BR112013029348B1 - USE OF AN OSTEOGENESIS ACCELERATING AGENT FOR PREVENTION AND TREATMENT OF BONE DISEASES - Google Patents

Abstract

PROMOTER OF OSTEOGENESIS. The purpose of the present invention is to provide an osteogenesis promoter for directly promoting osteogenesis by osteoblasts, and an agent for preventing and treating bone disease. The present invention is characterized in that a semaphorin 4D and plexin B1 binding inhibitory substance is used. For the binding inhibitory substance, suitable examples include anti-semaphorin 4D antibody, anti-plexin B1 antibody, and protein comprising extracellular domain of plexin B1.

Description

Technical Field

[001] The present invention relates to an osteogenesis accelerator and a preservative and therapeutic agent. It relates more particularly to an osteogenesis accelerator and to a preservative and therapeutic agent having as an active ingredient a substance which inhibits the binding of 4D semaphorin and B1 plexin.

Background of the Invention

[002] As the Japanese population ages, there has been an increase in patients suffering from bone fractures, osteoporosis, joint rheumatism, low back pain, and other bone diseases. Tissue function in bone tissue is maintained by coordinating osteoblasts that support osteogenesis and osteoclasts that support bone resorption and by maintaining a balance between osteogenesis and bone resorption. The balance of bone metabolism is disrupted by age, a decline in ovarian function as well as other factors. When osteogenesis decreases or when you have abnormal bone resorption, the amount of bone (bone density) decreases and a variety of bone diseases occur. Bone diseases include fractures, bone deficiency, osteoporosis, osteomalacia, osteopenia, low back pain, Paget's disease of bone, tonic myelitis, articular rheumatism, deformative arthrosis and the like. In particular, when an elderly person is afflicted with a bone disease, it is difficult for this person to function at a level required for daily life and depending on the case, there is a risk that the patient will become bedridden. As a result, it is extremely significant in the prevention and treatment of bone diseases in modern society where an increasing number of people are elderly.

[003] Activated vitamin D3 preparations, bisphosphonates, calcitonin, hormone preparations containing estradiol, vitamin K2 and the like are commonly used as therapeutic agents for bone diseases. Estradiol derivatives, activated vitamin D3 derivatives, bisphosphonate derivatives and the like are being developed as more effective therapeutic agents as they have fewer side effects (see Patent Document 1). However, vitamin D3 has an action that increases the concentration of calcium in the blood and estradiol and bisphosphonate have an effect of inhibiting bone resorption. However, none of these have an effect that directly accelerates osteogenesis through osteoblasts. A multi-drug therapy is the treatment of choice in this case so there is a need to develop a new preventive and therapeutic agent having a different action from that of conventional preservative and therapeutic agents.

[004] Bones are continually being recreated in stages known as bone remodeling in which the stage of bone resorption and the following stage of osteogenesis are repeated. The transition from one stage to another must be controlled precisely by bone cell secretion as it contributes to osteoclast-osteoblast communication or by bone rebuilding factors released from the bone matrix. It is well known that TGF-β and IGF-1 released during bone resorption stimulate osteogenesis so they are known as a coupling factor. Although there is an abundance of in vitro data on candidate molecules that are extremely important for the coupling factor, there is as yet no in vitro evidence for this.

[005] Axon guidance molecules manifest widely outside the nervous system. Thus, amoeboid cell, immune response, tissue development and angiogenesis and the like are controlled (see Non-Patent Documents 1 and 2). Based on research carried out in recent years, it is suggested that the axon guidance molecules of semaphorin and ephrin and the like contribute to intercellular communication between osteoclasts and osteoblasts (see Non-Patent Documents 3 to 5).

[006] It is well known that Semaphorin 4D is secreted from oligodendrocytes and that it induces growth cone destruction in the central nervous system. It is also clear that semaphorin 4D is extremely important in maintaining the immune response and the homeostasis of the immune system. A well-known 4D semaphorin receptor is plexin-B1 (see Non-Patent Document 6). Furthermore, Non-Patent Document 7 suggests that osteoclast formation can be accelerated through the action of inhibiting the differentiation of osteoclasts as well as osteoblasts. Non-Patent Document 8 reveals that semaphorin 4D is not detected in osteoblasts, that it is present on the surface of osteoclasts and bone quantity has been confirmed to increase as compared to wild-type mice in sema4D-/- female mice where Semaphorin 4D (Sema 4D) is deficient in homo[zygotes]. Prior Art Documents Patent Documents Special Table in Publication 2005-509629. [Non-Patent Document 1] .J. Dickson, Science 298, 1959 (Dec 6, 2002) [Non-Patent Document 2].B. Huber, A. L. Kolodkin, D.D. Ginty, J. F. Cloutier, Annual Review Neuroscience 26, 509 (2003) [Non-Patent Document 3] N. Takagahara et al., Nat. Cell Biol 8,615 (Jun, 2006) [Non-Patent Document 4] N. Irie et al., Cell Metab 4, 111 (Aug, 2006) [Non-Patent Document 5] C. Zhao et al., Cell Metab 4, 111 (Aug, 2006) [Non-Patent Document 6] I. Oinuma et al. , Science, Vol. 305, No. 5685, pp. 862-865 (August 6, 2004) [Non-Patent Document 7] Ishida Masanari, Kaneda Toshio, Muto Akihiro, Yoshida Masashi, Meeting Program Abstracts of the Japan Society of Bone Metabolism, vol. 25, page 266, published June 2007, [Analysis of Bioaction in Bone Metabolism in Osteoclast-derived Semaphorin 4D] [Non-Patent Document 8] Romain Dacquin, Chantal Domenget, Pierre Jurdic, Irma Machuca-Gayet, ASBMR 2010 Annual Meeting Abstracts, Presentation Abstracts, Presentation Number MOO 152, "Physiological Control of Bone Resorption by Sema phorin 4D is Dependent on Ovarian Function"

Overview of the Invention Problems Which the Present Invention Is Intended to Solve

[007] It is an object of the present invention to provide an osteogenesis accelerating agent and a preventive and therapeutic agent for bone diseases involving direct promotion of osteogenesis using osteoblasts.

Means Used to Solve Problems

[008] After much hard work and research under conditions described in the above-mentioned prior art, the inventors find that (a) when 4D semaphorin which is derived from osteoclasts binds with the plexin B receptor on the steoblast, it activates the small G protein RhoA which inhibits osteoblast differentiation, decreases IRS signals (signals that promote osteoblast differentiation) and inhibits osteoblast differentiation so that osteogenesis is inhibited; and that (b) anti-semaphorin 4D antibodies and anti-plexin B1 antibodies directly accelerate osteogenesis using osteoblasts.

[009] This means that the present invention relates to (1) an osteogenesis accelerating agent having an active ingredient of a substance that inhibits the binding between semaphorin 4D and plexin B1; (2) an osteogenesis accelerating agent as described in (1) above wherein the binding-inhibiting substance is an anti-4D semaphorin antibody; and (3) an osteogenesis accelerating agent as described in (1) above characteristic wherein the binding-inhibiting substance is an anti-plexin B1 antibody or a protein containing extracellular regions of plexin B1.

[0010] The present invention also relates to (4) an agent for preventing and treating bone diseases having as an active ingredient a substance that inhibits the binding of semaphorin 4D and plexin B1; (5) an agent for preventing and treating bone diseases as described in (4) above, wherein the binding-inhibiting substance is an anti-semaphorin 4D antibody; (6) an agent for preventing and treating bone diseases as described in (4) above, wherein the binding-inhibiting substance is an anti-plexin B1 antibody or a protein containing extracellular regions of plexin-B1; and (7) an agent for preventing and treating bone diseases as described in any of (4) to (6) above, characteristic in which bone diseases are selected from bone fracture, bone deficiency, osteoporosis, osteomalacia, osteopenia, low back pain, Paget's disease of bone, tonic myelitis, articular rheumatism and deformative arthrosis.

