JPWO2017175625A1 - Composition for regulating bone metabolism - Google Patents

Abstract

Drugs based on naturally occurring compounds capable of preventing and / or treating osteopenic diseases have been desired. There is provided a pharmaceutical composition for preventing and / or treating osteopenia containing a compound represented by the following chemical formula (I).
Embedded image

(In formula (I),
R1 is one functional group or 2 to 4 functional groups that are the same or different from each other, and is a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group,
(R2 and R3 are a hydrogen atom or a ketone group, and either R2 or R3 is a ketone group)

Description

  The present invention relates to a prophylactic / therapeutic agent for various osteopenic diseases based on the bone metabolism regulating action of diphenyl ether lactone.

  Osteopenia includes exogenous diseases such as traumatic fractures and fatigue fractures, as well as osteoporosis, osteoporosis, hypercalcemia, and hyperparathyroid hormone (hereinafter sometimes referred to as “PTH”) blood. , Bone Paget's disease, arthritis, rheumatism, bone metastasis of breast cancer, osteomalacia, bone tissue weakening due to malignant tumors and other diseases, and endogenous diseases such as bone tissue weakening due to nutritional disorders Broadly divided. In the present specification, the term “osteopenic disease” includes alveolar bone resorption and diseases caused by it, in addition to the above-mentioned exogenous diseases and intrinsic diseases.

  Human bones maintain homeostasis while constantly repeating bone formation by osteoblasts and bone resorption by osteoclasts. However, when homeostasis cannot be maintained due to aging or inflammation and bone resorption becomes excessive, pathological conditions such as osteoporosis and rheumatoid arthritis occur.

  Among these endogenous bone diseases, the number of patients with osteoporosis is increasing as the number of elderly people increases. In addition, it is said that the weakening of bone tissue is accelerated because the intake of calcium and the rate of colonization of bone tissue are reduced along with the change of dietary habits and the amount of exercise.

  A fracture refers to a state in which communication with a part of bone tissue is disconnected by an external force, and is accompanied by significant pain at the fracture site. In the case of a healthy person, for example, a fracture does not occur unless a considerable external force is applied due to a traffic accident, etc., but when the bone mass decreases and the bone tissue weakens, it falls while walking or running, etc. Even if an external force that is not so great is applied, a fracture occurs. In addition, when the bone mass is reduced due to an intrinsic disease such as osteoporosis as described above, a fracture may be caused by a slight external force applied when crawling or coughing on the stairs. .

  When a fracture occurs, the treatment method is generally to correct the position of the displaced bone by traction, etc., fix it with a bolt or pin, and then leave it to the patient's natural healing power Is adopted.

  On the other hand, in endogenous diseases such as osteoporosis, both bone calcareous and bone matrix decrease, so natural healing is often delayed. There are many cases where people are gone and become bedridden.

  Periodontal disease is a national disease that affects 80% of Japanese people over the age of 30. However, it does not feel pain unless it is severely ill. For this reason, it is regarded as a disease having characteristics that are likely to cause a serious disease. In advanced periodontal diseases, eating disorders due to pain and discomfort during chewing, bad breath with bad odor, and the like may result in poor nutrition and communication problems. In such a case, professional treatment by dentistry is performed, but the progression of symptoms cannot be stopped and extraction is often performed. On the other hand, it is known that healthy elderly people have a large number of remaining teeth.

  In recent years, periodontal diseases are spreading not only in humans but also in pets such as dogs and cats fed with soft food and raised in the home, and are regarded as a problem.

  When suffering from periodontal disease, the cementum, periodontal ligament and alveolar bone constituting the periodontal tissue are destroyed. For this reason, human periodontal disease has been conventionally treated by a method that promotes tissue healing by removing causative substances that cause inflammation such as tartar and necrotic cementum. However, regeneration of periodontal tissue that has already been destroyed by this method is difficult.

  Both osteoporosis and periodontal disease are said to have more than 10 million patients, and the potential market size is extremely large. In osteoporosis that is clinically applied, there are many bone resorption inhibitors, but they are not always satisfactory in terms of administration method, side effects, and effectiveness. Parathyroid hormone preparations have been put into practical use as osteogenesis promoters. Patent Document 1 discloses a method for increasing the toughness and stiffness of bone, preferably cortical bone, by administration of recombinant human parathyroid hormone, and / or the occurrence and / or severity of fractures. A method for reducing this is disclosed.

