Composition for promoting osteoblast differentiation comprising dock5 inhibitor and BMP and used thereof

Abstract

The present invention relates to a composition for promoting osteoblast differentiation, comprising a DOCK5 inhibitor and BMP, and uses thereof. More specifically, the DOCK5 inhibitor of the present invention enhances activation of the BMP signaling pathway, and when co-administered with BMP, synergistically promotes osteoblast differentiation and bone regeneration, thereby providing a novel combination therapeutic strategy for bone defect repair and bone regeneration treatment.

Description

Composition for promoting osteoblast differentiation comprising DOCK5 inhibitor and BMP and use thereof

[0001] The present invention relates to a composition for promoting osteoblast differentiation comprising a DOCK5 inhibitor and BMP and its use.

[0002] The development of bone regeneration technologies to effectively treat bone defects caused by traffic accidents, trauma, tumor resections, and infections, as well as degenerative bone diseases such as aging and osteoporosis, holds significant clinical significance. Tissue engineering-based approaches utilizing biomaterials, stem cells, and growth factors are actively being utilized, particularly for large defects and complex skeletal defects that are difficult to regenerate. However, these approaches still face challenges such as nonunion and inadequate function of the regenerated bone.

[0003] Bone tissue contains three representative cells that are deeply involved in bone metabolism: osteoblasts, osteoclasts, and osteocytes. The function and amount of bone tissue are closely regulated by the equilibrium between bone formation by osteoblasts and bone resorption by osteoclasts. If this equilibrium between bone formation and bone resorption is not maintained and bone resorption increases more than bone formation, bone disease occurs.

[0004] Recently, much research has focused on inducing osteogenesis, and scientific approaches are being developed based on the fundamental mechanisms that enhance osteogenesis. Therefore, promoting osteoblast differentiation, which plays a crucial role in increasing osteogenesis, could be an effective treatment for bone diseases.

[0005] Meanwhile, anabolic factors such as bone morphogenetic proteins (BMPs) promote osteoblast differentiation of mesenchymal stem cells (MSCs), thereby promoting bone formation and regeneration. In particular, recombinant human BMP2 (rhBMP2; BMP2) has been approved by the Ministry of Food and Drug Safety and is being used clinically as a treatment alternative to autologous transplantation. However, inducing adequate bone regeneration requires a dose of rhBMP2 exceeding physiological levels, which can lead to adverse effects such as inflammation, pain, adipose tissue formation, bone resorption, wound complications, and tumor formation. Therefore, precise control of BMP2 signaling can minimize adverse effects while achieving adequate bone regeneration.

[0006] Accordingly, the inventors of the present invention have made efforts to study the regulatory mechanism that activates the bone formation pathway by BMP in order to develop an effective treatment for bone disease, and as a result, they have completed the present invention by elucidating that osteoblast differentiation and bone formation are promoted (regulated) through the synergistic effect of combined treatment with a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP2.

[0007] Accordingly, one object of the present invention is to provide a pharmaceutical composition for promoting bone formation comprising a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

[0008] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating bone disease, which comprises a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

[0009] In addition, another object of the present invention is to provide a health functional food for preventing or improving bone disease, which contains a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

[0010] In addition, another object of the present invention is to provide a composition for promoting differentiation of osteoblasts and bone regeneration, which contains a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

[0011] In addition, another object of the present invention is to provide a method for producing a DOCK5 gene-deficient animal model other than a human, which comprises a step of inserting a gene trap vector between the first exon (exon1) and the second exon (exon2) of the DOCK5 (dedicator of cytokinesis 5) gene to delete the DOCK5 gene.

[0012] In addition, another object of the present invention is to provide a method for treating a bone disease, comprising a step of administering to a subject a pharmaceutical composition comprising a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

[0013] The terminology used herein is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0014] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0015]

[0016] Hereinafter, the present invention will be described in detail.

[0017]

[0018] According to one aspect of the present invention, the present invention provides a pharmaceutical composition for promoting bone formation, comprising a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients; and / or a pharmaceutical composition for preventing or treating bone diseases.

[0019] The present invention is characterized by achieving excellent osteoblast differentiation and bone formation promotion through the synergistic complementary effect of combined use of a DOCK5 inhibitor and BMP, thereby exhibiting preventive or therapeutic activity for bone diseases. Bone diseases that cause a decrease in bone density are caused by an imbalance in bone metabolism, i.e., the rate of bone formation, which creates new bone, and bone loss, which removes old bone. Therefore, the combined treatment of a DOCK5 inhibitor and BMP of the present invention, which can promote osteoblast differentiation, thereby reducing bone loss, and increasing bone formation, can be used as an osteogenesis promoter and / or a treatment for bone diseases.

[0020] The DOCK5 inhibitor of the present invention may use any DOCK5 inhibitor known in the art as long as it can achieve the purpose of the present invention, and includes, for example, one or more compounds selected from the group consisting of C21 (CAS No.: 54129-15-6), CPYPP (CAS No.: 310460-39-0), E197 (CAS No.: 2378515-04-7) and DOCK5-IN-1 (CAS No.: 149775-26-8), which are known to inhibit DOCK5. In one embodiment of the present invention, C21 is used, but the present invention is not limited thereto.

[0021] In addition, the DOCK5 inhibitor may be at least one selected from the group consisting of guide RNA, antisense oligonucleotide, siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), and ribozyme targeting the DOCK5 gene, and is not limited thereto as long as the purpose of the present invention can be achieved.

[0022] In addition, the DOCK5 inhibitor may be at least one selected from the group consisting of peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that specifically bind to the DOCK5 protein, and is not limited thereto as long as the purpose of the present invention can be achieved.

[0023] In addition, the BMP may be any BMP known in the art as long as it can achieve the purpose of the present invention, and may be, for example, at least one selected from the group consisting of BMP2, BMP3, BMP3b, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14, and BMP115. In one embodiment of the present invention, BMP2 is used, but the present invention is not limited thereto.