[0011] The present invention also relates to (8) a method used to determine a candidate active ingredient for an osteogenesis accelerating agent in which it is determined whether a substance being studied is a substance that inhibits the binding between semaphorin 4D and plexin B1, and if the above-mentioned substance being studied is the above-mentioned substance that inhibits binding, the above-mentioned substance being studied is determined to be a candidate active ingredient for an osteogenesis accelerating agent; (9) a method as described in (8) above characteristic wherein the method used to determine whether a substance being studied is a substance that inhibits the binding between semaphorin 4D and plexin B1 consists of the following steps (A) to (D): (A) a step in which semaphorin 4D and plexin B1 are brought into contact with each other in the presence of the substance being studied, (B) a step in which the degree of binding between semaphorin 4D and plexin B1 is measured , (C) a step in which the grade measured in step (B) above is compared with the grade when not in the presence of the substance being studied, and (D) a step in which the substance being studied is determined to be a substance that inhibits binding between 4D semaphorin and plexin B1 when the grade measured in step (B) above is less than the grade when not in the presence of the substance being studied; (10) a screening method for a candidate active ingredient for a characteristic osteogenesis accelerating agent wherein the methods described in (8) or (9) above are used to screen substances being studied for a candidate active ingredient for an osteogenesis accelerating agent.

[0012] The present invention can be used to directly promote osteogenesis using osteoblasts and to prevent and/or treat bone diseases.

Brief Explanation of the Figures

[0013] [Figure 1] A diagram indicating bone quantity in wild-type (WT) mice measured using microcomputed tomography (μCT) and semaphorin 4D knockout mice (Sema 4d-/-).

[0014] [Figure 2] A diagram indicating trabecular width in WT mice using μCT and Sema 4d-/- mice.

[0015] [Figure 3] A diagram indicating osteogenesis (osteoblast surface area (left); calcified bone surface area (center); and rate of osteogenesis (right)] measured using osteomorphogenesis analysis in WT mice and Sema4d-/- mice with dual calcein marker every 4 days.

[0016] [Figure 4] A diagram indicating osteoclast surface area and cell count (on the left and middle) as well as parameters (on the right) of bone resorption measured using osteomorphogenesis analysis in WT mice and Sema4d-/- mice.

[0017] [Figure 5] A diagram indicating the amount of bone from mice in which adoptive immune cell engraftment has been performed using bone marrow cells from WT mice and Sema4d-/- mice. The second graph from the left indicates the bone quantity of mice when grafted into WT mice and the second graph from the right indicates the bone quantity of mice when grafted into Sema4d-/- mice.

[0018] [Figure 6] A diagram indicating the number of bone nodules when Fc-sema4D (Semaphorin 4D recombined with the Fc area of IgG1) is added to calvarial cells cultured under conditions of osteogenesis.

[0019] [Figure 7] A diagram indicating the manifestation of Plexin type B and differentiation cluster mRNA 72 (CD72) during osteoblast differentiation.

[0020] [Figure 8] A diagram indicating analysis results using Fc-sema4D. The right panel indicates sample analysis results before co-precipitation (pull down) and the left panel indicates sample analysis results after co-precipitation.

[0021] [Figure 9] A diagram indicating the amount of bone in WT mice measured using μCT analysis and in Plexin B1 knockout mice (Plxnb1-/-)/

[0022] [Figure 10] A diagram indicating the width of the trabeculae in WT mice using μCT mice and Plxnb1-/- mice.

[0023] [Figure 11] A diagram indicating the surface area of osteoblasts in WT mice measured using osteomorphogenesis analysis.

[0024] [Figure 12] A diagram indicating bone surface area that has become calcified in WT mice measured using osteomorphogenesis analysis and Plxnb1-/- mice.

[0025] [Figure 13] A diagram indicating the rate of osteogenesis in WT mice measured using osteomorphogenetic analysis in Plxnb1-/- mice.

[0026] [Figure 14] A diagram indicating RA mutants (R1661/1662/1968A) of Plexin B1 and Plexin B1 having mutations in the GAP domain as well as the cut mutant for Plexia B1 in which the PDZ binding domain is absent (Δ PDZ).

[0027] [Figure 15] A diagram indicating bone quantity in WT mice (control mice) measured using μCT analysis and dominant RhoA negative mice (RhoA DNOB).

[0028] [Figure 16] A diagram indicating the trabeculae in WT mice measured using μCT analysis and RhoA DNOB.

[0029] [Figure 17] A diagram indicating formation [osteoblast surface area] in WT mice and RhoA SNOB mice measured using osteomorphogenesis analysis.

[0030] [Figure 18] A diagram indicating the formation [calcified bone surface area] of WT mice and RhoADNOB mice.

[0031] [Figure 19] A diagram indicating the formation (rate of osteogenesis) in WT mice and RhoA DNOB mice measured using osteomorphogenesis analysis.

[0032] [Figure 20] A diagram indicating the surface area of osteoblasts from mice with ovaries extracted (OVX) and OVX mice treated with anti-Sema4D bodies measured using osteomorphogenetic analysis.

[0033] [Figure 21] A diagram indicating the bone quantity of OVX mice and OVX mice treated with anti-semaphorin 4D antibodies (anti-Sema4D antibodies) measured using μCT analysis.

[0034] [Figure 22] A diagram indicating osteogenesis (rate of osteogenesis) in OVX mice and an OVX mice treated with anti-Sema4D antibodies as measured using osteomorphogenesis analysis.

[0035] [Figure 23] A diagram indicating the trabecular gaps in OVX mice and in OVX mice treated with anti-Sema4D antibodies measured using µCT analysis.

[0036] [Figure 24] The top panel in Figure 24 is a diagram indicating results of study of calcification formation of osteoclasts from mice cultured using osteoclasts from WT mice from SEma 4D-/- mice and when cultured in the presence of anti-Sema 4D antibodies. The lower panel in Figure 24 is a diagram indicating the results of studying the calcification of mouse osteoblasts when cultured in the presence of Fc-sema4d and/or anti-plexin Ba antibodies.

[0037] [Figure 26] A diagram indicating the surface area of osteoblasts from mice with ovaries extracted (OVX) and OVX mice treated with anti-semaphorin 4D antibodies (anti-Sema4D antibodies) 6 weeks after OVX treatment measured using osteomorphogenesis analysis.

[0038] [Figure 27] A diagram indicating the bone quantity of OVX mice and OVX mice treated with anti-Sema4D antibodies 6 weeks after OVX treatment measured using μCT analysis.

[0039] [Figure 28] A diagram indicating osteogenesis (rate of osteogenesis) in OVX mice and one OVX mice treated with anti-Sema4D antibodies 6 weeks after OVX treatment measured using osteomorphogenesis analysis.

[0040] [Figure 29] A diagram indicating the trabecular gaps in OVX mice and in OVX mice treated with anti-Sema4D antibodies 6 weeks after OVX treatment measured using μCT analysis. Preferred Way of Working the Invention

[0041] There are no particular restrictions on [osteogenesis accelerator] in the present invention and [preventive and therapeutic agent for bone disease] (hereinafter referred to collectively as [agent in the present invention], as long as they have as the active ingredient a substance that inhibits the binding of 4D semaphorin and plexin B1 (hereinafter referred to simply as [binding in the inhibition of the substance in the present invention]) as the active ingredient, and there are no particular restrictions on the binding of the inhibitor in the present invention while it is the substance that inhibits the binding of vertebrate 4D semaphorin and B1 plexin. However, protein comprising the extracellular regions of anti-4D semaphorin antibodies, anti-B1 plexin antibodies, and B1 plexin can be cited as suitable examples. and by inhibiting the decrease of IRS signals in a way that is thought to accelerate osteogenesis using osteoblasts. In addition, in this Specification, the terms [inhibition] and [suppression] are used interchangeably.

