 and indirectly promote the function of the receptor And so on. The point of action of the compound can be verified by various control experiments. For example, a test compound is brought into contact with cells overexpressing the receptor by transforming the vascular endothelial growth factor receptor type 1 gene and control cells, and the differentiation of osteoclasts is significantly promoted in the overexpressed cells compared to the control cells. By examining whether the compound promotes the activity of vascular endothelial growth factor receptor type 1, it can be determined. In addition, osteoclasts in the presence of a protein containing an extracellular domain of vascular endothelial growth factor receptor type 1 (or a ligand binding domain), such as the VEGFR-1 / Fc chimeric protein and soluble VEGFR-1 described in the Examples. Is detected in the same manner as above, and if a compound having a significantly higher activity of promoting differentiation into osteoclasts is selected as compared with this control experiment, a compound acting as a ligand of vascular endothelial growth factor receptor type 1 is obtained. be able to. Alternatively, the same detection may be performed using cells lacking the vascular endothelial growth factor receptor type 1 gene or cells expressing a mutant lacking the kinase activity as a control to confirm whether the effect of the compound is suppressed. it can. In this way, a compound may be a vascular endothelial growth factor receptor 1
It is possible to determine whether to promote osteoclast differentiation through the mold. In addition, screening is performed in the presence of an antibody against c-Fms, which is a receptor for M-CSF, or using a cell in which c-Fms has been mutated, and a signal mediated by M-CSF / c-Fms is detected. It can also be turned off.

It is also possible to screen for a compound that inhibits osteoclast differentiation using the osteoclast differentiation induction system of the present invention. This screening is
(A) contacting cells expressing vascular endothelial growth factor receptor type 1 with vascular endothelial growth factor or placental growth factor type 1 in the presence of a test compound; (b) formation and survival of osteoclasts;
And / or detecting bone resorption; and (c)
Selecting a compound that inhibits the formation, survival, and / or bone resorption.

Specifically, for example, a test compound is added to a mouse bone marrow cell culture system in the presence of VEGF or PlGF-1,
After culturing for 7 to 10 days, the number of generated osteoclasts is measured. A compound is selected such that the addition significantly reduces the number of osteoclasts.

In the screening of the present invention, preparation of bone marrow cells and measurement of formation and / or survival of osteoclasts can be performed, for example, as follows. <Preparation of mouse bone marrow cells> 6-12 week old mice (C3H / He
J; CLEA Japan), the femur and tibia were aseptically removed, their epiphyses were cut off, and 1 ml of α-MEM medium (10% fetal serum, 1%
Bone marrow cells are extruded with 00 units / ml penicillin G and 100 μg / ml streptomycin), pipetted well, wait until the bone residue precipitates, and collect the supernatant.
It is further washed 1-2 times with fresh medium to prepare bone marrow cells for the assay. <Osteoclast differentiation formation method> The above bone marrow cells were suspended in an α-MEM medium containing 10 ^-8 M of active vitamin D and [1,25 (OH) ₂ D ₃ ], and 2 × 10 ⁶ Adjust to a concentration of individual cells / ml, add 180 μl to a 96-well plate and 20 μl of the test sample solution, and add
% C0 ₂ under culturing one or two weeks. During that time, 3/4 of the medium is replaced with a new medium at intervals of 3 to 4 days, and the same amount of the test sample solution is newly added. <Method of identifying osteoclasts> TR, a marker enzyme for osteoclasts
AP (tartrate-resistant acid phosphatase) is stained with a substrate. That is, after the above cultured bone marrow cells were fixed with an acetone-citrate buffer, the substrate (Naphthol AS) was added in the presence of tartaric acid.
-MXphosphate) and pigment (Fastredviolet LB salt) 37
Stain by reacting at 1 ° C for 1 hour (Takahashi,
N. et al., Endocrinology, 122, p1373 (1988)).

In addition, bone resorption was measured using a pit using ivory.
For example, it can be performed as follows using a forming method. Prepare a dentin slice 6 mm in diameter and 1 mm thick from ivory and sterilize it by sonication in 80% alcohol. After washing with α-MEM medium, each slice was
The cells are transferred to the well bottom of a well plate, and then osteoclasts are induced to differentiate from bone marrow cells according to the above-mentioned “Osteoclast differentiation forming method”. One or two weeks later, the osteoclasts on the dentin slice are stained by the TRAP staining method, treated with 0.25% trypsin-0.02% EDTA overnight, and the cells on the slice are scraped off with a silicon scraper. Pit on dentin slice
The bone resorption activity (osteolytic activity) of cells induced to differentiate from bone marrow cells can be examined by observing the (resorption pit) under a microscope and measuring the number or the number of meshes per pit.