[0024] The above BMP may include a BMP production inducer, examples of which include, but are not limited to, statin compounds such as lovastatin, simvastatin, and compactin.

[0025] In addition, the DOCK5 inhibitor of the present invention increases the expression level or activity of MKK3 / 6 and p38.

[0026] The above bone disease can be used for the treatment of any bone disease known in the art, as long as the composition of the present invention is effective, and may be, for example, at least one selected from the group consisting of osteoporosis, fracture, rheumatoid arthritis, periodontitis, osteomalacia, osteopenia, bone atrophy, osteoarthritis, bone defect, osteolysis, osteonecrosis, osteodystrophy, Paget disease, and metastatic bone cancers, but is not limited thereto.

[0027] In this specification, “treatment” is used to mean alleviation, alleviation, or stabilization of a disease state or symptoms, partial or complete recovery, prolongation of survival, reduction in the extent of the disease, delay or palliation of disease progression, and other beneficial therapeutic outcomes. “Prevention” is used to mean all mechanisms and / or effects that act on a subject who does not have a specific disease to prevent the development of the specific disease, delay the onset of the disease, or reduce the frequency of the disease.

[0028] The content of the active ingredient in the pharmaceutical composition provided in this specification can be appropriately adjusted according to the form and purpose of use, the condition of the subject of use, the type and severity of the symptom, etc., and is 0.001 to 99.9 wt%, 0.001 to 90 wt%, 0.001 to 75 wt%, 0.001 to 50 wt%, 0.01 to 99.9 wt%, 0.01 to 90 wt%, 0.01 to 75 wt%, 0.01 to 50 wt%, 0.1 to 99.9 wt%, 0.1 to 90 wt%, 0.1 to 75 wt%, 0.1 to 50 wt%, 1 to 99.9 wt%, 1 to 90 wt%, 1 to 75 wt%, 1 to 50 wt%, 5 to 99.9 wt%, based on the solid weight. It may be, but is not limited to, 5 to 90 wt%, 5 to 75 wt%, 5 to 50 wt%, 10 to 99.9 wt%, 10 to 90 wt%, 10 to 75 wt%, or 10 to 50 wt%.

[0029] In addition, the content of the effective ingredient may be 0.001 to 400 mg / kg (body weight; BW), 1 to 400 mg / kg, 50 to 400 mg / kg, 100 to 400 mg / kg, 0.001 to 300 mg / kg, 1 to 300 mg / kg, 50 to 300 mg / kg, 100 to 300 mg / kg, 0.001 to 200 mg / kg, 1 to 200 mg / kg, 50 to 200 mg / kg, 100 to 200 mg / kg, 0.001 to 100 mg / kg, 1 to 100 mg / kg or 50 to 100 mg / kg based on the body weight of the subject to whom the pharmaceutical composition is administered, but is not limited thereto.

[0030] The pharmaceutical composition can be administered to a subject selected from among primates such as humans and monkeys, rodents such as mice, rats and rabbits, mammals including dogs, cats, cows, pigs, sheep, horses and goats, birds including chickens, ducks and geese, reptiles including snakes, lizards, turtles and crocodiles, amphibians and vertebrates such as fish through various routes, but is not limited thereto.

[0031] The pharmaceutical composition may be administered via any commonly used route, including intravenous, intramuscular, or subcutaneous injections, or parenteral routes such as local infusion. In one example, the pharmaceutical composition may be administered to a site requiring bone formation or a site suffering from bone disease (e.g., by injection, etc.), but is not limited thereto.

[0032] The above pharmaceutical composition can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., or parenteral formulations such as injections (e.g., sterile injection solutions, etc.) according to conventional methods, but is not limited thereto.

[0033] In addition to the active ingredient described above, the above pharmaceutical composition may be administered mixed with one or more auxiliary agents selected from the group consisting of pharmaceutically and / or physiologically acceptable carriers, excipients, and diluents, which are generally selected in consideration of the administration method and standard pharmaceutical practice. For example, the type of the pharmaceutically and / or physiologically acceptable carrier is not particularly limited, and any carrier commonly used in the relevant technical field can be used, and may include at least one selected from the group consisting of saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, glycerol, ethanol lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto, and may be any carrier commonly used in the pharmaceutical field.

[0034] The above pharmaceutically and / or physiologically acceptable diluent and / or excipient may be at least one selected from the group consisting of all fillers, bulking agents, binders, wetting agents, disintegrants, lubricants, surfactants, etc., commonly used in the appropriate formulation of pharmaceutical compositions, but is not limited thereto.

[0035] The composition of the present invention can be manufactured by including, in addition to the above-mentioned effective ingredient, one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier must be compatible with the effective ingredient of the present invention, and may be used as a mixture of saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, or emulsion. Furthermore, the composition can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing company, Easton PA).

[0036] The content and administration method of the active ingredients, etc. included in the composition of the present invention can be determined by a person skilled in the art based on the symptoms and severity of the disease of a typical patient. Furthermore, the composition can be formulated in various forms, such as powders, tablets, capsules, liquids, injections, ointments, and syrups, and can also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles.

[0037] The composition of the present invention can be administered orally or parenterally. The route of administration of the composition according to the present invention is not limited to these, but for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, enteral, sublingual, or topical administration is possible. The dosage of the composition according to the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate, or disease severity, and can be easily determined by a person skilled in the art. In addition, the composition of the present invention can be formulated into a suitable dosage form using known techniques for clinical administration.

[0038] The present invention provides a pharmaceutical composition for preventing or treating bone disease, comprising a DOCK5 inhibitor, which is administered together with a pharmaceutically effective amount of BMP.