[0042] The above-mentioned anti-4D semaphorin antibodies and anti-plexin B1 antibodies (hereinafter referred to as [antibodies in this invention] can be polyclonal antibodies and can be monoclonal and any functional fragment thereof. However, monoclonal antibodies are preferred given their high specificity. The above-mentioned anti-4D semaphorin antibodies and anti-plexin B1 antibodies can be produced using a well-known conventional method using 4D semaphorin and B1 plexin. The functional fragments that are the antibodies in the present invention indicate fragments of antibodies that bind specifically relative to (a) semaphorin 4D which is an antigen that the antibodies in the present invention specifically bind to, and to (b) plexin B. T.J. International Ltd.). Such antibody fragments can be obtained using a conventional method such as digestion of antibody molecules using papain, pepsin and other proteases or by using a well-known genetic engineering method.

[0043] The antibodies in the present invention also include human antibodies. Here, [human antibodies] such as those relating to the present invention indicate antibodies that are manifested products of human-derived antibody genes. Human antibodies can be obtained by introducing a human antibody gene locus and administering semaphorin 4D and plexin B1 into transgenic animals that are capable of producing human-derived antibodies. A mouse is an example of said transgenic animal. Mice that are capable of producing human antibodies, for example, are deficient and endogenous mouse immunoglobulin (Ig) heavy chains and mouse K light chains and mice can be used that simultaneously retain a 14th chromosome fragment (SC20) comprising the human Ig heavy chain genes as well as human IgK chain transgenes (KCo5). These mice are produced by breeding an A strain of mice having a human IgK chain gene locus and B strain mice having a human IgK chain transgene. The mouse strain A is homozygous for both Ig heavy chain and K light chain destruction and it is a mouse strain (Tomizuka, et al., Proc Nati Acad Sci USA, 200 Vol 97:722) that retains a fragment of the 14th chromosome (SC20) that is capable of apomorphic transmission. Strain B is also a homozygote for both mouse Ig heavy chain and K light chain deficiency and is a mouse strain (at Biotechnol., 1996 Vol 14:845) that retains a human IgK chain transgene (KCo5).

[0044] The polyclonal antibodies in the present invention can be produced using the method given below. They can be obtained by using semaphorin 4D and plexin B1 and if necessary an immunoactivating agent (Freund's Adjuvant and the like) in mice, rabbits, goats, horses and other non-human mammals. The monoclonal antibodies in the present invention are used to produce hybridoma from antibody-producing cells from immunosensitized animals and myeloma cells that do not produce their own antibodies. The hybridoma is cloned and clones are selected that produce monoclonal antibodies indicating specific affinity for the antigens used for immunization. Said hybridoma can be produced based on the method of Keller and Millstein (Nature, 1975 Vol. 256:495-497). Selection of the hybridoma clone that produces monoclonal antibodies is performed by culturing the hybridoma in a microtiter plate. The response to immunoantigens in the culture supernatant in wells seen by proliferation can be measured using ELISA and other enzyme-linked immunoassay methods, radioimmunoassay, the fluorescent antibody method, and other immunological methods.

[0045] Production of monoclonal antibodies from hybridomas can be performed by culturing the hybridoma in vitro and isolating from the supernatant. They can also be grown in vivo in the pleural fluid of mice, rats, guinea pigs, hamsters or rabbits and the like and isolated from the pleural fluid. Genes encoding monoclonal antibodies from hybridoma and other antibody-producing cells are cloned, recombined into a suitable vector, and these are introduced into the host (e.g., Chinese hamster ovary (CHO) cells and other mammalian cell stock, Escherichia coli, yeast cells, insect cells, plant cells, and the like) and recombinant antibodies are produced using gene recombination technology (.J. Delves, Antibody Production Essential Techniques, 1997, Wiley , P. Shepherd and C. Dean, Monoclonal Antibodies, 2000, Oxford University Press, J.W. Goding, Monoclonal Antibodies: Principles and Practice, 1993, Academic Press).

[0046] Transgenic cows, goats, sheep or pigs in which the desired antibody genes that are recombined into endogenous genes using transgenic animal production technology are produced and large amounts of antibodies derived from the antibody genes are obtained from the milk of these transgenic animals.

[0047] The produced antibodies can be refined using a method well known in the field such as a combination of protein A column chromatography, ion exchange chromatography, hydrophobic chromatography, the sulfate analytical method, gel filtration, affinity chromatography and the like.

[0048] The protein comprising the aforementioned extracellular region of plexin B1 can inhibit the binding of semaphorin 4D and plexin B1 by binding semaphorin 4D. There are no particular restrictions on the protein comprising the extracellular region plexin B1, however, a protein which fuses to the constant region of antibodies (preferably any Fc fragment of immunoglobulin) is a suitable example.

[0049] The protein comprising the aforementioned semaphorin 4D, plexin B1 and extracellular regions plexin B1 can produce a manifestation vector comprising this sequence based on the sequence information of these proteins. The manifestation vector is subjected to phenotypic transformation and, in suitable host cells, the target protein is produced within the cells and the target protein can be introduced using an isolation method or other well known method. For example, human semaphorin 4D DNA sequence (sequence number 1) 3 and amino acid sequence (sequence number 2) are disclosed in GenBank Accession Number NM_001142287 and human plexin B1 DNA sequence (sequence number 3) and amino acid sequence (sequence number 4) are disclosed in GenBank Accession Number NM_001130082. Furthermore, the human plexin B1 extracellular region corresponds to amino acid numbers 1 to 1490 of the above-mentioned amino acid sequence of Accession Number NM_001130082.

[0050] We measure by immunoblotting analysis the binding between 4D semaphorin and B1 plexin in the presence of and not in the presence of the substance to see whether a certain substance is a substance that inhibits the binding of 4D semaphorin and B1 plexin and study whether the binding between them has decreased in the presence of that substance so that it can be easily confirmed.

[0051] The effect of accelerating osteogenesis referred to in the present invention indicates the effect of accelerating osteogenesis and more precisely includes the effect of accelerating osteogenesis by osteoblasts by inhibiting the suppression of differentiation of osteoblasts. Whether or not a certain substance has an accelerating effect on osteogenesis can be confirmed by administering the substance to vertebrates having a lower than usual bone quantity (preferably patients with osteoporosis and vertebrate model of osteoporosis) and finding out whether or not the bone quantity increases.

[0052] The prevention and treatment effect of bone disease in the present invention indicates the effect of preventing and/or treating any of the bone disease or the effect of ameliorating the symptoms in the present invention. Whether or not a certain substance has a therapeutic effect on bone disease can be confirmed by administering the substance to a patient or a vertebrate with a bone disease (preferably a patient with osteoporosis or a vertebrate model of osteoporosis) and then studying whether the bone disease is cured or ameliorated.

[0053] The formulation of the present invention may contain only the binding inhibiting substance in the present invention, however, a usual pharmacologically permissible vehicle, binding agent, stabilizer, excipient, diluent, pH buffer, disintegrant, solubilizing agent, solubilization aid, isotoner and other composition component adjusting agents can be added. The reducing agent formulation in the present invention may be a powder formulation, granular formulation, a capsule agent or other solid preparation. It can also be a preparation solution, an emulsion, a suspension or other liquid formulation. Such preparations can be used as suitable for the binding inhibiting substance in the present invention by treatment using the regular method.