Special table 2002-523375

  However, parathyroid hormone preparations still have problems such as high cost and restrictions on long-term use. If a low-molecular-weight drug that has not only an osteogenesis promoting action but also a bone resorption inhibiting action is developed, it may become a revolutionary new drug.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a drug based on a naturally occurring compound or a method for preventing and / or treating osteopenia using the drug.

  The inventors of the present application conducted research on a method for preventing and / or treating osteopenic disease using a naturally occurring compound with high safety, and the compound represented by the following chemical formula (I) is an osteopenic disease. Has been found to prevent and / or treat the disease, and the present invention has been completed.

According to the present invention,
There is provided a pharmaceutical composition for preventing and / or treating osteopenia containing a compound represented by the following chemical formula (I).
(In formula (I),
R1 is one functional group or 2 to 4 functional groups that are the same or different from each other, and is a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group,
(R2 and R3 are a hydrogen atom or a ketone group, and either R2 or R3 is a ketone group)

  As demonstrated in the examples described later, the compound represented by the above chemical formula (I) is an osteoblast differentiation promotion, osteoclast differentiation inhibition, bone formation promotion, bone resorption inhibition and tooth or alveolar bone Showed growth and formation. Therefore, osteopenia can be prevented and / or treated by using this pharmaceutical composition.

According to the present invention,
There is provided a pharmaceutical preparation for regulating bone metabolism comprising a compound represented by the following chemical formula (I).
(In formula (I),
R1 is one functional group or 2 to 4 functional groups that are the same or different from each other, and is a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group,
(R2 and R3 are a hydrogen atom or a ketone group, and either R2 or R3 is a ketone group)

  As demonstrated in the examples described later, the compound represented by the above chemical formula (I) is an osteoblast differentiation promotion, osteoclast differentiation inhibition, bone formation promotion, bone resorption inhibition and tooth or alveolar bone Showed growth and formation. Therefore, osteopenic diseases can be prevented and / or treated by using this pharmaceutical preparation for regulating bone metabolism.

According to the present invention,
There is provided a therapeutic agent for osteopenia, comprising the pharmaceutical preparation for regulating bone metabolism.

  As demonstrated in the examples described later, the compound represented by the above chemical formula (I) is an osteoblast differentiation promotion, osteoclast differentiation inhibition, bone formation promotion, bone resorption inhibition and tooth or alveolar bone Showed growth and formation. Therefore, osteopenic diseases can be treated by using a therapeutic agent containing a pharmaceutical preparation for bone metabolism regulation containing the compound represented by the above chemical formula (I). Therefore, it is possible to treat osteopenia by applying this therapeutic agent to patients suffering from osteopenia.

According to the present invention,
A method for preventing and / or treating osteopenic disease, the method comprising:
There is provided a method comprising administering to a subject suffering from the osteopenic disease a pharmaceutical composition containing a compound represented by the following chemical formula (I) or a pharmaceutical preparation for regulating bone metabolism.
(In formula (I),
R1 is one functional group or 2 to 4 functional groups that are the same or different from each other, and is a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group,
(R2 and R3 are a hydrogen atom or a ketone group, and either R2 or R3 is a ketone group)

  As demonstrated in the examples described later, the compound represented by the above chemical formula (I) is an osteoblast differentiation promotion, osteoclast differentiation inhibition, bone formation promotion, bone resorption inhibition and tooth or alveolar bone Showed growth and formation. By administering a pharmaceutical composition containing a compound represented by the above chemical formula (I) or a bone metabolism regulator, osteopenic disease was improved. Therefore, it is possible to prevent or ameliorate osteopenic diseases by applying this method to patients suffering from osteopenic diseases.

FIG. 1 shows the structural formula of combretastatin D-4. FIG. 2 is a graph showing ALP activity of MC3T3-E1 cells treated with combretastatin D-4. FIG. 3 is a graph showing the amount of osteocalcin in the culture supernatant of MC3T3-E1 cells treated with combretastatin D-4. FIG. 4 is a photograph showing ALP staining and alizarin staining of combretastatin D-4 treated MC3T3-E1 cells. FIG. 5 is a graph showing the ALP activity of MC3T3-E1 cells treated with each sample. FIG. 6 is a photograph of alizarin staining of MC3T3-E1 cells treated with each sample. FIG. 7 is a photograph showing the effect of combretastatin D-4 on osteoclast differentiation of RAW264 cells. FIG. 8 is a photograph showing the effect of combretastatin D-4 on osteoclast differentiation by co-culture of bone marrow cells and osteoblast cell lines. FIG. 9 is a photograph showing the effect of combretastatin D-4 on the formation of bone resorption pits associated with osteoclast differentiation by co-culture on bone fragments. FIG. 10 is a graph showing the effect of combretastatin D-4 and its derivatives on osteoclast differentiation of RAW264 cells. FIG. 11 is a μCT image 3 weeks after tooth germ transplantation with PBS and combretastatin D-4.