[0039] In one embodiment of the present invention, BMP can be administered simultaneously with the DOCK5 inhibitor, or can be administered separately or sequentially, and the effective amount means an amount that can exhibit a desired effect at the application site, and can vary depending on factors such as the formulation method of the pharmaceutical composition, the administration method, the patient's age, weight, sex, pathological condition, administration time, administration route, excretion rate, and response sensitivity, and a person of ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment.

[0040]

[0041] In addition, according to another aspect of the present invention, the present invention provides a food composition for preventing or improving bone disease, comprising the above-described DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients; or a health functional food comprising the same.

[0042] In the present invention, 'food' means a natural product or processed product containing one or more nutrients, preferably a product that has gone through a certain degree of processing to become directly edible, and in its conventional sense includes all foods, food additives, health functional foods, and beverages.

[0043] The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients with functional properties useful to the human body, as defined by the Health Functional Food Act. "Functionality" refers to ingestion for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients for the structure and functions of the human body or physiological functions.

[0044] The food composition of the present invention may include conventional food additives, and its suitability as the "food additive" is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0045] In addition, the composition of the present invention can be used as a food additive for health supplements for the prevention or improvement of bone diseases. Foods to which the composition of the present invention can be added include various foods, such as beverages, gum, tea, vitamin complexes, health supplements, etc., and can be used in the form of pills, powders, granules, infusions, tablets, capsules, or beverages.

[0046] In the present invention, the food refers to a food having a bioregulatory function such as prevention and improvement of bone disease, biodefense, immunity, and recovery after illness, and must be harmless to the human body when consumed over a long period of time.

[0047] When the food composition of the present invention is a food additive, the effective ingredient may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. The amount of the effective ingredient mixed may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the effective ingredient of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the effective ingredient may also be used in an amount exceeding the above range.

[0048] There are no specific restrictions on the types of the above foods. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all health functional foods in the conventional sense.

[0049] The health functional food composition according to the present invention may be in various forms, such as a health drink. If the health functional food composition of the present invention is in the form of a health drink, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates mentioned above may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g, per 100 ml of the composition of the present invention.

[0050] In addition to the above, the food composition or health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0051]

[0052] In addition, according to another aspect of the present invention, the present invention provides a composition for promoting in vitro osteoblast differentiation and bone regeneration, comprising the above-described DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients. In addition, a method for promoting in vitro osteoblast differentiation is provided, comprising a step of treating target cells in vitro with the DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins).

[0053] Since the composition and method of the present invention utilize the effective ingredient of the present invention described above, redundant descriptions are omitted to avoid excessive complexity of the present specification.

[0054]

[0055] In addition, according to another aspect of the present invention, the present invention provides a method for producing a non-human DOCK5 gene-deficient animal model, including a step of inserting a gene trap vector between the first exon (exon1) and the second exon (exon2) of the DOCK5 (dedicator of cytokinesis 5) gene to completely delete the DOCK5 gene; and a non-human DOCK5 gene-deficient animal model produced thereby.

[0056] The DOCK5 (dedicator of cytokinesis 5) gene can be used without limitation as long as it is a mammalian DOCK5 (dedicator of cytokinesis 5) gene, and can be derived from, for example, chimpanzee, monkey, mouse (e.g., mouse DOCK5 gene NCBI GeneID: 68813), cow, dog, cat, sheep, horse, or human (e.g., human DOCK5 gene NCBI GeneID: 80005).

[0057] In one embodiment of the present invention, exon 1 (Genbank numb. NC_000080.7: c68170881-68170741) and exon 2 (Genbank numb. NC_000080.7: c68118731-68118648) of the mouse Dock5 gene were used.

[0058] The inventors of the present invention first discovered that when the DOCK5 (dedicator of cytokinesis 5) gene is knocked out in a mouse model, the differentiation rate of osteoclasts in the mouse decreases and the differentiation rate of osteoblasts increases, thereby increasing bone density, and this led to the invention.

[0059] Accordingly, the animal of the present invention, for example, a mouse model, can exhibit resistance to osteoporosis by suppressing the occurrence of osteoporosis due to a decrease in bone density by suppressing DOCK5.

[0060] Therefore, by using the mouse model of the present invention as an osteoporosis research model, it can be utilized for functional research on osteoporosis-related genomes, target genes of osteoporosis drugs can be discovered, the regulatory action point of osteoporosis can be analyzed and a new signal transmission system can be identified, and it can be utilized for suggesting the regulatory mechanism of osteoporosis, so it can be usefully utilized.

[0061] In order to avoid excessive complexity of this specification, description of the method of the present invention is omitted.

[0062]

[0063] In addition, according to another aspect of the present invention, the present invention provides a method for treating a bone disease, comprising administering to a subject a pharmaceutical composition comprising a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

[0064] Since the pharmaceutical composition of the present invention promotes differentiation of osteoblasts, which play an important role in increasing bone formation, administration of the pharmaceutical composition of the present invention can be an effective method for treating bone diseases.

[0065] Since the method of the present invention utilizes the pharmaceutical composition of the present invention, description of redundant content is omitted to avoid excessive complexity of this specification.

[0066] The combined use of a DOCK5 inhibitor and BMP according to the present invention is expected to be useful for improving and treating bone defects and bone diseases, as it has excellent efficacy in promoting osteoblast differentiation and increases the desired bone regeneration effect.

[0067] Figures 1a-1e show the effect of C21 on osteoblast differentiation. (a) MC3T3-E1 cells were treated with rhBMP2 and 25 or 50 μM C21 for 12 and 15 days. Mineral deposition was visualized with Alizarin Red S solution (n = 3). (b) Expression of bone formation-specific marker genes was measured by RT-qPCR in cells undergoing osteoblast differentiation on days 4 and 7 (n = 3). Expression of each gene was normalized to Gapdh expression. (c) Protein expression of OSX, BSP, and RUNX2 was measured by Western blotting on days 4, 7, and 10 of osteoblast differentiation (n = 4-8). (d) hBMSCs treated with 50 ng / mL rhBMP2 and 10 or 25 μM C21 for 12 days. Mineral deposition was visualized with Alizarin Red S solution (n = 6). (e) Expression analysis of osteoblast-specific marker genes in hBMSCs undergoing osteoblast differentiation on days 4 and 7 (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA and Tukey's multiple comparison post-hoc test.