The screening system of the present invention is not limited to the screening of drugs for inducing osteoclast differentiation, but also isolates compounds that promote or suppress various signal transduction via vascular endothelial growth factor receptor type-1. Useful to For example, the screening system of the present invention can be used for screening for a drug that promotes or suppresses vascular induction. Since vascular endothelial growth factor receptor type 1 is involved in angiogenesis, compounds that modulate this activity can be used as agents to control vascular induction. Since it is complicated to directly assay the vascular inducing activity, screening can be performed more easily by using the osteoclast-inducing system of the present invention. The actual screening involves osteoclast formation, survival,
And / or screening for compounds that promote or inhibit bone resorption. The vascular induction promoter obtained by this screening is useful as an agent for regenerating blood vessels lost due to diseases such as myocardial infarction and cerebral infarction. Conversely, vascular induction inhibitors are useful as anticancer agents. Cancers of interest include all solid tumors.

[0028] 3. Promoter or inhibitor of osteoclast differentiation (1) Promoter In the present invention, administration of vascular endothelial growth factor (VEGF) to op / op mice results in formation of osteoclasts and replacement of VEGF. In addition, osteoclasts were formed similarly to VEGF by administering placental growth factor type 1 (PlGF-1). That is, it was shown that the VEGF signal was mediated through vascular endothelial growth factor receptor type 1. This fact indicates that a compound targeting vascular endothelial growth factor receptor type 1 is an agent that regulates osteoclast formation and survival, and furthermore, bone resorption.

That is, the present invention relates to an agent for promoting the formation, survival and / or bone resorption of osteoclasts, comprising a compound that activates vascular endothelial growth factor receptor type 1 as an active ingredient. Examples of the compound that activates vascular endothelial growth factor receptor type 1 as an active ingredient of a drug include those that bind to and activate vascular endothelial growth factor receptor type 1 (receptor agonists), Compounds that promote the binding between growth factor receptor type 1 and its ligand are included. Suitable compounds include, for example, ligands for vascular endothelial growth factor receptor type 1. Known vascular endothelial growth factor receptor type 1 ligands include vascular endothelial growth factor (VEGF) and placental growth factor type 1 (PlGF-1). Further, a compound obtained by the above screening may be used. Such compounds are useful in vivo, e
It is used as an agent to induce osteoclast formation and maintain its survival in vivo or in vitro. It can also be administered to a living body to promote bone resorption. When administering proteins such as vascular endothelial growth factor (VEGF) and placental growth factor type 1 (PlGF-1), in addition to administering these proteins directly or ex vivo, nucleic acids encoding these proteins may be used. Gene therapy, etc., is also conceivable. Diseases to be treated include bone marble disease and low-rotation osteoporosis, and can be used for promoting fracture healing.

(2) Inhibitor The present invention inhibits the formation, survival, and / or bone resorption of osteoclasts, which comprises a compound that inhibits activation of vascular endothelial growth factor receptor type 1 as an active ingredient. For drugs. Such compounds include, for example, those that bind to vascular endothelial growth factor receptor type 1 and inhibit its activation (receptor antagonists) and those that bind vascular endothelial growth factor receptor type 1 to its ligand. Inhibitors and the like are included.
As shown in the Examples, administration of an antibody against a vascular endothelial growth factor receptor type 1 ligand inhibited the binding of the ligand to vascular endothelial growth factor receptor type 1 and reduced the number of osteoclasts. (Example 5). Thus, antibodies against vascular endothelial growth factor receptor type 1 or its ligands are useful as agents of the invention for inhibiting osteoclast formation, survival, and / or bone resorption. When used for antibody therapy, human antibodies or human antibodies are preferred. In addition, administration of soluble vascular endothelial growth factor receptor type 1 protein, such as a chimeric protein of vascular endothelial growth factor receptor type 1 and Fc, has a ligand neutralizing effect (Example 3). . Soluble forms of the receptor also exist in nature. Such proteins are also suitably used for the drug of the present invention. Further, a compound obtained by the above screening may be used.

Such compounds can be used in vivo, ex vivo,
Alternatively, it is used as an agent for suppressing the formation and / or survival of osteoclasts in vitro, and can suppress bone resorption when administered to a living body. Diseases to be treated include bone metastatic cancer, osteosarcoma, high-turn osteoporosis, hypercalcemia, bone destruction in rheumatoid arthritis, and the like.