Definition Osteopenic diseases Osteopenic diseases are exogenous diseases such as traumatic fractures and fatigue fractures, osteoporosis, osteoporosis, hypercalcemia, hyperparathyroid hormone (hereinafter referred to as “PTH”). ) Bone tissue due to blood loss due to blood loss, Paget's disease, arthritis, rheumatoid arthritis, bone metastasis of cancer, osteomalacia, malignant tumor, periodontal disease, other diseases, and nutritional disorders It is broadly divided into intrinsic diseases such as weakening of the body. In the present specification, the term “osteopenic disease” includes alveolar bone resorption and diseases caused by it, in addition to the above-mentioned exogenous diseases and intrinsic diseases. Osteoblast differentiation promotion, osteoclast differentiation inhibition, bone formation promotion, bone resorption inhibition, tooth and alveolar bone growth and formation, alveolar bone and periodontal tissue destruction prevention and bone and tooth regeneration , Included in causes for preventing, treating and / or ameliorating “osteopenia”.

Bone metabolism regulating action The bone metabolism regulating action includes an osteoclast differentiation inhibiting action and an osteoblast differentiation promoting action. Osteoclasts differentiate from myeloid progenitor cells that have undergone various extracellular stimuli. Osteoclasts absorb the bone matrix and melt the bone, so that when bone resorption by osteoclasts is abnormally enhanced, the bone becomes brittle. On the other hand, osteoblasts differentiate from mesenchymal progenitor cells and play a role in bone formation. In bone metabolism, osteoclasts and osteoblasts cooperate to repeat bone resorption and bone formation and maintain homeostasis.

Overview Hereinafter, embodiments of the present invention will be described in detail. In addition, in order to avoid the repetition complexity about the same content, description of omission is abbreviate | omitted.

<Embodiment 1: Pharmaceutical composition>
According to the present invention,
There is provided a pharmaceutical composition for preventing and / or treating osteopenia containing a compound represented by the following chemical formula (I).
(In formula (I),
R1 is one functional group or 2 to 4 functional groups that are the same or different from each other, and is a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group,
(R2 and R3 are a hydrogen atom or a ketone group, and either R2 or R3 is a ketone group)
By using this pharmaceutical composition, osteopenia can be prevented and / or treated.

According to the present invention,
Provided is a pharmaceutical composition for preventing and / or treating osteopenia, wherein the compound represented by the chemical formula (I) is a compound selected from the group of the following chemical formulas (II) to (V): The
By using this pharmaceutical composition, osteopenia can be prevented and / or treated.

  The compounds represented by the chemical formulas (II) to (V) are combretastatin D-4 (CAS = 126191-24-0) and O-methyl combretastatin D-4 (CAS = 1358550-64-7, respectively). ), Isocorniclatride A (CAS = 1360058-54-3) and O-methylisocornicrate A (CAS-1358550-62-5). In the present specification, the chemical formula (II) and combretastatin D-4 can be substituted with each other, and the chemical formula (III) and O-methyl combretastatin D-4 can be substituted with each other. ) And isocorniclate A can be substituted for each other, and chemical formula (V) and O-methylisocornicrate A can be substituted for each other.

  The above compound may be a purified compound or a synthesized compound. Moreover, the form contained in the crude extract of a plant may be sufficient as the said compound. Examples of the plant include Getonia floribunda (containing combretastatin D-4), Aegiceras corniculatum (combretastatin D-4, O-methyl combretastatin, isocorniclatride A and O-methylisocorniclatride A) and Celastrus hindsii (containing isocorniclatride A). In addition, any compound isolated from the above-described plant may be chemically treated to synthesize the compound. The compound may be a hydrate or a salt.