[0054] There are no particular restrictions on the method used to administer the agent in the present invention as long as it has the desired preventive or therapeutic effect on bone disease and it can be administered orally or non-orally. The non-oral method used to administer the same includes vascular administration, muscular administration, hypodermic administration, transdermal administration, nasal administration, transpulmonary administration. Of these, transdermal and intravenous administration can be used to particular advantage. The dose of preparation in the present invention, as well as the number of times administered and the concentration can be adjusted according to the patient's body weight, the type of bone disease and the symptoms of bone disease.

[0055] The patient for the present invention can be vertebrates such as mammals and animals belonging to the bird family. These may include humans, monkeys, mice, rats, hamsters, guinea pigs, cows, pigs, horses, rabbits, sheep, goats, cats, dogs, chickens, quail and other suitable animals. Of these, humans and domestic animals, and birds are particularly suitable. The binding-inhibiting substance contained in the agent of the present invention is suitable from the point of view of obtaining an optimal osteogenesis accelerating effect and the bone disease prevention and treatment effect if the vertebrate type derived from semaphorin 4D and plexin B1 that brings the binding-inhibiting action coincides with the vertebrate type that is a candidate for administration of the agent in the present invention. Furthermore, a vertebrate 4D semaphorin derivative and the type of vertebrate B1 plexin derivative may be the same or different.

[0056] There are no particular restrictions on the type of bone disease that can be treated using the present invention as long as it is a bone disease having a factor that brings about a decline in osteogenesis or a bone disease related to a decline in osteogenesis. However, bone fractures, bone deficiency, osteoporosis, osteomalacia, bone deficiency, low back pain, Paget's disease of bone, tonic myelitis, articular rheumatism, dysosteogenesis, and deformative arthrosis are all suitable candidates. Of these, osteoporosis, osteomalacia, osteopenia and dysosteogenesis are particularly suitable.

[0057] The method in the present invention is a method used to determine a candidate active ingredient for an osteogenesis accelerating agent in which it is determined whether a substance being studied is a substance that inhibits the binding between semaphorin 4D and plexin B1, and if the above-mentioned substance being studied is the above-mentioned substance that inhibits binding, the above-mentioned substance being studied is determined to be an active candidate ingredient for an osteogenesis accelerating agent. In the aforementioned method of determination, the following steps (A) to (D) are a suitable example of the method used to determine whether a substance being studied is a substance that inhibits binding between semaphoria 4D and plexin B1: (A) a step in which semaphorin 4D and plexin B1 are brought into contact with each other in the presence of the substance being studied; (B) a step in which the degree of binding between 4D semaphoria and B1 plexin is measured; (C) a step in which the degree of measurement in step (B) above is compared with the degree when not in the presence of the substance being studied; (D) a step in which the substance being studied is determined to be a substance that inhibits binding between semaphorin 4D and plexin B1 when the grade measured in step (B) above is less than the grade when not in the presence of the substance being studied.

[0058] In the above-mentioned determination method, the following can be used to measure the degree of binding: for semaphorin 4D, extracellular region labeled with semaphorin 4D or fusion protein of extracellular region of semaphorin 4D and an Fc immunoglobulin region etc, and for plexin B1, a cell expressing plexin B1 on its surface. The following can also be used to measure the degree of binding: for plexin B1, labeled extracellular region of plexin B1 or fusion protein of extracellular region of plexin B1 and Fc region of immunoglobulin etc, and for semaphorin 4D, a cell expressing semaphorin 4D on its surface.

[0059] There are no particular restrictions on the substance being studied in the determination method in the present invention and it can be a substance predetermined to have an activity that inhibits the binding of 4D semaphorin and plexin B1 and it can be any substance whose activity is unknown. It is also possible to simultaneously use multiple substances being studied. When simultaneously using multiple substances being studied, it is possible to simultaneously use individual substances being studied each in a separate sample, to simultaneously use multiple substances being studied in a single sample, or to simultaneously use multiple individually prepared samples with multiple substances being studied. When simultaneously using multiple substances being studied in a single sample, it may not be possible in one test to determine which of the substances being studied inhibit the binding of 4D semaphorin and plexin B1, but it is possible to determine which of these substances being studied are an inhibitor of binding by running the test multiple times and reducing the substances being studied in stages. The determination method in the present invention is characterized by screening substances being studied for a candidate active ingredient for an osteogenesis accelerating agent, and the method can be used to select a candidate active ingredient for an osteogenesis accelerating agent.

[0060] Furthermore, other modes of the present invention may involve (a) use of the binding inhibiting substances in the present invention in the production of the preventive and therapeutic agent for the aforementioned osteogenesis accelerating agent and bone diseases; (b) a binding inhibiting substance in the present invention for use as an osteogenesis accelerating agent and in the prevention and treatment of bone diseases; (c) a method of using binding-inhibiting substances for accelerating osteogenesis and preventing and treating bone diseases; (d) a method of promoting osteogenesis by administering the binding inhibiting substance of the present invention; and (e) a method of preventing and treating bone diseases by administering the binding inhibiting substance in the present invention.

[0061] Next we describe the present invention in detail, referring to practical examples thereof, however, it is not intended to be construed that the present invention is restricted to these practical examples. [Practical Examples 1] [Analysis of Mice and Bone Phenotypes]

[0062] We produced the mice Sema4d-/-,Plxnb1-/-, plexin B2 knockout (Plxnb2-/-), CAT-RhoA DN and α 1 (I)-Cre according to the method described in the literature (ov, 2000); R.H. Friedel et al., J Neurosci 27, 3921 (Apr 4, 2007); HR Friedel et al., Proc Natl Acad Sci USA 102, 13188 (Sep 13, 2006); K. Kobayashi et al., J. Neurosci 24, 3480 (Apr 7, 2004); R. Dacquin, M. Starbuck, T. Schinke; G. Karsenty, Dev Dyn 224, 245 (June 2002)]. All of the mice were backcrossed 8 or more times with C57BL/6 mice. All mice were kept in a condition where there was no specific pathogenic fungus. All animal experiments were authorized by the Experimental Animal Committee of Tokyo Medical and Dental University and complied with related guidelines and laws. Analysis of the bone phenotype involved in the control of respectively the genetically altered mice and the mice of the same box and we analyzed at least 8 males and 8 females. Three-dimensional micro CT (μCT) analysis and histomorphological measurement analysis were performed according to the methods described in the literature (K. Nishikawa et al., J Clin Invest 120, 3455 (Oct 1, 2010); T. Koga et al., Nature 428, 758 (Apr 15, 2004).

[Mice Bone Marrow Chimera]

[0063] We produce bone marrow chimera from mice by changing part of the method described in the literature (B. Zhao et al., Nat Med 15, 1066 (Sep, 2009). This means that we collect donor bone marrow cells (C57BL6-Ly5, 2 strains) from wild-type Sema 4d-/- mice from the same box. We inject intravenously 2x 106 cells obtained from each donor in the tail vein of wild type recipient mice (3 weeks old, C57BL/6-Ly5, 1 strain) exposed to lethal radiation or Sema 4d-/- mice. Eight weeks after bone marrow transplantation, a high donor level chimerism (>95%) was achieved.