(3) Formulation A compound that regulates osteoclast differentiation, survival, and / or bone resorption, or a compound that regulates vascular induction is used as a regulator (such as an accelerator or a promoter) of bone resorption in vitro or in vivo. It is useful as an agent such as an inhibitor) or a regulator of blood vessel induction (an accelerator or an inhibitor). The drug of the present invention can be used as a reagent in test research and the like, and as a drug for preventing or treating various diseases. The agents of the present invention can also be formulated into compositions with other solutes or solvents. When a compound that regulates osteoclast differentiation, survival, and / or bone resorption or a compound that regulates vascular induction is used as a medicament, the compound itself may be directly administered to a patient, or a known pharmaceutical method may be used. And can be administered. For example, pharmacologically acceptable carriers or vehicles, specifically, sterilized water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, can be formulated and administered as appropriate in combination with stabilizers and the like. Conceivable. Administration to a patient can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, or the like, or intranasally, transbronchially, ironically, intramuscularly, or orally by a method known to those skilled in the art. . When administered by the in vivo method, the composition is generally prepared as an injection or the like, and if necessary, a conventional carrier may be added.
In the case of liposome or membrane-fused liposome (Sendai virus (HVJ) -liposome etc.),
Liposome preparations such as suspensions, cryogens, and centrifugal concentrated cryogens can be prepared. The dose varies depending on the purpose of treatment, the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
If the compound can be encoded by DNA, the DNA may be incorporated into a gene therapy vector to perform gene therapy. The dose and the method of administration vary depending on various factors such as the weight and age of the patient, the symptoms, the activity of the active ingredient, and the like, and can be appropriately selected by those skilled in the art. The dosage is usually 0.0001-100 mg, preferably
It is believed to be in the range of 0.001-10 mg.

[0033]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples. [Example 1] Effect of VEGF on osteoclast formation In osteopetrotic (op / op) mice that have lost M-CSF / CSF-1 activity, VEGF complements functional M-CSF deficiency, and osteoclasts To see if it can support cell formation,
First, rhM-CSF, rhVEGF165, rhVEGF121, or rhPlGF-
An experiment was performed in which 1 was administered to 12-day-old op / op mice. op / o
p mice and their normal littermates (+ /?) were prepared as previously described (Kodama, H. et al. 1991. J. Exp. Med. 1
73: 269; Kodama, H. et al. 1993. J. Bone Miner. Re
s. 8:45). Whether the mice were of the op / op genotype was identified by the absence of incisor eruption at 11 days of age. Administration of cytokines and / or antibodies to op / op mice was performed as follows. 5 μg of rhM-CSF (Austral Biologicals, San Ramo
n, CA), rhVEGF165 (Genzyme, Cambridge, MA), rhVE
GF121 (Genzyme) or rhPlGF-1 (R & D Systems, Min
neapolis, MN) was intraperitoneally administered to 12-day-old op / op mice and sacrificed 3 days after administration. AFS98 rat anti-mouse c-Fm
s monoclonal antibody (Sudo, T. et al. 1995. Oncogen
e. If 11: 2469) is administered, 2
Twenty hours before and 24 hours after cytokine administration, mutant mice were intraperitoneally administered at a dose of 750 μg / mouse and sacrificed 3 days after cytokine administration. From sacrifice to counting of osteoclasts, the procedure was as follows. Op / op mice were anesthetized with ether and perfused with a 4% periodate-lysine-paraformaldehyde fixative (pH 7.4) from the descending aorta.
The femur was decalcified in 10% EDTA (pH 7.0) for 10 days and embedded in paraffin. A longitudinal section (7 μm thick) of the center of the sample containing the entire femur was made and stained for tartrate-resistant acid phosphatase (TRAP) activity as previously described (Kodama, H. et al. 1993. J. Bone Mine
r. Res. 8:45; Niida, S. et al. 1994. J. Bone Mine
r. Res. 9: 873), and counterstained with hematoxylin. TRAP positive cells with two or more nuclei were counted as osteoclasts. Six sections obtained from three mice were counted, and the mean ± standard deviation was determined. Some sections were subjected to Mallory Azan staining.

As shown in Table 1, any of the above factors
A single dose of 5 μg was sufficient for the formation of osteoclasts in mutant mice, but not in rhVEGF (rhVEGF165 and rhVGF165).
The formation by EGF121) and rhPlGF-1 was 60-70% as compared with that by rhM-CSF. An anti-c-Fms monoclonal antibody, AFS98 (Sudo, T. et al.
1995. Oncogene. 11: 2469) reduced the formation of osteoclasts by rhM-CSF to about 25%, but not rhVEGF or rhPlGF-1.
Did not reduce formation. From this, c-Fms
Mediates the response of osteoclast precursor cells to M-CSF, but not VEGF or PlGF-1.

[0035]

Table 1 rhM-CSF, rhVEGF, and op / op mice
The ability of rhPlGF-1 to form osteoclasts

Example 2 Immunohistochemical Staining of VEGFR The femurs of 2-3 week old + /? Mice or op / op mice were fixed in the same manner as in Example 1 and embedded in paraffin. A section (5 μm thick) of a femur was prepared using a rabbit anti-mouse VEGFR-1 polyclonal antibody (Santa Cruz Biotechnology) or AVAS12.
Rat anti-mouse VEGFR-2 monoclonal antibody (Kataoka,
H. et al. 1997. Develop. Growth Differ. 39: 729) for immunohistochemical staining. Counterstaining was performed with hematoxylin using a Vectastain elite ABC kit (Vector Laboratories, Burlingame, CA). Normal rabbit IgG (Santa Cruz Biotechnology) and rat IgG
2a (Santa Cruz Biotechnology) was used as a control for polyclonal and monoclonal antibodies, respectively.