[0068] rhBMP2: recombinant human bone morphogenetic protein 2, RT-qPCR: reverse transcription-quantitative polymerase chain reaction, Gapdh: glyceraldehyde-3-phosphate dehydrogenase, OSX / Osx: osterix, BSP / Bsp: bone sialoprotein, RUNX2 / Runx2: runt-related transcription factor 2, Alp: alkaline phosphatase, Oc: osteocalcin, Col1a1: collagen, type I, alpha 1, hBMSCs: human bone marrow mesenchymal stem cells

[0069] Figures 2a-2i show bone characteristics of Dock5 KO mice. (a) Representative micro-CT images showing the cross-sectional trabecular bone area of ​​WT and Dock5 KO mice. (b) Quantitative analysis of bone morphometric indices in WT and Dock5 KO mice. Bone volume per volume (BV / TV), bone mineral density (BMD), bone mineral content (BMC), trabecular bone number (Tb.N), trabecular bone separation (Tb.Sp), and trabecular bone thickness (Tb.Th) were analyzed (WT, n = 8; KO, n = 18). (c) Hematoxylin and eosin (H&E) staining of tibial trabecular bone and von Kossa staining of lumbar spine. Boxed areas are indicated at higher magnification next to low-magnification images. (d) TRAP staining of tibial trabecular bone and the number of TRAP-positive osteoclasts. (e) Osteoclast differentiation and number of TRAP-positive osteoclasts in Dock5 KO and WT mouse BMMs treated with M-CSF and RANKL. (f) Cells were stained with rhodamine-phalloidin (red), DAPI (blue), and anti-Nfatc1 antibody (green) to detect actin, nuclei, and Nfatc1, respectively. (g) Quantification of osteoclast-specific gene expression by RT-qPCR. (h) Bone resorption assay. BMMs were seeded on bone slices and cultured under osteoclast-inducing conditions for 5 days. Pits formed by bone resorption were stained with hematoxylin (n = 3). The bone resorption area per bone slice was calculated using ImageJ software. p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, analyzed by two-tailed unpaired Student's t-test. (i) Calcein labeling on days 1 and 6 of 8-week-old mice. Two days after the second injection, mice were sacrificed and analyzed. Representative calcein-labeled lumbar spine images from WT (top panel) and Dock5 KO (bottom panel) mice are shown on the left. Enlarged images are shown on the right. Mineral deposition rates (MAR) were calculated (n = 29 for WT and 31 for KO). *p < 0.05, **p < 0.01, ***p < 0.001, analyzed by one-way ANOVA and Tukey's multiple comparison post-hoc test or unpaired Student's t-test.

[0070] Dock5: dedicator of cytokinesis 5, KO: knockout, micro-CT: microcomputed tomography, WT: wild-type

[0071] Figures 3a–c show the effects of Dock5 deletion on osteoblast differentiation. (a) Osteoblast differentiation of mBMSCs isolated from WT and Dock5 KO mice induced with OS or OS + BMP2. Mineral deposition was assessed by alizarin red S staining (n = 4). (b) RT-qPCR for osteoblast-specific marker gene expression on day 3 (n = 4). (c) Osteocalcin immunohistochemistry of tibial trabecular bone (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons post-hoc test or unpaired Student's t-test.

[0072] Dock5: dedicator of cytokinesis 5, KO: knockout, WT: wild-type, mBMSCs: mouse bone marrow mesenchymal stem cells, OS: osteogenic induction, BMP2: bone morphogenetic protein 2, Oc: osteocalcin, Bsp: bone sialoprotein, Runx2: runt-related transcription factor 2, Osx: osterix, Alp: alkaline phosphatase, RT-qPCR: reverse transcription-quantitative polymerase chain reaction

[0073] Figures 4a-h show the effects of Dock5 gene deletion and chemical inhibition on bone regeneration in animal models. (a) Representative micro-CT images of calvarial defect regeneration in WT and Dock5 KO mice are shown. (b) Analysis of bone morphometric markers by micro-CT (n = 7-10 per group). (c) BMP2-induced ectopic bone formation beneath the calvarial periosteum in WT, Dock5 heterozygous, and Dock5 KO mice. Representative micro-CT images are shown. (d) Analysis of bone parameters by genotype group. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA and Tukey's multiple comparisons post-hoc test. (e) Cross-sections of calvarial ossicles stained with H&E and Masson's trichrome. Scale bar = 200 μm. (f) Subcutaneous ectopic bone formation induced by BMP2 or BMP2 + C21 in C57BL6 mice. Micro-CT images of ectopic bone are shown (n = 5). (g) Analysis of bone morphometric indices by micro-CT according to treatment group. *p < 0.05 and **p < 0.01, one-way ANOVA followed by Tukey's multiple comparison post-hoc test. (h) Ossicle staining with H&E (top) and Masson's trichrome (bottom). Scale bar = 100 μm.