[GeneChip Analysis]

[0064] We performed GeneChip analysis according to the method described in the literature (K. Nishikawa et al., J Clin Invest 120, 3455 (Oct 1, 2010)). This means that after cDNA synthesis is performed using reverse transcription using all the RNA, we synthesize cRNA that has been subjected to biotinylated labeling by in vitro transcription. After we fragmented the cRNA, we performed hybridization using the mouse A430 GeneChip (made by Affymetrix) according to the method described in the literature (T. Koga et al., Nat Med 11, 880 (Aug, 2005); H. Takayanagi et al., Dev Cell 3, 889 (Dec, 2002).

[Induction of Osteoblast Differentiation in vitro]

[0065] Induction of osteoblast and osteoclast differentiation in vitro was performed according to the method described in the document (T. Koga et al., Nat Med II, 880 (Aug, 2005); and H. Takayanagi et al., Dev Cell 3, 889 (Dec, 2002). This means that we performed differentiation induction by performing culture of calvaria-derived cells in an osteogenesis culture medium ( 50 μM ascorbic acid, 10 mM dextrasone, and 10 mM β-glycerophosphate) and the induction of differentiation was confirmed by alkaline phosphatase (ALP) assay (after 7 days) and osteonodosity analysis (21 days later, staining with alizarin red). ) were added every 3 days. Osteoclast supernatant was recovered from each of the wild-type and Sema4d -/- cell culture solutions after stimulation of receptor ligand-activated intranuclear factor K B (RANKL; made by Peprotech). We cultured the osteoclast cultures on a collagen coding plate (made by IWAKI) and recovered using trypsin two days after RANKL challenge. The osteoclast supernatant was used as an osteogenesis culture medium containing the above-mentioned reagent and we added the same to the cultured osteoclasts (1 x 150 cells/24-well plate) every 3 days.

[Real-Time Quantitative RT-PCR Analysis]

[0066] Real-time quantitative RT-PCT was performed according to the product protocol using a luminescent cycler device (made by Roche) and SYBR Green (made by Toyobo). We use the following starters. Plxnbl sense: 5' tgggtcatgtgcagtaccgat-3' (sequence number 5), Plxnbl antisense: 5'-cactgctctccaggttctcc-3' (sequence number 6), Plxnb2 sense: 5' aggggagcctctctctacaagc-3' (sequence number 7), Plxnb2 antisense: 5' tcgatcccttcat cctgaac-3' (sequence number 8), Plnxb3 sense: 5' atatgctgagcgtgccttct-3' (sequence number 9), Plnxb3 antisense: 5'-tgctgttgagcaaattggag-3' (sequence number 10), CD72 sense: 5'-gccttctcctgtcctgtctg-3' (sequence number 1) 1), CD72 antisense: 5' cctcctggaactgctgagac-3' (sequence number 12), Alpl sense: 5'-aacccagacacacaagcattcc-3' (sequence number 13) Alpl sense: 5'-gcctttgaggtttttggtca-3' (sequence number 14) Bglap sense: 5' gcgctctgtctctctgacc t-3' (sequence number 15), Bglap antisense: 5' accttattgccctcctgctt-3' (sequence number 16), Coll1l sense: 5' gagcggagagtactggatcg-3' (sequence number 17), Collll antisense: 5' gttcgggctgatgtaccagt-3' (sequence number 18) Gapdh sense: 5' acccagaaagactgtggatgg-3' (sequence number 19), [Introduction Genetics of Adenoviruses and Retroviruses]

[0067] The method of production of the adenovirus vector carrying the active type configuration (CA) or RhoA (Myc-V14Rho) and Racl (jRacl V12) and the dominant negative type (DN) of RhoA (Myc-N19TRho) and Racl (hRacl V12) and the method of introduction of these were carried out according to the method described in the literature (Bito, H. t al. A Critical Role for a Rho-Associated Kinase, p16 OROCK, in Determining Axon Outgrowth in Mammalian CNS Neurons, Neuron 26, 431-441(2000).Producing retroviral vectors (pMXs-Plexin-B 1-EGFP, pMXs-Plexin-B 1 RA-EGFP and pMXs-Plexin-B 1 DPDS-EGF where the mutant [Plexin-B1 DPDZ-EGFP) manifests or in which activation of [Plexin-B1 RA] and RhoA may not be activated was performed by inserting the cDNA fragment of respectively Plexin-B1, Plexin-B1 RA ((I. Oinuma, Y. Ishikawa, H. Katoh, M. Negishi, Science 305, 862 (Aug 6,2004) and Plexin-B1 DPDZ (V. Perrot, J. Vazquez-Prado, J. S Gutkind, J Biol Chem 277, 43115 (Nov 8, 2002)) in pMXs-IRES-EGFP Production of a recombinant retrovirus was carried out according to the method described in the literature (. Morita, T. Kojima, T. Kitamura, Gene Ther 7, 1063 (Jun, 2000). This means that retrovirus packaging was performed by introducing the retrovirus vector produced in Plat-E cells.

[Anti-Sema4D Antibody Processing for OVX-Induced Decline in Bone Quantity]

[0068] OVX-induced osteoporosis production model in mice (extracted ovaries) was performed according to the method described in the literature (M. Shinohara et al., J. Bio Chem 282, 17640 (Jun 15, 2007). This means that we performed an ovary extraction operation or pseudo-operation in 7-week-old female mice. more than 6 mice in each group. We validate this using the method given below to find whether or not there is a preventive effect on the decline in bone quantity. This means that we intravenously inject 20 μg of Sema4D antibodies (made by MBL) or a saline solution from the tail vein once a week. We sacrifice all mice 8 weeks after surgery and perform μCT analysis and osteomorphogenesis analysis. We also perform validation using the following study method whether or not there is an acceleration effect on the amount of bone that has been decreased. This means that after 6 weeks we intravenously injected 20 μg of anti-Sema4D antibodies (made by MBL) from the tail vein for 3 weeks every 3 days into OVX mice. We sacrificed all mice 9 weeks after the operation and used them for μCT analysis. [Immunobloting Analysis, Co-precipitation Analysis and Immunofluorescence Staining]

[0069] We cultured calvaria cells for two days in an osteogenesis culture medium (50 μM ascorbic acid, 10 nM dexamethasone and 10 mM β-glycerophosphate) and then stimulated them with Fc-Sema4D. We used purified human IgG (Fc part) (made by Beckman Coulter) for native control for pull down analysis (time 0). We harvest cells at the indicated point and perform immunoblotting analysis or pull down analysis using anti-Plexin-B1 antibodies (clone A-8, made by Santa Cruz); anti-PDZ-RhoGEF antibodies (polyclonal, made by Protein Express); anti-LARG antibodies (polyclonal, made by Lifespan Biosciences); anti-phospho-Akt antibodies (Tgr308) (polyclonal, made by Cell Signaling); anti-Akt antibodies (polyclonal, made by Cell Signaling); ERK anti-phosphorus antibodies (Thr202/Tyr204) (polyclonal, made by Cell Signaling); anti-ERK antibodies (polyclonal, made with Cell Signaling); anti-Met antibodies (clone 25H2) (made by Cell Signaling); anti-ErbB2 antibodies (clone 29D8) (made by Cell Signaling); anti-Raci antibodies (clone 102/Rae 1) (made by BD Transduction Laboratories); anti-RhoA antibodies (clone 55/Rho) (made by BD Transduction Laboratories); anti-cadherin-11 antibodies (polyclonal, made by Invitrogen); anti-IRS 1 antibodies (clone 53-10C-31) (made by Millipore); and anti-b-actin antibodies (clone AC40) (made by Sigma-Aldrich). Phosphorylation of plexin B1, Met, ErbB2 and IRS1 was detected by anti-phosphotyrosine antibodies (4G10, made by Upstate) after immunoprecipitation using respectively specific antibodies for these. We incubated a dissolved cell product with Fc-sema4D (500 ng) that bound with protein A-agarose beads in order to detect plexin B1 that bound semaphorin 4D and performed immunoblotting analysis using anti-Plexin-B1 antibodies. Detection of GTPase activation was performed according to the description in the literature (M. Shinohara et al., J Bio Chem 282, 17640 (Jun 15, 2007). This means that we treat calvarial cells with Fc-sema4D and collect them at the point where they appear. We incubate the dissolved cell product with GST-RBD (using RhoA) or GST-PAK1 (using Racl) (2 μg) which bound with glutathione sepharose and we performed immunoblot analysis using respectively anti-RhoA antibodies or anti-Rac1 antibodies. We fixed the cells with a 4% paraformaldehyde for immunofluorescence staining, performed the permeabilization process and then stained using secondary antibodies labeled with Alex Fluor 488 and phalloidin-conjugated rhodamine (made by Molecular Probes).