<Embodiment 2: Formulation>
According to the present invention,
There is provided a pharmaceutical preparation for regulating bone metabolism comprising a compound represented by the following chemical formula (I).
(In formula (I),
R1 is one functional group or 2 to 4 functional groups that are the same or different from each other, and is a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group,
(R2 and R3 are a hydrogen atom or a ketone group, and either R2 or R3 is a ketone group)

  When the above-mentioned compound, hydrate or salt of the above-mentioned compound is used as a pharmaceutical composition or pharmaceutical preparation, the crystals may be treated according to a conventional method and mixed with the excipients described later. Pharmaceutical preparations containing such compounds as active ingredients include injections, suppositories, aerosols, transdermal absorption agents, ointments, plasters, sprays and other parenterals, tablets, powders, granules, powders, capsules Agents, pills, troches, liquids and other oral preparations.

  Here, the tablets include sugar-coated tablets, coated tablets, and buccals, and the capsules include both hard capsules and soft capsules. Granules also include coated granules. The above liquid preparations include suspensions, emulsions, syrups, elixirs and the like, and syrups include dry syrups. Each of the above-mentioned preparations includes not only those that are not sustained-released but also those that are sustained-released. These preparations are in accordance with known pharmacological manufacturing methods, such as bases, carriers, excipients, binders, disintegrants, lubricants, colorants, etc. described in the Japanese Pharmacopoeia that are pharmacologically acceptable when manufacturing the preparations. Can be used. Examples of such carriers and excipients include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, and gentian powder.

  Examples of the pharmaceutical composition or binder include starch, tragacanth gum, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, and the like. Examples of the disintegrant include starch, agar, gelatin powder, sodium carboxymethylcellulose, carboxymethylcellulose calcium, crystalline cellulose, calcium carbonate, methylcellulose, ethylcellulose, carboxymethylcellulose, and the like. Examples of the lubricant include talc and magnesium stearate. The colorant can be used as long as it is allowed to be added to pharmaceuticals, and is not particularly limited. In addition to these, flavoring agents, flavoring agents, and the like can be appropriately used as necessary.

  For tablets or granules, sucrose, gelatin, purified shellac, glycerin, sorbitol, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl as necessary Coating may be performed using a methacrylate, a methacrylic acid polymer, or the like, and coating may be performed with a plurality of layers. Furthermore, granules and powders can be packed into capsules such as ethyl cellulose and gelatin to form capsules.

  When preparing an injection using a pharmaceutical composition or pharmaceutical preparation containing the above compound or a pharmaceutical composition or pharmaceutical preparation containing a hydrate of the above compound, a pH adjuster, buffer material, A solubilizer, solubilizer, and the like can also be added.

  The pharmaceutical preparation for bone metabolism regulation of this embodiment may be used for preventing or treating osteopenic diseases. The osteopenic disease may be accompanied by subjective symptoms or not, or may be based on a doctor's diagnosis.

<Embodiment 3: Therapeutic>
According to the present invention,
There is provided a therapeutic agent for osteopenia, comprising the pharmaceutical preparation for regulating bone metabolism.
By using this therapeutic agent, it becomes possible to treat osteopenic diseases. Certain osteopenic diseases can be used for the prevention and / or improvement of osteopenic diseases.

  The therapeutic agent, whether alone or in combination with other therapeutic agents, is administered in an effective amount to treat osteopenic disease. However, the total dose of the compound is determined by the attending physician within the scope of sound medical judgment. The effective amount for a patient suffering from an osteopenic disease is the severity of the osteopenic disease; the patient's age, weight, general health, sex and diet; administration time; route of administration; elimination rate of the compound; Depending on the drug used in combination with or concurrently with the above therapeutic agents. The dose of the compound does not have to be constant for each administration. For example, the dose may be administered at a dose lower than that required to achieve the desired therapeutic effect, and the dose may be gradually increased until the desired effect is obtained.

  If necessary, the effective daily dose may be divided into multiple doses depending on the purpose of administration. Those skilled in the art will be able to easily optimize the effective dose and the combined dosage regimen, depending on good medical practice and individual patient clinical symptoms.

  The daily dose of the above therapeutic agent (daily dose) is calculated in terms of combretastatin D-4, O-methyl combretastatin D-4, isocorniclatride A and / or O-methylisocornicrate A 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 150 and 200 mg / kg body weight may be within a range between two points. Administration forms include oral, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, parenteral, intranasal or intradermal administration. Alternatively, the therapeutic agent may be contained in a base material (for example, collagen or agarose gel) and implanted (implanted) in a patient (particularly, the affected area of the patient). The therapeutic agent is preferably administered continuously over a certain period of time, ie 3 days or longer, preferably 1 week or longer, more preferably 2 weeks or longer, even more preferably 1 month or longer, for example 6 months or 1 year or longer. The therapeutic agent is preferably administered daily, but may not be administered daily as long as it is continuously administered throughout the period. The above-mentioned therapeutic agent may be administered once a day, or may be administered in several divided daily doses. Administration of the therapeutic agent may be terminated by a doctor's judgment or may be terminated by the patient's self-judgment.