As shown in FIG. 1A, osteoclasts were strongly stained with a rabbit anti-mouse VEGFR-1 polyclonal antibody, whereas endothelial cells were weakly positive for VEGFR-1. On the other hand, osteoclasts are AVAS12 anti-mouse VEGFR-2 monoclonal antibodies (Kataoka, H. et al. 1997. Develop. Growth Diff.
er. 39: 729), but the endothelial cells were VEGF
Positive for R-2 staining (FIG. 1B). Normal rabbit Ig
G (FIG. 1C) and rat IgG2a did not stain either cell type. The osteoclasts of op / op mice induced by rhM-CSF also showed the same staining pattern as above. These results demonstrated that osteoclasts, like cells of the monocyte / macrophage lineage, mainly expressed VEGFR-1 (Berleon, B. et al. 1996. Blood. 87: 3336). ; Claus
s, M. et al. 1996. J. Biol. Chem. 271: 17629). VEGF
121 does not bind to neuropilin-1 (Neufeld, G. et a
l. 1999. FASEB J. 13: 9). PlGF-1 binds to VEGFR-1, but not VEGFR-2 or neuropilin-1 (Neufel
d, G. et al. 1999.FASEB J. 13: 9; Berleon, B. et a
l. 1996. Blood. 87: 3336; Clauss, M. et al. 1996.J.
Biol. Chem. 271: 17629; Park, JE et al. 1994. J.
Biol. Chem. 169: 25646; Sawano, A. et al. 1996. Ce
ll Growth Differ. 7: 213). Both rhVEGF121 and rhPlGF-1 showed the same activity as rhVEGF165 in supporting osteoclastogenesis (Table 1), indicating that the response of osteoclast precursor cells to VEGF is mediated by VEGFR-1. I was assured.

Example 3 Influence of Neutralization of VEGF on Osteoclast Formation and Survival Next, we administered endogenous VEGFR-1 / Fc chimeric proteins to op / op mice VEGF produced in
Were tested for the ability of VEGF and M-CSF to support the survival of mature osteoclasts. First, add a 12-day-old op / op mouse
Pretreatment was performed in which hM-CSF was administered once. 4 of this preprocessing
After 5 days, 5 μg of VEGFR-1 / Fc chimeric protein (R & D System
s) and / or rhM-CSF was administered intraperitoneally 6 times every 12 hours. Mice were sacrificed 7 days after pretreatment. As a control for the chimeric protein, 5 μg of human IgG (ICN Pharmaceutical
s, Aurora, OH) were administered as above. The number of osteoclasts was counted in a longitudinal section at the center of the entire femur. The results were expressed as the mean ± standard deviation of six sections obtained from three mice (Table 2).

The present inventors have previously reported (Kodama, H. et a
l. 1993. J. Bone Miner. Res. 8:45; Niida, S. et a
l. 1994. J. Bone Miner. Res. 9: 873)
Three days after the single administration of M-CSF, the number of osteoclasts reached a plateau and was maintained until day 7 (Tables 1 and 2). Continuous administration of the chimeric protein every 12 hours on days 4-6 reduced osteoclasts to about 25%. Administration of human IgG1 did not change the number of osteoclasts (Table 2). On the other hand,
When rhM-CSF was administered together with VEGFR-1 / Fc, the number of osteoclasts increased to the same level as when rhM-CSF alone was administered continuously. These results indicate that osteoclasts formed after a single dose of rhM-CSF are supported by VEGF produced endogenously in op / op mice,
M-CSF has been shown to be able to support the survival of mature osteoclasts without the help of VEGF.

[0040]

[Table 2] Effect of VEGFR-1 / Fc chimeric protein administration on the survival of osteoclasts of op / op mice formed with rhM-CSF

The present inventors further concluded that op / op mice that received only one dose of rhM-CSF, or one dose of rhM-CSF, and a continuous dose of VEGFR-1 / Fc and rhM-CSF were given. Op /
Op mice were examined for bone resorption in the femur. Osteoclasts from the former group of mice can function with the support of endogenous VEGF, whereas the latter group will rely on exogenous rhM-CSF.

As previously reported (Kodama, H. et al. 199
3. J. Bone Miner. Res. 8:45), after one dose of rhM-CSF
On day 7, a large amount of bone trabec
ulae) and its replacement with bone marrow were observed (FIGS. 2A and 2B). Bone resorption was similarly observed in the latter group of mice (FIG. 2C). These observations indicated that both M-CSF and VEGF can support osteoclast bone resorption.