[Flow Cytometry]

[0070] We stain a single-cell float solution using monoclonal antibodies conjugated with 8 types of fluorescent dyes (anti-CD45.2 antibodies conjugated with PerCP.Cy5.5; anti-CD45.2 antibodies (clone 104) conjugated with FITC; anti-CD11b antibodies (clone M1/70) conjugated with eFLuor450; anti-CD105 antibodies (clone MJ7/18) conjugated with PE; anti-CD10 antibodies (clone 4.29E+02) conjugated with Alex Fluor 647; (anti-CD44 antibodies conjugated with APC- Alexa Fluor 750; anti-CD44 antibodies (clone IM7) conjugated with AC-Cy7; and anti-Sca-1- antibodies (clone D7) (made by eBioscience) conjugated with APC in order to analyze bone marrow-derived cells and calvarial cells Then, we performed flow cytometry using FACSCanto II using Diva program (made by BD Biosciences).

[Cell Proliferation Analysis]

[0071] We culture the bone marrow-derived interstitial cells in an osteogenesis condition culture medium and analyze the cell proliferation rate before osteoblast differentiation (Day 0) or in the process of osteoblast differentiation (Day 14) using semaphorin4D stimulation using partial Fc human IgG or using cell proliferation ELISA kit (made by Roche) in presence of Fc-Semar4D and detecting 5-bromo-2 incorporation 'deoxyuridine (BrdU). [Colony Forming Unit (CFU) Analysis]

[0072] We plated the bone marrow cells at 3x106 cells per well in a 24-well plate. Then, we cultured them for 3 days using α-MEM culture medium containing 10% fetal bovine serum and then replacing it with an osteogenesis condition culture medium. The colony forming unit alkaline phosphatase (CFU-ALP) was detected as an ALP positive colony on day 7 and the CFU-osteoblast (CFU-Ob) was detected as an alizarin red positive colony on day 21. The aggregate number of colonies (CFU-fibroblasts (CFU-F)) was detected by staining with toluidine blue on day 7 and day 21.

[Statistical analysis]

[0073] All data are reported as mean ± SEM (n=15). Statistical analysis was performed using the Student ANOVA test and when possible the Bonferroni test (*p<0.005; **p<0.01; ***p<0.005; n.s., not significant). Results are shown in representative examples of 4 or more individual experiments.

[Results]

[0074] We performed genome-wide mRNA screening in osteoclasts and osteoblasts using GeneChip analysis to study whether or not semaphorin, ephrin, slits, and netrin which are axon-guided molecules contribute to bone remodeling. This means that after analysis of 20 types of semaphorin, 16 types of ephrin, 6 types of slits and 6 types of netrin, an extremely high manifestation of 4D semaphorin was confirmed in osteoclasts. Meanwhile, this manifestation has not been confirmed in osteoblasts. These results indicate that 4D semaphorin had a selectively induced manifestation in the process of osteoclast formation.

[0075] We performed a functional analysis of the skeletal system using Sema4d-/- mice to study 4D semaphorin function with selectively induced manifestation in the process of osteoclast formation. Bone quantity (Figure 1) and trabecular width (Figure 2) in Sema4d-/- mice was shown to be significantly increased compared to wild-type mice (WT mice) by using μCT analysis and osteomorphogenesis. The surface area of osteogenesis in Sema4d-/- mice (Figure 3, left), the surface area of calcified bone (Figure 3, middle), and the rate of osteogenesis (Figure 3, right) increased conspicuously as compared to wild-type mice. However, there were no changes in the parameters (osteoclast surface areas (Figure 4, left), the number of osteoclasts (Figure 4, center) and the eroded bone surface area (Figure 4, right) indicating osteoclast bone resorption. We also observed that in vitro osteoclast formation in Sema4d-/- cells was normal. Despite the fact that 4D semaphorin manifested specifically in osteoclasts, these results suggest that the high bone density phenotype in SEma4d-/- mice increased due to osteogenesis via osteoblasts.

[0076] We performed adoptive immune cell engraftment using bone marrow cells that included osteoclast precursor cells to find whether or not the bone phenotype of the Sema4d-/- mice was based on abnormalities in the bone marrow cell system. As a result, the amount of bone increased when Sema4d-deficient bone marrow cells were engrafted into wild-type mice as compared to when wild-type bone marrow cells were engrafted (see two graphs on the left in Figure 5). Meanwhile, when wild-type bone marrow cells were engrafted into Sema4d-/- mice, bone quantity decreased and returned to normal as compared to when Sema4d-deficient bone marrow cells were engrafted (see two graphs on the right of Figure 5). Based on these results, it was identified that the bone phenotype in the Sema4d-/- mice was such that abnormalities in the cells of the hematopoietic system comprising osteoclasts were the cause. Furthermore, no clear increase in osteonodes of Sem4d-/- calvary cells was confirmed in osteonodes of Sema4d-/- calvaries cells. These results suggest that 4D semaphorin manifested in osteoclasts functions as a bone remodeling factor by inhibiting osteogenesis through osteoblasts.

[0077] We produced Fc-sema4D (I. Ishida et al., mt Immunol 15, 1027 (Aug, 2003) to study in detail the effect of 4D semaphorin inhibition on osteoblasts. Then, we added Fc-sema4D to calvarial cells that were cultured under conditions of osteogenesis. Addition of Fc-sema4D inhibited concentration-dependent activation of ALP and manifestation of osteocalcin (Bglap] and collagen type I (Colla1]). These results indicate that osteonodosity is inhibited as a result of Sema4D inducing osteoblast differentiation. We also cultured calvarial cells in the presence of osteoclast culture supernatant or in the presence of osteoblasts to see whether or not osteoclast-derived 4D semaphorin contributed to the regulation of osteogenesis. osteoclasts or with osteoclasts, there was no effect on osteonodosidase although when pairing the culture with Sema4d-/- osteoclast culture supernatant or when pairing the culture with osteoclasts, osteonodosidase was clearly accelerated. Based on these results, osteoclasts are indicated as osteogenesis inhibitors through 4D semaphorin produced as at least partially soluble. It should also be pointed out that one or more factors are contained in the osteoclast supernatant. The effect of accelerating osteogenesis brought about by these factors is thought to be brought to the fore when 4D semaphorin is not present.