<Embodiment 4: Biocompatible material>
According to the present invention,
There is provided a biocompatible material containing a compound represented by the above chemical formula (I) or containing at least one compound selected from the group of the above chemical formulas (II) to (V).
By using this biocompatible material, it is possible to directly contact the affected part of the osteopenic disease.

  In the present specification, “biocompatibility” means that a negative reaction such as an inflammatory reaction, an immune reaction, or an addiction reaction is not caused to a living tissue or cell, or such a reaction is small. “Biocompatible materials” include biodegradable materials and non-biodegradable materials. A “biodegradable material” is a material that can be degraded in the living body or absorbed in the living body. For example, collagen, atelocollagen, hyaluronic acid, hydroxyapatite, TCP, polylactic acid (PLLA), polyglycolic acid ( PGA), PLGA, polyethylene glycol (PEG), MPC polymer, and agarose gel. The “non-biodegradable material” is a material that is not decomposed and absorbed in vivo or is not substantially decomposed and absorbed, and examples thereof include titanium, a titanium alloy, stainless steel, and ceramics. The biocompatible material may be a cell scaffold (scaffold), or may be a scaffold (for example, a cell sheet or cell mass) to which desired cells are adhered. When using a non-biodegradable material as a biocompatible material, the said biodegradable material containing the said compound may adhere to the surface of the non-biodegradable material, for example. The biocompatible material may have sustained release properties by methods known to those skilled in the art.

  The biocompatible material can be embedded in the affected area of osteopenic disease, can be used for the formation or regeneration of bones and teeth (including alveolar bone), periodontal tissue regeneration, fracture site repair, It can also be used for pretreatment of implant tooth placement.

<Embodiment 5: Functional food or health food>
According to the present invention,
A functional food for preventing or ameliorating osteopenia, comprising the compound represented by the chemical formula (I) or at least one compound selected from the group of the chemical formulas (II) to (V) Or health food is provided.
By appropriately adding the above compound or a hydrate of the above compound as necessary, it is possible to provide a functional food or health food having an effect of preventing or improving osteopenia.

  In the present specification, the “functional food” refers to a food containing ingredients that can provide more benefits than can be provided to those who have consumed the food by the nutrients that the food itself originally contains.

  In addition, the “health food” in this specification is a general term for foods that are generally sold and used as foods that contribute to the maintenance and promotion of health, and includes supplements that assist in the intake of nutrients that are often insufficient.

  The above compound or a hydrate of the above compound, for example, bread, cookies and biscuits, cooked wheat and millet, udon, buckwheat, pasta, other noodles, cheese, yogurt and other dairy products, jam, mayonnaise, miso, Made from soy sauce and other soy products, tea, coffee and cocoa, soft drinks, fruit drinks and other non-alcoholic drinks, medicinal liquors, other alcoholic drinks, candy, chocolate and other snacks, chewing gum, rice crackers, sheep's straw and other soybeans It can be added to confectionery and the like to make a functional food. Alternatively, it can be added to animal feed to make a functional mixed feed. In addition, when adding to said yogurt, soy sauce, a drink, etc., in order to prevent the compound of this invention from crystallizing and precipitating in these, a dissolution aid and a stabilizer can also be added suitably. .

  Moreover, it can be set as a health food by making the said compound or the hydrate of the said compound individually or in mixture of 2 or more types, and setting it as a powder agent, a granule, a tablet, a capsule according to a conventional method. Here, in order to make the compound of the present invention into a powder, the extract obtained in the production process is concentrated and dried using a method such as freeze drying, spray drying, vacuum drying, sample mill, blender, mixer The dry solid may be pulverized by, for example. Moreover, you may add corn starch, potato starch, dextrin, oyster shell powder, etc. as needed.

  Further, the powder obtained as described above may be appropriately added with the above-described binder and compressed into tablets. After making into tablets, the above-described coating agents such as sucrose or gelatin may be used to form sugar-coated tablets, or other coating agents may be used to make enteric solvents and the like. Further, the powder obtained as described above can be made into granules according to a conventional method to produce granules. Moreover, it can also be set as a capsule by filling the above-mentioned capsule with the above-mentioned powder and granule in an appropriate amount.