As described above, it was found that endogenous VEGF was produced in an amount sufficient for the survival of mature osteoclasts and the expression of their functions. Then, we next examined whether rhM-CSF could form osteoclasts without the help of endogenous VEGF. At 12 days of age, treatment of op / op mice was started. 5 μg without pretreatment
1 dose of rhM-CSF alone, or 5 μg of rhM-CSF alone, or 6 doses of 5 μg of VEGFR-1 / Fc consecutively every 12 hours, and mice were sacrificed 3 days after the start of treatment did. The number of osteoclasts was counted in a longitudinal section at the center of the entire femur. The results were expressed as the mean ± standard deviation of six sections obtained from three mice. As shown in Table 3, twice the number of osteoclasts was formed when rhM-CSF was administered a plurality of times compared to when the rhM-CSF was administered once. Administration of VEGFR-1 / Fc simultaneously with rhM-CSF had no effect on osteoclast formation. These results clearly demonstrate for the first time that M-CSF has the ability to support osteoclast differentiation in vivo.

[0044]

[Table 3] Effect of VEGFR-1 / Fc chimeric protein administration on rhM-CSF-induced osteoclast formation in op / op mice

[Example 4] VE in an in vitro culture system
Generation of osteoclasts by GF M-CSF enhances osteoclast differentiation ODF / OPGL / TRANCE / RANK
L (osteoclast differentiation factor / osteoproteg
erin ligand / TNF-related activation-induced sytok
ine / receptor activator of nuclear factor κB lig
and) in support of it (Y
asuda, H et al. 1998. Proc. Natl. Acad. Sci. USA.
95: 3597; Lacey, DL et al. 1998. Cell. 93: 165).
Therefore, rhVEGF165 is a rhM-CSF
Was investigated using an in vitro culture system of non-adherent bone marrow cells. rhVEGF165 and rhM-CSF were dissolved in fetal bovine serum (FBS) at concentrations of 2 μg / ml and 200 ng / ml, respectively. Each well of a 96-well plate was coated with 5 μl of either cytokine solution or FBS and air-dried for 30 minutes. Bone marrow cells obtained from the tibia and femur of 5 to 8 week old male ddY mice (obtained from Saitama Experimental Animal Supply Co., Ltd. (Sugito-cho, Kita-Katsushika-gun, Saitama)) were subjected to Ly and Mishell (Ly,
IA and RI Mishell. 1974. J. Immunol. Methods.
5: 239) as described in Sephadex G-10 (Amersham
(Pharmacia Biotech, Uppsala, Sweden). Non-adherent cells are seeded in cytokine-coated wells at a density of 1 × 10 ⁵ cells / well, and 100 ng / ml of recombinant human RANK ligand (rhRANKL, PeproTec, London, U.S.A.)
K) in the presence or absence of α-MEM with 15% FBS
Cultured for 7 days. Final concentrations of rhVEGF165 and rhM-CSF were 100 ng / ml and 10 ng / ml, respectively. Cultured cells are fixed with 4% paraformaldehyde as described above, and TRAP
Stained. Non-adherent bone marrow cells were placed on a 5 mm diameter dentin slice, placed in a 24-well plate, and cultured for 7 days in the same manner as above. Slices are backscattered (backscatt
Observed with a microscope as described previously (Amano, H. et al. 1998. J. Bone Miner. Re
s.13: 846).

Previous findings (Yasuda, H et al. 1998.
Proc. Natl. Acad. Sci. USA. 95: 3597; Lacey, DL.
Consistent with et al. 1998. Cell. 93: 165), no TRAP-positive cells were observed even when rhM-CSF or rhRANKL alone was present. RhVEGF165 alone did not support osteoclast differentiation (FIG. 3A). The combination of rhVEGF165 and rhRANKL supported the generation of TRAP positive cells (FIG. 3B). The size of the cells was significantly smaller than cells generated in the presence of rhM-CSF and rhRANKL (FIG. 3C). As a result, osteoclasts supported by rhVEGF165 and rhRANKL are:
Small resorption pits formed compared to those with rhM-CSF and rhRANKL (FIGS. 3D and 3E). These results demonstrated that VEGF could certainly support osteoclast differentiation in cooperation with ODF / OPGL / TRANCE / RANKL.

Example 5 Effect of Endogenous VEGF on Age-Related Increase in Osteoclasts in Op / op Mice In this example, osteoclasts in op / op mice increased with aging, and (Marks, SC, Jr., and
PW Lane. 1976. J. Hered. 67:11; Marks, SC, Jr.
1982. Am. J. Anat. 163: 157; Begg, SK et al. 199
3. J. Exp. Med. 177: 237) shows that endogenously produced V
We investigated whether it was due to EGF. 100 μg of goat anti-mouse VEGF polyclonal antibody (R & DS
ystems) were administered 5 times continuously every 12 hours. As a control, goat IgG (SantaCrus Biotechnology, Santa Cru
z, CA) was administered as above. Another group of mice received a single dose of 5 μg of rhVEGF165. All these mice were sacrificed 3 days after the start of treatment.