[0078] We analyzed (a) the manifestation of the B-type plexin which is well known as the D semaphorin receptor in non-lymphatic cells and lymph nodes and (b) the C72 RNA (K. Suzuki, A. Kumanogoh, H. Kikutani, Nat Immunol 9, 17 (Jan, 2008) using real-time quantitative RT-PCR in order to specify the semaphorin 4 receptor D where osteoclasts manifested. As a result, the amount of manifested plexin B1 increased conspicuously during differentiation into osteoblast (Figure 7). Meanwhile, the amount of manifested plexin B2 was very low and there was virtually no manifestation of manifested plexin B3 and CD72 (Figure 7). After performing co-precipitation analysis using 4D Fc-sema, it was indicated that 4D semaphorin interacted with B1 plexin (Figure 8). it has been indicated that Fc-sema 4D induces phosphorylation of plexin B1.It is well known that when semaphorin 4D binds with plexin B1, a conjugate is formed with ErbB2 tyrosine kinase and ErbB2 phosphorylates itself and plexin B1 is dependent on semaphorin B4. In osteogenesis, after inhibition of ErbB2 kinase activation, phosphorylation declines dependent on 4D semaphorin stimulation of plexin B2. Based on these results, it is suggested that plexin B1 acts as the main receptor for 4D semaphorin in osteoblasts. Then, we analyzed the bone phenotype of Plxbl-/- mice. Like Sema4d -/- mice, bone quantity (Figure 9) and trabecular width (Figure 10) of Plxnb1 -/- mice increased conspicuously as compared to wild-type mice due to an increase in osteoblast osteogenesis. The surface area of osteoblasts in Plxnbl -/- mice (Figure 11), the surface area of calcified bone (Figure 12) and the rate of osteogenesis (Figure 13) also increased respectively as compared to wild-type mice. However, there is no change in parameters (osteoclast surface area, osteoclast cell count and erosion of bone surface area) indicating bone resorption in osteoclasts. Based on these results, it is suggested that the high bone density phenotype of Plxnbl-/- mice, like the high bone density phenotype of Sema4d-/- mice, is caused by an increase in osteogenesis brought about by osteoblasts. No stimulation effect on osteonodosity by osteoblasts confirmed by wild-type Sema4d-/- osteoclasts was confirmed with Sem4d-/- osteoclasts. Thus, this suggests that plexin B1 recognizes osteogenesis primarily through 4D semaphorin.

[0079] How does semaphorin 4D-plexin B1 transform to inhibition signals in osteoclasts? It is well known that semaphorin-pleoxin system regulates osteomorphogenesis and amoeboid cell by regulating bone cell actin rearrangement. It is also well known that when semaphorin 4D is combined with plexin B2, RhoA activity is stimulated in the presence of tyrosine kinase ErbB2 although semaphorin 4D has a counteracting action in the presence of another tyrosine kinase Met (M. Swiercz, T. Worzfeld, S. Offermanns, J Bio Chem 283, 1893 (Jan 25, 2008). the possibility that 4D semaphorin activates RhoA. When we analyzed the amount of ErbB2 and Met manifested in osteogenesis, it was suggested that ErbB2 manifested in considerably greater amounts than Met. Although 4D semaphorin stimulation induced ErbB2 phosphorylation, Met phosphorylation was not induced. Furthermore, based on immunoblotting analysis and co-precipitation analysis, 4D semaphorin increased the type of GTP-combinant activation for RhoA and it is indicated that this activity is particularly inhibited by Plxnb1-/0 cells. Meanwhile, Racl activity which is in another Rho family did not affect 4D semaphorin stimulation. Coinciding with these results, when we introduced RhoA-like structured activation using an adenovirus, osteonodosidase in calvarial cells was inhibited although when dominant negative RhoA was introduced, osteonodosidase was accelerated. Meanwhile, Racl-like structured activation and dominant negative Racl did not affect osteogenesis. Based on these results, it is suggested that RhoA selectively mediates the inhibition effect for osteogenesis of semaphorin 4D.plexin B1. Plexin B1 has two RhoGTPase regulatory domains, that is, a GTPase-activated protein (GAP) domain and a PDZ-binding domain that binds to Rho-GEF. Oinuma, Y. Ishikawa, H. Katoh, M. Negishi, Science 305, 862 (Aug. 6, 2004), J.M. Swiercz, R. Kuner, J. Behrens, S. Offermanns, Neuron 35, 51 (July 3, 2002), V. Perrot, J. Vazquez-Prado, J. S. Gutkind, J Bio Chem 277, 43115 (November 8, 2002), M.H. Driessens, C. Olivo, K. Nagata, M. Inagaki, J.G. Collard, FEBS Left 529, 168 (Oct 9, 2002). Based on co-precipitation analysis and immunoblotting analysis, PDZ-RhoGEF and LARG (RhoGEF-associated leukemia) which are known as Rho-GEG were indicated as plexin B1 ligands even in osteoclasts. Thus, the aforementioned GAP domain and PDZ binding domain produced two types of plexin B1 mutants (Plexin B1-Δ PDZ and Plexin B1 RA) to determine whether osteoporosis was significantly inhibited ( Oinuma, Y. Ishikawa, H. Katoh, M. Negishi, Science 305, 862 (6 Aug. 2004) ( Figure 14 ). Wild-type plexin B1 (WT-Plexin B1) were respectively used to produce overexpressing Plxnbl-/- calvarial cells, we stimulated these using Fc-sema 4D and evaluated the effect on inhibition of osteoblast differentiation. however, it was not recovered in the manifestation of excess Plexin B1-ΔPDZ. These results suggest that RhoA mediates osteogenesis inhibition specifically by 4D semaphorin.

[0080] We crossed CAT-RHoA D transgenic mice (K. Kobayashi et al., J. Neurosci 24, 3480 (April 7, 2004) and α 1 (J)-Cre transgenic mice (R. Dacquin, M. Starbuck, T. Schinke, G. Karsenty, Dev. Dyn 224, 245 (Jun, 20 02)) and bred mice in which dominant negative RhoA was specifically expressed (RhoA DNOB) in osteoblasts to study the role of RhoA in osteoblasts in vivo. Bone quantity (Figure 15) and trabecular width (Figure 16) of RhoA DNOB mice increased as compared to wild-type mice when osteogenesis was accelerated by osteoblasts. In addition, osteoblast surface area (Figure 17) of Rho mice The DNOB, the surface area of bones (Figure 18) that had calcification, and the rate of osteogenesis (Figure 19) increased conspicuously for wild-type mice, however, there were no changes in the parameters that indicated osteoclast bone resorption (surface area, number of osteoclasts, and surface area of eroded bone). The bone phenotype was the same as the bone phenotype for Sema4d-/- and Plxbl-/- mice. As expression of RhoA DNOB increased during osteoblast differentiation, expression of osteonodosidase and osteoblast marker genes (Alpa, Bglap, and Collal) increased markedly in calvarial cells originating in RhoA DNOB mice. The osteonodosidase that increased in RhoA DNOB cells was not inhibited by Fc-sema4d so it is suggested that RhoA mediates downstream inhibition signals from semaphorin 4D-plexin B1.