[0074] Dock5: dedicator of cytokinesis 5, KO: knockout, WT: wild-type, micro-CT: microcomputed tomography, BMP2: bone morphogenetic protein 2, H&E: hematoxylin and eosin, BMD: bone mineral density, TV: tissue volume, BV: bone volume, BV / TV: bone volume per tissue volume, Tb.Th: trabecular thickness, Tb.N: trabecular number, Tb.Sp: trabecular separation, TS: tissue surface, BS: bone surface

[0075] Figures 5a to 5d show analysis of the MKK3 / 6 and p38 signaling pathways through Dock5 gene deletion or chemical inhibition. (a) mBMSCs isolated from WT or Dock5 KO mice stimulated with BMP2 in OS medium. Phosphorylation of signaling molecules was analyzed at the indicated times (n = 2). (b) MC3T3-E1 cells induced with OS + BMP2 or OS + BMP2 + C21. Phosphorylation of signaling molecules was analyzed at the indicated times (n = 3). (c) MC3T3-E1 cells induced as in b. Phosphorylation of signaling molecules was analyzed at the indicated times (n = 3). (d) hBMSCs induced for osteoblast differentiation with OS + BMP2 or OS + BMP2 + C21. Phosphorylation of signaling molecules was analyzed at the indicated times (n = 3). Statistical analysis was performed only at each identical time point, and comparisons with other time points were not performed. *p < 0.05, **p < 0.01, ***p < 0.001, analysis by two-tailed unpaired Student's t-test.

[0076] TAK1: transforming growth factor-β-activated kinase 1, MKK3 / 6: mitogen-activated protein kinase kinase 3 / 6, SMAD1 / 5 / 9: mothers against decapentaplegic homolog 1 / 5 / 9, Dock5: dedicator of cytokinesis 5, mBMSCs: mouse bone marrow mesenchymal stem cells, WT: wild-type, KO: knockout, BMP2: bone morphogenetic protein 2, OS: osteogenic induction, hBMSCs: human bone marrow mesenchymal stem cells

[0077] Figures 6a to 6c show the results of analysis of the TAK1 and Rac1 signaling pathways through chemical inhibition of DOCK5. (a) Rac1 activity in MC3T3-E1 cells treated with BMP2 or BMP2 + C21. A dose-dependent inhibitory effect of C21 on Rac1 activity was observed (n = 3). (b) Effects of GEF inhibitors (C21 and NSC23766) on ALP activity and staining. MC3T3-E1 cells were treated as indicated, and ALP staining and activity assays were performed (n = 3). (c) BMP2-induced phosphorylation assay in hBMSCs. hBMSCs were pretreated with 10 μM HS-276 (a TAK1 inhibitor) for 24 h and stimulated with BMP2 or BMP2 + C21 in OS medium in the presence of HS-276 for the indicated times. Phosphorylation of signaling molecules was analyzed (n = 3). p < 0.05, **p < 0.01, ***p < 0.001, analyzed by two-tailed unpaired Student's t-test (n = 3).

[0078] TAK1: transforming growth factor-β-activated kinase 1, MKK3 / 6: mitogen-activated protein kinase kinase 3 / 6, SMAD1 / 5 / 9: mothers against decapentaplegic homolog 1 / 5 / 9, Rac1: ras-related C3 botulinum toxin substrate 1, BMP2: bone morphogenetic protein 2, ALP: alkaline phosphatase, GEF: guanine nucleotide exchange factor, hBMSCs: human bone marrow mesenchymal stem cells, OS: osteogenic induction

[0079] Figure 7 schematically shows the process of creating a Dock5 gene-deficient mouse model (Dock5 null mice).

[0080] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0081]

[0082] Materials and Methods

[0083] Osteoblast differentiation analysis

[0084] Mouse MC3T3-E1 cells were cultured in α-MEM medium without ascorbic acid, supplemented with 10% FBS (Welgene), 100 U / mL penicillin, and 100 μg / mL streptomycin (P / S; Gibco BRL). Mouse bone marrow-derived mesenchymal stem cells (mBMSCs) and human bone marrow-derived mesenchymal stem cells (hBMSCs, PT-2501; Lonza, Basel, Switzerland) were cultured in α-MEM medium containing 10% FBS (Gibco BRL) and P / S. For osteoblast induction of MC3T3-E1 cells and mBMSCs, 10 μM β-glycerophosphate and 50 μg / mL ascorbic acid were added to the medium (OS medium; osteoblast induction medium). For hBMSCs, 10 nM dexamethasone was additionally added to the mouse OS medium. All cells were cultured in a humidified atmosphere at 37°C with 5% CO2.

[0085] To induce osteoblast differentiation, MC3T3-E1 cells were seeded at 5 X 10 in a 24-well plate. 4 Cells were seeded at a density of 10 cells / cm². After 1 day, the cells were treated with OS medium for 3 days, and then treated with OS medium containing 25 or 50 μM C21 (Sigma-Aldrich) and 20 ng / mL rhBMP2 (Cowellmedi). On days 12 and 15 after differentiation induction, mineralization was confirmed by Alizarin Red S staining. For staining, the cells were washed with 1X PBS and fixed with 70% ethanol for 20 minutes at room temperature. After washing twice with distilled water, they were treated with 40 mM Alizarin Red S solution adjusted to pH 4.2 for 10 minutes. Nonspecific staining was removed by washing five times with distilled water.

[0086] For osteogenic differentiation of hBMSCs, cells were seeded at 1 X 10 4Cells were seeded in 24-well plates at a density of 10 cells / cm². The following day, OS medium was administered for 3 days. From day 3, differentiation was continued by adding 10 or 25 μM C21 together with 50 ng / mL rhBMP2 to the medium. The OS medium containing rhBMP2 and C21 was replaced every 3 days.

[0087] mBMSCs were isolated from mouse bone marrow according to a previously reported method and seeded at 2.5 X 10 4 Cells were seeded in 24-well plates. When 50% confluent was reached, they were treated with OS medium or OS + 50 ng / mL rhBMP2 medium. Staining with Alizarin Red S was performed on day 6.

[0088] Western blotting and phosphorylation analysis

[0089] To determine the signaling phosphorylation level in MC3T3-E1 cells, 4.8 X 10 5 Cells were seeded in 35 mm dishes. After 24 h, the medium was replaced with medium containing 0.3% FBS and cultured for another 24 h. Under OS conditions, cells were treated with vehicle or 100 μM C21, and the reaction was stopped after 0, 5, 15, 30, and 60 min by washing twice with ice-cold PBS. Under OS + BMP2 conditions, MC3T3-E1 cells were cultured in OS medium for 3 days, then treated with OS medium containing 0.3% FBS for 24 h, and then treated with 50 ng / mL of rhBMP2 and vehicle or 100 μM C21 at each time point.