Although the size of the femur section of the aged mouse was about 1.6 times that of the young mouse, as shown in FIG. 4A, the femur of the 2-week-old op / op mouse (Table 1) Compared with (3) and (3), a significantly large number of small osteoclasts having 2-3 nuclei were observed in 2-month-old op / op mice (28 ± 1 osteoclasts / section). In addition, TRAP-positive mononuclear cells were often observed in the marrow space.

100 μg of goat anti-VEGF polyclonal antibody
When administered 5 times every 12 hours, the number of osteoclasts was significantly reduced (4 ± 2 osteoclasts / section) (FIG. 4B). Goat IgG administration did not change the number of osteoclasts. When VEGFR-1 / Fc was administered in the same manner as in the 2-week-old mutant mouse (Table 2), no change was observed in the number of osteoclasts. 5 μg rhVE
A single administration of GF165 caused further formation of osteoclasts (64 ±
5 osteoclasts / section) (FIG. 4C), indicating that VEGF levels in the femur of 2-month-old op / op mice are still insufficient to maximize osteoclast formation. . These results demonstrated that VEGF causes spontaneous osteoclast formation in op / op mice lacking functional M-CSF. The number of osteoclasts changes with age,
Increased levels of VEGF production in aged mice due to differences in the amount of VEGFR-1 / Fc required to neutralize endogenous VEGF in 2-week-old and 2-month-old mice Is suggested. However, it cannot be denied that the sensitivity of osteoclast precursor cells to VEGF has changed with aging.

Example 6 Micromorphology of Osteoclasts Induced by VEGF As described above, recombinant human VEGF was produced in op / op mice.
Administration of (rhVEGF) restored osteoclastogenesis and improved osteopetrosis symptoms. This means that r
hVEGF-induced osteoclasts suggest that their function is also restored. Therefore, in order to confirm the morphology of the osteoclasts induced in this experimental system, observation using an electron microscope was performed. Four days after intraperitoneal administration of 5 μg / body rhVEGF to the op / op mice, the op / op mice were treated with 2% paraformaldehyde-2% glutaraldehyde (in 0.1 M cacodylat
e buffer, pH 7.4), fix the perfusion and remove the femur 1%
Post fixation with osmic acid was performed. Then with 10% EDTA
Perform decalcification for 2 weeks, embed bone tissue in Epon812 resin,
An electron microscope sample was used.

Most of the VEGF-induced osteoclasts were small and had a small number of nuclei. However, the wavy border (ruffled border: FIG. 5, rb) developed on the side in contact with the bone surface and the bright zone surrounding it (Clear zone: Fig. 5, cz). In addition, many mitochondria (mt) were observed in the cytoplasm, and a Golgi apparatus was observed between them, indicating the characteristic of active osteoclasts. These findings indicate that osteoclasts induced by VEGF are small but have morphological features to fulfill their function.

[Example 7] Examination of the effect of VEGF on mature osteoclasts In order to examine the effect of VEGF on mature osteoclasts, an experiment was carried out in a culture system using isolated mature osteoclasts. went. Isolation of osteoclasts is described in Chambers et al. (Chambers TJ and Magnus C.
J .: J. Pathology 136, 27-39, 1982)
It was collected from rat lower limb bone. Briefly, a 1-day-old Wistar rat was sacrificed by decapitation, left and right tibias and femurs were taken out, placed in a Petri dish containing M199 medium, and bone attachments such as muscle were removed using a scalpel. The bone was further minced in a fresh M199 medium, and the supernatant (a suspension containing bone cells) was collected and centrifuged to obtain bone cells.
This bone cell was adjusted with 25 mM HEPES buffer (pH 7.0) 1
The suspension was suspended again in an M199 medium containing 0% FBS, placed on a cover slip, and allowed to adhere in a constant temperature incubator at 37 ° C. in the air for 1 hour. After washing with M199 medium containing 10% FBS, osteoclasts and their progenitor cells were isolated from other cells such as osteoblasts due to differences in adhesion. This isolated osteoclast is IB containing 15% FBS
The cells were cultured in a medium. The reaction of this culture system was compared and observed between a group to which 20 ng / ml of M-CSF was added and a group to which 100 ng / ml of VEGF was added.