  The amount of combretastatin D-4, O-methyl combretastatin D-4, isocorniclatride A and / or O-methylisocorniclatride A contained in one functional food and health food of It can be adjusted to be within a range between two points selected from the group consisting of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 150 and 200 mg / kg body weight.

<Embodiment 6: Method for preventing or treating osteopenia>
According to the present invention,
A method for preventing or treating osteopenic disease, the method comprising:
Administering a pharmaceutical composition containing a compound represented by the following chemical formula (I) or a pharmaceutical preparation for regulating bone metabolism to a subject suffering from the osteopenic disease:
A method is provided.
When the above pharmaceutical composition or pharmaceutical preparation for regulating bone metabolism is administered to a patient, the dosage varies depending on various conditions such as the severity of the patient's symptoms, age, weight, PSA value, urine flow rate, and health status. . In general, the dosage and usage described above may be administered once or more times a day, and the number and amount of administration may be appropriately increased or decreased according to the above conditions.

  The daily dosage, administration period, and number of administrations of the pharmaceutical composition or the pharmaceutical preparation for regulating bone metabolism may be the same as those of the therapeutic agent described above. The administration of the pharmaceutical composition or the pharmaceutical preparation for controlling bone metabolism may be terminated by a doctor's judgment or by the patient's self-judgment.

  As mentioned above, although embodiment of this invention was described, these are illustrations of this invention, Various structures other than the above can also be employ | adopted: For example, many hard tissues (bone and teeth) reduction of a human and an animal Food compositions, health foods, pharmaceutical compositions, hygiene products and methods for the prevention and treatment of sexual diseases (osteoporosis, rheumatoid arthritis, periodontal disease, Paget's disease, bone metastasis of cancer, etc.); fractures, Inducing and promoting agents for bone formation and bone regeneration in hard tissue (bone and teeth) reduction and defect sites due to accidents, surgery, and other factors, and methods and methods; Alveolar bone formation and promotion of alveolar bone regeneration as pretreatment for implant placement Agent and method; prevention and regeneration agent for destruction of alveolar bone and periodontal tissue in periodontal disease and method thereof; bone, tooth and periodontal tissue regeneration-promoting cells prepared using the above composition and use and preparation thereof A method is mentioned.

  EXAMPLES Hereinafter, although an Example and drawing demonstrate this invention further, this invention is not limited to these. The instrument operation and kit use were in accordance with the manufacturer's manufacturer's protocol.

  Combretastatin D-4 (CAS = 126191-24-0), O-methylcombretastatin D-4 (CAS = 1358550-64-7), isocornicrate (as test samples) isocorniculatolide) A (CAS = 1360058-54-3) and O-methylisocorniculatolide A (CAS = 1358550-62-5) were synthesized in Nishiyama Laboratory, Keio University.

<Example 1>
Osteoblast differentiation promoting action (1) Material
MC3T3-E1 cells (mouse fetal skull-derived preosteoblast cell line, cell number RCB1126) were purchased from RIKEN Cell Bank. Α-MEM was purchased from INVITROGEN (catalog number 11900-024). The 96-well microplate was purchased from Nunc (catalog number 161093).

  p-Nitrophenyl phosphate, naphthol AS-MX phosphate, Fast Blue BB salt and Alizarin Red S were purchased from SIGMA. Ascorbic acid, β-glycerophosphoric acid, Tris-hydrochloric acid, methanol, magnesium chloride, sodium hydroxide and ethanol were purchased from Wako Pure Chemical Industries, Ltd. An EIA kit for measuring the amount of osteocalcin was purchased from Biomedical Technologies Inc.

(2) Examination of osteoblast differentiation
MC3T3-E1 cells are wrapped in α-MEM containing 10% fetal calf serum and rolled to each well of a 96-well microplate at 4 × 10 ³ cells / well, 37 ° C., 5% CO Pre-cultured for 2 days in ₂ incubators. On the third day from the start of preculture, the culture medium was changed to α-MEM containing 50 μg / ml ascorbic acid and 10 mM β-glycerophosphoric acid, and main culture was performed for 7 to 21 days. During the main culture, the medium was changed every 3 to 4 days.

  After completion of the culture, the cells were fixed with methanol and the dish was dried. Then, the activity of alkaline phosphatase (ALP), an early differentiation marker enzyme of osteoblasts, was measured, and ALP staining was performed. Osteoblast calcification was confirmed by alizarin red S staining. In addition, the expression level of osteocalcin, which is known as a marker for osteoblasts with advanced differentiation, was measured using the above EIA measurement kit.