[0090] Additionally, hBMSCs (1 X 10 5 cells) and mBMSCs (2 X 10 5 After seeding cells in a 35 mm dish, they were cultured in OS medium for 3 days and treated with C21 and BMP2 under the same conditions as above.

[0091] For cell lysis, cells were washed with ice-cold 1X PBS, and 1X gel loading buffer (GLB; 50 mM Tris-HCl pH 6.8, 2% SDS, 6% glycerol, 0.01% bromophenol blue) was added. Then, the cells were sonicated for 43 s at 20% intensity with 1 s on / 2 s off cycles using a microprobe sonicator (Qsonica). Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific), and 5% β-mercaptoethanol was added to 30 μg of the sample. Western blot was performed according to a previously reported method. Each antibody was purchased from Cell Signaling Technology, Santa Cruz Biotechnology, Abcam, ABclonal, and Sigma-Aldrich.

[0092] ALP staining and activity measurement

[0093] MC3T3-E1 cells were induced to undergo osteogenic differentiation as previously described. To examine the Rac1 inhibitory effect, cells were pretreated with 50 μM NSC23766 (MedChemExpress) for 14 h and then treated with OS medium containing DMSO or 50 μM C21 and 20 ng / mL rhBMP2. On day 7, ALP staining was performed using an ALP staining kit (EMD Millipore Corp.) according to the manufacturer's instructions. To assess the effect of C21 on TAK1 signaling, cells were pretreated with 10 μM HS-276 (TAK1 inhibitor; Sigma-Aldrich) in OS medium for 24 h, and then treated with 2 μM HS-276 and 50 ng / mL rhBMP2, either alone or in combination with C21 (25 μM). To measure ALP activity, cells were washed twice with ice-cold 1X PBS on day 7 and measured according to the manufacturer's instructions (Abcam). The measured pNPP concentration was normalized to protein concentration.

[0094] Rac1 activity assay

[0095] MC3T3-E1 cells were seeded in 12-well plates and cultured for 3 days in OS medium, replacing the regular medium. Afterwards, starvation was performed for 24 h in OS medium containing 0.3% FBS. Cells were treated for 1 h under the following conditions: OS + vehicle, OS + rhBMP2 (20 μg / mL), and OS + rhBMP2 + C21 (25 or 50 μM, 0.3% FBS). After cell lysis, Rac1 activity assay was performed according to the manufacturer's instructions (Cytoskeleton Inc.).

[0096] Dock5 null mice

[0097] Dock5-deficient mice were designed and custom-made (Texas Institute for Genomic Medicine) using a gene trap method that inserts a beta-Geo cassette between exon 1 (Genbank numb. NC_000080.7: c68170881-68170741) and exon 2 (Genbank numb. NC_000080.7: c68118731-68118648) of the Dock5 gene, resulting in complete deletion of DOCK5 (Fig. 7).

[0098] Micro-CT analysis

[0099] The specimens were fixed in 10% formalin / PBS (pH 7.4) solution at 4°C for 24 h and then washed several times with PBS. Subsequently, micro-CT analysis was performed on the tibia, skull, and ectopic bone using a Skyscan 1272 scanner (Bruker).

[0100] Histomorphometry and calcein analysis

[0101] Skeletons of 9-week-old mice were prepared according to a previous report. The tibia and lumbar vertebrae were fixed in 10% formalin at 4°C for 24 h, decalcified in 0.5 M EDTA (pH 8.0) for 2–3 weeks, and embedded in paraffin. Paraffin sections were sectioned at 6 μm thickness and stained with H&E and Masson's trichrome (Polyscience). The skull and ectopic bones were processed in the same manner. The stained samples were observed under a light microscope (Leica Microsystems GmbH). von Kossa staining was also performed on the fixed lumbar vertebrae. For MAR analysis, 8-week-old mice were injected with calcein (20 mg / kg) on ​​days 1 and 6, respectively, and sacrificed 2 days after the second injection.

[0102] Animal testing and care

[0103] All animal experiments were performed in accordance with the approved guidelines of Kyungpook National University. Dock5 KO mice were housed under sterile conditions at 22–24°C and 50–60% humidity on a 12-h day / night cycle, with water and solid food provided ad libitum. Euthanasia was performed by setting the CO2 inflow rate at 40–60% of the chamber volume / min. BMMs were obtained from 4–6-week-old male BL6 mice and housed under identical conditions. Euthanasia was performed by intraperitoneal injection of 480 mg / kg avertin followed by cervical dislocation under anesthesia.

[0104] Model of calvarial defect and heterotopic bone formation

[0105] A calvarial defect model was created using WT (n = 6) and Dock5 KO (n = 10) mice. Mice were anesthetized with 240 mg / kg avertin, and the skull was exposed through a midline incision and periosteum dissection. A circular defect was created in the left parietal bone using a 3-mm trephine burr and a low-speed dental handpiece (Surgic XT) washed with sterile PBS. The periosteum was then covered, and the skin was sutured with 5-0 dissolvable silk sutures. Mice were sacrificed after 8 weeks.

[0106] In the heterotopic bone formation model, after the same incision procedure, a 3 mm collagen sponge soaked in rhBMP2 (1 μg) was placed on the skull. The periosteum was covered, the skin was sutured, and the mice were sacrificed after 4 weeks. In the subcutaneous transplantation model, 3 μL of a mixture of rhBMP2 (1.5 μg) + DMSO or rhBMP2 (1.5 μg) + C21 (100 μg) was absorbed into a 3 mm collagen sponge, and the sponge was implanted subcutaneously into 6-week-old male C57 / BL6J mice. The implanted scaffolds were excised and analyzed after 6 weeks. Euthanasia was performed by intraperitoneal injection of avertin (480 mg / kg) as described previously.