The osteoclasts isolated and transferred into the culture broth showed a relatively small cell morphology (FIG. 6a). In the M-CSF-added group, cell extension started immediately after the addition, and fusion between cells was also observed. Extension occurred throughout the cell perimeter. One minute after the addition, the cells turned into large cells having many nuclei (FIG. 6b). Furthermore, no change was observed in the morphology even after 30 minutes and 60 minutes (Fig. 6c-d). On the other hand, VEGF
In the added group, no change was observed even after 10 minutes from the addition (FIG. 6f), and extension was observed in a part of the cell periphery 20 minutes after the addition (FIG. 6g, arrow). 6 more
After 0 minutes, the extension became clearer (FIG. 6h, arrow). In view of the fact that osteoclast extension in vitro is homologous to the activity of bone resorption function, VE
Although GF does not have immediate effect like M-CSF, it suggested that GF acts slowly on mature osteoclasts and expresses bone resorption function. This phenomenon suggests that osteoclasts gradually appear with age in op / op mice, and that they are closely related to their small size.

[0054]

According to the present invention, it has been revealed that VEGFR-1 is closely involved in the formation and survival of osteoclasts and bone resorption. Also, by controlling the activation of VEGFR-1, new methods and agents for regulating osteoclast formation and survival and controlling bone resorption have been provided. The present invention also provides a method for screening a novel compound that controls bone resorption. The agent of the present invention can be used for prevention and treatment of various diseases accompanied by abnormal bone resorption. VEGFR-1 and its ligands VEGF and PlG
Since the expression level of F-1 is deeply involved in bone metabolism, it may be possible to diagnose abnormal bone metabolism by examining the levels of these proteins. In addition, it is also possible to screen for a drug that controls vascular induction by the screening method of the present invention.

[Brief description of the drawings]

FIG. 1 is a diagram showing immunohistochemical staining of a femoral section for VEGFR. Three-week old + /? Longitudinal sections of the femurs of the mice were subjected to anti-VEGFR-1 polyclonal antibody (A), AVAS12 anti-VFGF
The cells were stained with an R-2 monoclonal antibody (B) or rabbit IgG (C). Arrowheads indicate osteoclasts and arrows indicate endothelial cells. 238 times magnification.

FIG. 2 shows bone resorption of osteoclasts in op / op mouse femoral trabeculae by osteoclasts supported by endogenous VEGF or exogenous rhM-CSF. Treatment of the mice started at 12 days of age and was sacrificed at 19 days of age. Longitudinal sections of the femur were stained with Mallory's Azan. Each micrograph shows a group of femurs obtained from three individuals. (A) No administration; (B) 5 μg of rhM-CSF at 12 days of age
Single administration; (C) 5 μg of rhM-CSF was administered once at the age of 12 days,
~ 18 days old VEGFR-1 / Fc chimeric protein and rhM-CSF 5μ each
g, 6 consecutive doses every 12 hours. Magnification is 20x.

FIG. 3 shows the ability of VEGF to support in vitro generation of osteoclasts. Non-adherent bone marrow cells were plated on a 96-well plate (AC) or dentin slices (D, E) with rhVEGF1
The cells were cultured for 7 days in the presence of 65 alone (A), rhVEGF165 and rhRANKL (B and D), or rhM-CSF and rhRANKL (C and E). Cultures were stained for TRAP activity (A-C) or observed using reflection electron microscopy (D, E). Arrows in (D) indicate small absorption fossa. Magnification 24x (AC).
Bars in (D) and (E) are 50 μm.

FIG. 4 shows the dependence of osteoclasts on endogenously produced VEGF in 2-month-old op / op mouse femurs. Mice were sacrificed 3 days after the start of treatment. Longitudinal sections of the femur were stained with TRAP activity and counterstained with hematoxylin. Each micrograph shows a group of femurs obtained from three individuals. The arrow in (B) is a mononuclear TRAP.
Shows positive cells. (A) No administration; (B) 100 μg of anti-VEGF polyclonal antibody administered continuously 5 times every 12 hours; (C) 5 μg of rhVEGF165 administered once. Magnification 103 times.

FIG. 5 is a diagram showing the micromorphology of osteoclasts induced by VEGF. Four days after rhVEGF was intraperitoneally administered to op / op mice, the femur was excised and observed with an electron microscope. Bones (Bon
e), nucleus (Nu), mitochondria (mt), wavy edge (ruffl
ed border: rb) and light zone (clear zone: cz).

FIG. 6 shows the effect of VEGF on mature osteoclasts. Osteoclasts were isolated from 1-day-old rat lower extremity bones and cultured in IB medium containing 15% FBS. The reaction of this culture system was compared and observed between a group to which 20 ng / ml of M-CSF was added and a group to which 100 ng / ml of VEGF was added. Before (a) and after 1 (b), 30 (c), and 60 after addition of M-CSF
(D) minutes, before VEGF addition (e), and after addition 10 (f),
The osteoclasts (oc) after 20 (g) and 60 (h) minutes are shown. .