[0081] In order to study whether or not inhibition of Sema 4D function was effective for osteoporosis, we performed validation using the postmenopausal osteoporosis mouse model treated for OVX (excised ovaries). This means that we intravenously administered anti-Sema4D antibodies to mice on a weekly basis following OVX treatment and studied whether there was a preventive effect for a decrease in bone quantity. After analyzing the bone tissue, bone quantity decreased when anti-Sema4d antibodies were not administered although when anti-Sema4d antibodies were administered, acceleration in osteogenesis (increase in osteoblast surface area [Figure 20], bone quantity [Figure 21] and rate of osteogenesis (Figure 22] and decrease in trabecular gaps [Figure 23]) was confirmed. Meanwhile, there were no changes in parameters indicating osteoclast bone resorption (number of osteoclasts and surface area of eroded bone). Based on these results, when the function of semaphorin 4D is inhibited by treatment with anti-Sema 4D antibody, a preventive effect on the decrease in bone quantity in osteoporosis is indicated. We also studied whether or not the inhibition of semaphorin 4D function was effective even for treatment of bone quantity that had already decreased. This means that after OVX treatment, we administered the anti-Sema4d antibodies from the caudal vein for three weeks three times a week, for 6 weeks after the amount of bone has decreased. The results of analysis of parameters indicating osteogenesis (osteoblast surface area, increase in bone quantity, increase in rate of osteogenesis and decrease in trabecular gaps) confirmed acceleration in osteogenesis (increase in osteoblast surface area [Figure 26], bone quantity [Figure 27] and rate of osteogenesis [Figure 28] and decrease in trabecular gaps [Figure 29]). Thus, it becomes clear that once the bone quantity has decreased back to the same level as when Sema4D antibodies were administered on a weekly basis after the above mentioned OVX treatment. This means that there was a return to the same level as when the decrease in bone quantity was prevented. Meanwhile, there were no changes in parameters indicating bone resorption from osteoclasts (surface area of eroded bone). We also studied the effect of functional inhibition of 4D semaphorin and B1 plexin on osteonodosidase. When mouse osteoblasts and mouse osteoclasts WT were used, osteonodosidase was accelerated as a function of anti-Sema 4D antibody concentration (plates 1 to 3 from the left in the upper panel of Figure 24). Meanwhile, osteonodosidase that was inhibited by Fc-sema4d was accelerated as a function of anti-plexin B1 antibody concentration (bottom panel in Figure 24 ). Based on these results, treatment or treatment with anti-plexin B1 antibody, not only is the decrease in bone quantity in osteoporosis inhibited, but an effect that promotes an increase in bone quantity is indicated as well. We also studied the effect of functional inhibition of 4D semaphorin in human osteoblasts on osteonodosidase. This means that when we performed the validation using osteoclasts that were differentiated from peripheral blood monocytes derived from CD14 positive cells (Figure 25, bottom row) and the culture supernatant thereof (Figure 25, top row) and when we added osteoclasts and culture supernatant thereof and added anti-Sema4d antibodies to the osteoblasts, osteonodosidase was accelerated as a function of the concentration of the anti-Sema antibodies. 4d (Figure 25). These results indicate that the results in the aforementioned murine model of postmenopausal osteoporosis were sustained in human cells and that inhibition of the 4D semaphorin-plexin B1 interaction is a novel strategy in promoting osteogenesis.

[0082] We did a detailed analysis of the action of inhibition of osteoblast differentiation semaphorin RD. This means that when we compared the number of hematopoietic cells in the bone marrow (Scal-1+CDIO5+CD106+CD44+CD45, 2-CD1 lb) in Sema4d-/- mice with wild-type mice by performing flow cytometry analysis, there were no differences. We also performed cell proliferation analysis and although there was somewhat an increase due to Sema4d prior to osteoblast differentiation (Day 0), there were no changes due to 4D semaphorin stimulation in the process of osteoblast differentiation (Day 14). Furthermore, based on CFU analysis, osteogenesis indicated in the ALP and alizarin manifestation was inhibited due to 4D semaphorin stimulation. These results suggest that 4D semaphorin acts on osteoblast differentiation stages.

[0083] Insulin receptor substrate (IRS) signals caused by insulin-like growth factors (IGF)-1 are known to accelerate osteoblast differentiation. Thus, we studied Akt and ERK phosphorylation related to IRS signals with 4D semaphorin stimulation and the level of phosphorylation declined. Tyr phosphorylation which is the indicator of IRS activation also declined. These results suggest that 4D semaphorin stimulation decreases IRS signals. It is also clear that activated RhoA type decreases Akt and ERK phosphorylation and conversely Y-27632 and RKI which are RhoA inhibitors promote this phosphorylation. It is also clear that the RhoA inhibitor induces the promotion of phosphorylation-type activation in IRS signals. These results suggest that 4D semaphorin induces a decline in IRS signals by activating RhoA and inhibiting osteoblast differentiation.

[Summary]

[0084] Based on the above-mentioned experiments, we have identified osteoclast-derived 4D semaphorin as an extremely important mediator for osteoclast-osteoblast transmission in bone reconstruction. Bone reconstruction is carried out in cycles done by three stages (starting from bone resorption due to osteoclasts, transition to new osteogenesis due to osteoblasts and termination of new bone synthesis (K. Matsuo, N. Irie, Arch Biochem Biophys 473, 201 (May 2008). Semar4d manifests in osteoclast functions until the time of osteoclast resorption is completed as an osteogenesis inhibiting factor at the early stage where osteoblast differentiation is inhibited. In addition, activation of RhoA is also indicated as an inhibitor of osteoblast osteogenesis in vivo. EphrinB2/EpfB4 indicated as a contributor to the transitional stage also makes use of RhoA to regulate osteogenesis (C. Zhao et al., Cell Metab 4, 111 (August, 2006) which suggests that the low molecular weight GTPase of the Rho family acts as a coordinator for unique transmission of reconstruction bone GEFA-specific RhoA Arfgef 12 containing PDZ (LARG) ( J.M. Swiercz, R. Kuner, J. Behrens, S. Offermanns, Neuron 35, 51 (Jul 3, 2002 ); V. Perrot, J.Vazquez-Prado, J.S. Gutkind, J Bio Chem 277, 43115 (Nov 8, 2002)) has been shown to be highly expressed in osteoblasts in the GeneChip analysis. These results support the importance of the 4D semaphorin-plexin B1-RhoA pathway. Furthermore, based on analysis using immunofluorescence staining, a downregulation for cadherin 11 has been observed after Fc-ema Rd stimulation and the contribution of the binding function of the regulated space is suggested. Intermittent parathyroid hormone (PTH) treatment is the only effective method that has been proven to increase osteogenesis. Anti-Sost antibodies that are currently under development have attracted attention as a new agent of osteogenesis while antibodies with target factors related to semaphorin pathway 4D-plexin B1 - RhoA and inhibitors and the like can be expected to be new therapeutic agents for patients with osteopenia.

[Industrial Applicability]

[0085] The present invention can be used to take effect in accelerating osteogenesis and in preventing and treating bone diseases. [Sequence Table] Patent Application P11-052011-108642_0.app [Document Name]

Claims (6)

1. Use of an anti-SEMA 4D antibody or antigen-binding fragment thereof or an anti-plexin B1 antibody that inhibits binding to SEMA 4D or plexin B1, respectively CHARACTERIZED by the fact that it is for the manufacture of a medicine for the prevention and treatment of bone diseases, where the bone diseases are selected from bone fracture, bone deficiency, osteoporosis, osteomalacia, osteopenia, low back pain, Paget's disease of bone, tonic myelitis, articular rheumatism and deformative arthrosis. 2. Use according to claim 1, CHARACTERIZED by the fact that the antibody is an anti-semaphorin 4D antibody. 3. Use according to claim 1, CHARACTERIZED by the fact that the antibody is an anti-Plexin B1 antibody. 4. Use, according to any one of claims 1 to 3, CHARACTERIZED by the fact that the antibody or fragment thereof is monoclonal. 5. Use according to any one of claims 1 to 4, CHARACTERIZED by the fact that the antibody fragment is an F(ab') 2 fragment, a Fab' fragment, a Fab fragment, a FIT fragment, a disulfide-linked FV fragment, a single-chain FV fragment (scFV) or a polymer of one or more of said fragments. 6. Use, according to any one of claims 1 to 3, CHARACTERIZED by the fact that the antibody or fragment thereof is a human antibody.