[0107] Statistical analysis

[0108] All experiments were performed at least three times. Results are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using a two-tailed nonparametric Student's t-test or a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p < 0.05 was considered statistically significant.

[0109]

[0110] Example 1. Confirmation of BMP2-mediated osteoblast differentiation promotion by DOCK5 inhibition.

[0111] The present inventors sought to determine the effects of DOCK5 inhibition and BMP2 on osteoblast differentiation.

[0112] First, MC3T3-E1 cells were treated with C21 (25 or 50 μM), a representative example of DOCK5 inhibitors, and osteoblast differentiation was analyzed. C21 was not cytotoxic in MC3T3-E1 cells at concentrations ranging from 1 to 1,000 μM and in hBMSCs at concentrations ranging from 5 to 100 μM. When treated with C21 in OS medium, mineral formation was increased at a concentration of 100 μM. DOCK5 is known to be involved in cell migration and actin polymerization in osteoclasts, a bone resorbing cell lineage. In the initial stage of osteoblast transformation, MC3T3-E1 cells were cultured in OS medium for 2 to 3 days to allow sufficient growth and migration, and then treated with rhBMP2 (20 ng / mL) and C21, either together or alone, for 12 and 15 days, respectively.

[0113] As a result, mineralization significantly increased depending on the C21 treatment concentration on days 12 and 15 of differentiation (Fig. 1a).

[0114] In addition, analysis of the expression of osteoblast marker genes showed that the expression of most marker genes increased, and in particular, Osx (osterix) and Bsp (bone sialoprotein) were significantly increased on the 4th day in the BMP2 + 50 μM C21 treatment group. The expression of Oc (osteocalcin) and Col1a1 (collagen type I alpha 1) was also significantly increased in the C21 combination group compared to the BMP2 alone treatment group (Fig. 1b).

[0115] Even at the protein level, OSX expression was significantly increased in the C21 combination group compared to the BMP2 alone group, and OSX expression was induced even under BMP2-untreated conditions, suggesting that DOCK5 inhibition can increase the expression of osteoblast marker genes / proteins (Fig. 1c).

[0116] In hBMSCs, co-treatment with C21 (0, 10, 25 μM) and BMP2 (50 ng / mL) resulted in a dose-dependent increase in mineral deposition, as confirmed by Alizarin Red S staining (Fig. 1d). Expression analysis of osteoblast marker genes also revealed that the Osx gene was significantly induced (Fig. 1e).

[0117] In mBMSCs, mineral deposition and osteoblast marker gene expression were also increased by BMP2 and C21.

[0118] This suggests that DOCK5 inhibition through C21 can synergistically induce osteoblast differentiation together with BMP2.

[0119]

[0120] Example 2. Confirmation of bone formation promotion through increased osteoblast differentiation when the Dock5 gene is missing.

[0121] To evaluate the in vivo function of Dock5, the present inventors generated knockout (KO) mice by inserting a gene trap immediately after exon 1 of the Dock5 gene (Fig. 7). This resulted in translational disruption in the SH3 domain of the DOCK5 protein.

[0122] Analysis of skeletal traits in Dock5 KO mice revealed that, compared to WT mice, the long bones (micro-CT) showed a 13% increase in BV / TV, an 11% increase in BMD, a 17% increase in BMC, a 28% increase in Tb.N, and a 36% decrease in Tb.Sp (Fig. 2a, b). H&E and von Kossa staining also confirmed increased bone mass (Fig. 2c).

[0123] The increase in bone mass can be interpreted as a result of decreased osteoclast activity or increased osteoblast activity. TRAP staining did not show a significant change in osteoclast number (Fig. 2d), but inhibition of osteoclast differentiation and decreased bone resorption activity were observed in Dock5 KO-derived bone marrow-derived macrophages (BMMs) (Fig. 2e-h).

[0124] In addition, double calcein labeling revealed that newly mineralized bone and MAR were significantly increased in the spine of Dock5 KO mice (Fig. 2i). This suggests that Dock5 deficiency may have contributed to increased bone mass by increasing osteoblast activity.

[0125]

[0126] Example 3. Confirmation of promotion of osteoblast differentiation by BMP2 in Dock5 deficiency

[0127] To exclude osteoclast activity and confirm the direct effect of Dock5 deficiency on osteoblast differentiation, we induced differentiation of BMSCs derived from WT and Dock5 KO mice under OS or OS + BMP2 conditions.

[0128] In response to BMP2, mineral deposition was significantly increased in Dock5 KO BMSCs at day 6 (Fig. 3a). Furthermore, expression of Oc, Bsp, Runx2, Osx, and Alp genes was significantly increased in Dock5 KO compared to WT (Fig. 3b). OC protein immunostaining and intensity analysis also showed increased expression in the trabecular bone of the tibia of Dock5 KO mice (Fig. 3c).

[0129] These results indicate that Dock5 deficiency synergistically induces osteoblast differentiation with BMP2.

[0130]

[0131] Example 4. Confirmation of bone regeneration promotion through Dock5 gene deletion and Dock5 inhibition by C21.

[0132] We used a calvarial defect model to evaluate the effects of DOCK5 inhibition or deletion on in vivo bone regeneration, as it promotes osteoblast differentiation in osteoblasts and human / mouse BMSCs.

[0133] After observing regeneration for 8 weeks after a 3 mm calvarial defect, micro-CT analysis showed that Dock5 KO mice had a 2-fold increase in BMD, BV, and BV / TV, a 1.2-fold increase in Tb.Th, a 1.6-fold increase in Tb.N, and a 4% decrease in Tb.Sp compared to WT (Fig. 4a, b).