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61P 43/00 101 43/00 101 105 105 C12Q 1/04 C12Q 1/04 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 X 33/566 33/566 A61K 37/24 (72) Inventor Norihiko Maeda 6-9-305 Ushida Honcho, Higashi-ku, Hiroshima City, Hiroshima, Japan

Claims (23)

[Claims] 1. A method for inducing osteoclast differentiation, comprising the steps of: causing a cell expressing vascular endothelial growth factor receptor type 1 in the presence of a compound that activates vascular endothelial growth factor receptor type 1; A method comprising culturing. 2. The method according to claim 1, wherein the compound that activates vascular endothelial growth factor receptor type 1 is a ligand for vascular endothelial growth factor receptor type 1. 3. The method of claim 2, wherein the ligand for vascular endothelial growth factor receptor type 1 is vascular endothelial growth factor or placental growth factor type 1. 4. The method according to claim 1, wherein the cells expressing vascular endothelial growth factor receptor type 1 are non-adherent bone marrow cells. 5. A method for screening a compound that promotes or inhibits osteoclast differentiation, survival, and / or bone resorption, comprising: (a) vascular endothelial growth factor receptor type 1 in the presence of a test compound; Contacting endothelial growth factor or placental growth factor type 1; (b) detecting the binding between the vascular endothelial growth factor receptor type 1 and the vascular endothelial growth factor or placental growth factor type 1; Selecting a compound that promotes or inhibits binding. 6. A method of screening for a compound that promotes osteoclast differentiation, survival, and / or bone resorption, comprising: (a) contacting a test compound with a cell that expresses vascular endothelial growth factor receptor type 1; (B) detecting osteoclast differentiation, survival, and / or bone resorption;
(C) selecting a compound that promotes said differentiation, survival, and / or bone resorption. 7. A method for screening a compound that inhibits osteoclast differentiation, survival, and / or bone resorption, comprising: (a) a cell that expresses vascular endothelial growth factor receptor type 1 in the presence of a test compound; Contacting vascular endothelial growth factor or placental growth factor type 1 with (b) osteoclast differentiation;
A method comprising: detecting survival and / or bone resorption; and (c) selecting a compound that inhibits said differentiation, survival, and / or bone resorption. 8. The method according to claim 6, wherein the cells expressing vascular endothelial growth factor receptor type 1 are non-adherent bone marrow cells. 9. A method for screening a compound that promotes vascular induction, comprising: (a) contacting a test compound with a cell that expresses vascular endothelial growth factor receptor type 1;
(B) detecting osteoclast differentiation, survival, and / or bone resorption; and (c) selecting a compound that promotes the differentiation, survival, and / or bone resorption. 10. A method for screening a compound that inhibits vascular induction, comprising: (a) in the presence of a test compound,
Contacting cells expressing vascular endothelial growth factor receptor type 1 with vascular endothelial growth factor or placental growth factor type 1,
(B) detecting osteoclast differentiation, survival, and / or bone resorption; and (c) selecting a compound that inhibits the differentiation, survival, and / or bone resorption. 11. The vascular endothelial growth factor receptor type 1-expressing cells are non-adherent bone marrow cells.
The method described in. 12. An agent for promoting osteoclast differentiation, survival, and / or bone resorption, comprising a compound that activates vascular endothelial growth factor receptor type 1 as an active ingredient. 13. The agent according to claim 12, wherein the compound that activates vascular endothelial growth factor receptor type 1 is a ligand for vascular endothelial growth factor receptor type 1. 14. The agent according to claim 13, wherein the ligand for vascular endothelial growth factor receptor type 1 is vascular endothelial growth factor or placental growth factor type 1. 15. The drug according to claim 12, wherein the compound that activates vascular endothelial growth factor receptor type 1 is a compound isolated by the screening method according to claim 5 or 6. 16. The agent according to claim 12, which is used for treating a disease or injury selected from the group consisting of marble disease, low-rotation osteoporosis, and fracture. 17. An osteoclast differentiation, comprising a compound that inhibits activation of vascular endothelial growth factor receptor type 1 as an active ingredient.
An agent for inhibiting survival and / or bone resorption. 18. The compound that inhibits activation of vascular endothelial growth factor receptor type 1 is a compound that inhibits binding between vascular endothelial growth factor receptor type 1 and a ligand for vascular endothelial growth factor receptor type 1. The medicament of claim 17. 19. The agent according to claim 17, wherein the compound that inhibits activation of vascular endothelial growth factor receptor type 1 is an antibody against vascular endothelial growth factor. 20. The compound that inhibits activation of vascular endothelial growth factor receptor type 1 is a compound isolated by the screening method according to claim 5 or 7.
7. The drug according to 7. 21. The method according to claim 17, which is used for treating a disease selected from the group consisting of high-rotation type osteoporosis, bone metastatic cancer, osteosarcoma, hypercalcemia, and bone destruction in rheumatoid arthritis. The drug according to any one of the above. 22. A blood vessel induction promoter comprising a compound isolated by the method according to claim 9 as an active ingredient. 23. An anticancer agent comprising a compound isolated by the method according to claim 10 as an active ingredient.