  For the measurement of ALP activity, a pH 8.5 buffer solution containing 6.7 mM p-nitrophenyl phosphate as a substrate and containing 2 mM magnesium chloride and 100 mM Tris-HCl was used. 100 μl of this buffer solution was added to each well and reacted at 37 ° C. for 30 minutes. Then, 100 μl of 0.1N sodium hydroxide was added to stop the reaction. Absorbance at 405 nm was measured with a plate reader, and the absorbance was defined as ALP activity.

  For ALP staining, 50 μl of a pH 8.5 buffer containing 0.1 mg / ml naphthol AS-MX phosphate, 0.6 mg / ml Fast Blue BB salt and containing 2 mM magnesium chloride and 100 mM Tris-HCl is added to each well. Each was put in, reacted at room temperature for 30 minutes to 1 hour, and stained.

  Osteoblast calcification was confirmed by alizarin red S staining. After completion of the culture, cells were fixed with 70% ethanol, the dish was dried, and the deposited calcium was stained with 1% alizarin red S solution.

  For measurement of osteocalcin concentration, the culture supernatant of MC3T3-E1 cells obtained by culturing for 7 to 21 days under the above conditions was used. The amount of osteocalcin in the culture supernatant was measured using the above-described EIA kit according to the method in the attached instruction manual.

(3) Results Combretastatin D-4 (Fig. 1) is dependent on the concentration of alkaline phosphatase (ALP), a known marker of osteoblast differentiation, in MC3T3-E1 cells, a mouse preosteoblast cell line. (Fig. 2, control was untreated with combretastatin D-4). Combretastatin D-4 significantly increased the protein expression of osteocalcin, a substrate protein known as a marker of late differentiation (FIG. 3, control was untreated with combretastatin D-4). In addition, combretastatin D-4 also markedly increased the mineralization of red stained by alizarin red staining (Fig. 4, control was untreated with combretastatin D-4). However, it has been shown to promote early and late differentiation of osteoblasts to induce calcification and promote bone formation.

<Example 2>
Osteoblast differentiation promoting action of derivatives (1) Materials MC3T3-E1 cells (purchased from RIKEN), a mouse-derived preosteoblast cell line, were used for osteoblast differentiation experiments. For culturing MC3T3-E1 cells, α-MEM containing 10% fetal calf serum was used. 10% fetal bovine serum was purchased from Asahi Techno Glass Co., Ltd. (IWAKI). The other materials used here are the same as those used in Example 1.

(2) Osteoblast differentiation induction test As a method for evaluating the effect on osteoblast differentiation, MC3T3-E1 cell osteoblast differentiation induction was used. In this test, in order to evaluate the effect on osteoblast differentiation, we measured the activity of alkaline phosphatase (ALP), a marker enzyme of early osteoblast differentiation, and performed staining using this activity (ALP staining). It was.

(3) Osteoblast differentiation
MC3T3-E1 cells were inoculated into a 96-well microplate at 4 × 10 ³ cells / well and cultured in a 37 ° C., 5% CO ₂ incubator for 2 days until confluent. Subsequently, in order to induce osteoblast differentiation, the cells were cultured for 7 or 14 days in α-MEM containing 50 μg / ml L-ascorbic acid and 10 mM β-glycerophosphate and a test sample having a concentration. At that time, the medium was changed every 3 or 4 days.

(4) Evaluation of differentiation induction of osteoblasts
Measurement of ALP activity
The ALP activity was measured as follows. First, MC3T3-E1 cells were cultured for 7 days under the same conditions as described above, the culture solution was removed, the wells were washed with PBS, the cells were fixed with methanol, and the dishes were dried. Next, 100 μL of 2 mM magnesium chloride and 100 mM Tris-HCl buffer (pH 8.5) containing 6.7 mM p-nitrophenyl phosphate as a substrate was added to each well and reacted at 37 ° C. for 30 minutes.

  The reaction was stopped by adding 100 μl of 0.1N sodium hydroxide, the absorbance at 405 nm was measured with a plate reader, and the released p-nitrophenol was measured to determine ALP activity. The result of the osteoblast differentiation test was expressed as a value (%) relative to the control group when the ALP activity of the control group was taken as 100.