[0134] To minimize osteoclast influence, we used an ectopic bone formation model by inserting a BMP2-soaked collagen sponge under the periosteum of the skull. Trabecular bone increased in the ectopic bone formed in Dock5 KO mice, and BV, BV / TV, and Tb.Th were all increased (Fig. 4c, d). New bone maturation was also confirmed by H&E and trichrome staining (Fig. 4e).

[0135] Furthermore, in a subcutaneous transplantation model, the BMP2 + C21 treatment group showed increased ectopic bone mass compared to the BMP2 alone group, and tissue volume (TV), bone volume (BV), tissue area (TS), and bone surface (BS) were all increased by C21 (Fig. 4f-h). This confirmed that DOCK5 inhibition strongly promoted BMP2-induced bone formation.

[0136]

[0137] Example 5. Confirmation of BMP2-induced MKK3 / 6 and p38 signaling promotion by Dock5 gene deletion or inhibition.

[0138] To determine whether Dock5 deletion or inhibition affects BMP2 signaling, we analyzed phosphorylation in BMSCs and MC3T3-E1 cells after BMP2 stimulation.

[0139] In Dock5 KO-derived BMSCs, TAK1 phosphorylation slightly increased upon BMP2 treatment, whereas MKK3 / 6 phosphorylation increased approximately 9-, 13-, 5-, and 1.7-fold, respectively, compared to WT at each time point (0, 5, 15, and 30 min) (Fig. 5a). p38 phosphorylation also increased 2- to 3-fold. Phosphorylation of SMAD1 / 5 / 9 was also increased, which may be related to the increased SMAD1 protein level induced by BMP2.

[0140] In MC3T3-E1 cells, MKK3 / 6, MEK1 / 2, ERK1 / 2, and p38 phosphorylation increased over 5-60 min when treated with BMP2 + C21, but SMAD1 / 5 / 9 phosphorylation remained unchanged and AKT phosphorylation decreased (Fig. 5b).

[0141] C21 had a weaker inhibitory effect on TAK1 phosphorylation in OS-treated osteoblasts than in naïve MC3T3-E1 cells. In long-term analyses (days 4, 7, 10, and 13), MKK3 / 6 and p38 phosphorylation continued to increase in the C21-treated group, whereas TAK1 phosphorylation decreased on day 10 and slightly increased on day 13 (Fig. 5c).

[0142] In hBMSCs, MKK3 / 6 and p38 phosphorylation were significantly increased between 5 and 60 min upon BMP2 + C21 treatment (Fig. 5d).

[0143] In addition, when Rac1 activity was measured, there was no increase in activity when BMP2 was treated alone, but when BMP2 + C21 was combined, it decreased by 10% at 25 μM and by about 13% at 50 μM (Fig. 6a).

[0144] NSC23766 (a Rac1 inhibitor) increased ALP activity by 1.5-fold when combined with C21 (Fig. 6b). This suggests that Rac1 activity can be regulated by DOCK5 in the presence of BMP2.

[0145] Although there are conflicting reports on the interaction between TAK1 and Rac1, in the present invention, inhibition experiments using HS-276, a TAK1 inhibitor, showed that BMP2-induced MKK3 / 6 and p38 phosphorylation were reduced, indicating that these are TAK1 downstream signals (Fig. 6c).

[0146] Consequently, our results suggest that DOCK5 inhibition may enhance osteoblast differentiation and bone regeneration through activation of MKK3 / 6 and p38 in the BMP2 signaling pathway.

[0147] Therefore, the present invention can be applied as an effective drug combination treatment for bone diseases.

[0148]

[0149] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for promoting bone formation containing a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

2. A pharmaceutical composition for preventing or treating bone disease, comprising a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

3. In paragraph 1, A pharmaceutical composition characterized in that the DOCK5 inhibitor is at least one compound selected from the group consisting of C21 (CAS No.: 54129-15-6), CPYPP (CAS No.: 310460-39-0), E197 (CAS No.: 2378515-04-7) and DOCK5-IN-1 (CAS No.: 149775-26-8).

4. In paragraph 1, A pharmaceutical composition characterized in that the DOCK5 inhibitor is at least one selected from the group consisting of guide RNA, antisense oligonucleotide, siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), and ribozyme targeting the DOCK5 gene.

5. In paragraph 1, A pharmaceutical composition characterized in that the DOCK5 inhibitor is at least one selected from the group consisting of a peptide, a peptide mimetic, a substrate analog, an aptamer, and an antibody that specifically binds to the DOCK5 protein.

6. In paragraph 1, A pharmaceutical composition, characterized in that the above BMP is at least one selected from the group consisting of BMP2, BMP3, BMP3b, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14 and BMP115.

7. In paragraph 1, A pharmaceutical composition characterized in that the composition promotes the production or differentiation of osteoblasts.

8. In paragraph 1, The above composition is a pharmaceutical composition characterized in that it increases the expression level or activity of MKK3 / 6 and p38.

9. In paragraph 2, A pharmaceutical composition characterized in that the bone disease is at least one selected from the group consisting of osteoporosis, fracture, rheumatoid arthritis, periodontitis, osteomalacia, osteopenia, bone atrophy, osteoarthritis, bone defect, osteolysis, osteonecrosis, osteodystrophy, Paget disease, and metastatic bone cancers.

10. A health functional food for the prevention or improvement of bone disease containing DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

11. A composition for promoting differentiation of osteoblasts and bone regeneration, comprising DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

12. A method for producing a DOCK5 gene-deleted animal model other than a human, comprising a step of inserting a gene trap vector between the first exon (exon1) and the second exon (exon2) of the DOCK5 (dedicator of cytokinesis 5) gene to completely delete the DOCK5 gene.

13. A method for treating a bone disease, comprising administering to a subject a pharmaceutical composition comprising a DOCK5 (dedicator of cytokinesis 5) inhibitor and BMP (bone morphogenetic proteins) as active ingredients.

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