ALP staining
For ALP staining, a buffer solution (pH 8.5) containing 0.1 mg / ml naphthol AS-MX phosphate, 0.6 mg / ml Fast Blue BB salt, 2 mM magnesium chloride, and 100 mM Tris-HCl was used as the staining solution. Using. After measuring ALP activity as described above, each well was washed with distilled water, 100 μL of the above ALP staining solution was added to each well, and the mixture was reacted at room temperature for 30 minutes to 1 hour for staining.

  Also, calcification of osteoblasts was confirmed by alizarin red S staining. After completion of the culture, cells were fixed with 70% ethanol, the dish was dried, and the deposited calcium was stained with 1% alizarin red S solution.

(5) Results Combretastatin D-4 (hereinafter referred to as D-4) and its isomer, isocornicrate tride A (hereinafter referred to as Iso-D-4), and derivatives in which the phenolic hydroxyl groups of these compounds are methylated For a total of 4 compounds of O-methyl combretastatin D-4 and O-methylisocorniclatride A (hereinafter referred to as Me-D-4 and Me-Iso-D-4, respectively) of MC3T3-E1 cells The effect on ALP activity under normal culture conditions was examined. As a result, D-4 showed a concentration-dependent ALP activity promoting action with a peak at 30 μM as before (FIG. 5). Iso-D-4, an isomer of D-4, showed an ALP activity promoting action with a peak at 100 μM (FIG. 5). Furthermore, Me-D-4 and Me-Iso-D-4, which are methylated derivatives of the 11-position hydroxyl group, significantly promoted ALP activity, although weaker than D-4 and Iso-D-4 ( Figure 5). In addition, all the compounds were strongly stained with alizarin red as compared with the control, and calcification was also promoted (FIG. 6). From the above results, it was shown that these four compounds all promote osteoblast differentiation and promote bone formation.

<Example 3>
Osteoclast differentiation inhibitory action (RAW264 cells)
(1) Material In the osteoclast early differentiation inhibition test, a macrophage RAW264 cell line (RIKEN, cell number RCB0535, hereinafter referred to as “MφRAW264”) that differentiates into osteoclast-like cells is used. did. For the cultivation of MφRAW264, α-MEM (manufactured by INVITROGEN, catalog number 11900-024) containing 10% fetal bovine serum (Asahi Techno Glass Co., Ltd. (IWAKI), catalog number IWK-500) was used. Moreover, the osteoclast differentiation inducer (RANKL) used PEPRO TECH INC, catalog number 310-01.

  Catalog numbers 161093 and 172958 made by INVITROGEN were used for the 96-well microplate and 100 mmφ dish, respectively.

  Ethanol, acetone, formaldehyde and sodium chloride were purchased from Wako Pure Chemical Industries.

(2) Initial differentiation inhibition test of osteoclasts As an in vitro test method for evaluating the suppression of bone resorption, an initial differentiation inhibition test of osteoclasts from the MφRAW264 cell line was performed. The osteoclast test was evaluated by staining with tartrate-resistant acid phosphatase (TRAP), which is an early differentiation marker.

The MφRAW264 cell line is suspended in α-MEM medium (hereinafter sometimes referred to as “10% FBS-MEM”) supplemented with 10% fetal bovine serum (FBS), and 1 × 10 ⁵ cells in a 100 mm diameter dish. The cells were inoculated at / 10 mL and cultured at 37 ° C. in the presence of 5% CO _{2 for} 3 days. After confirming that the cells were confluent in the dish, the culture was terminated. Subsequently, the confluent cells were treated with a Hanks buffer containing 0.05% trypsin and 0.53 mM EDTA (0.05% Trypsin-0.53 mM EDTA / HBSS (INVITROGEN, catalog number 25300-054) from the dish. The cells were peeled off, suspended in 10% FBS-MEM, inoculated into each well of a 96-well microplate at 0.4 × 10 ⁴ cells / 0.1 mL, and cultured for 1 day under the same conditions as described above.

  Osteoclast differentiation inducing factor (RANKL) was dissolved in 10% FBS-MEM to prepare a 100 ng / mL solution. Combretastatin D-4 dissolved in DMSO at a concentration of 10 mM was prepared as a stock solution and stored at −20 ° C. When performing treatment with combretastatin D-4, dilute the stock solution and prepare 100-fold solutions of each treatment concentration, and add each well so that the final concentration with respect to the solvent is less than 1%. (2 μL each) was added. The concentration of combretastatin D-4 in each well was 25, 50, 100 μM. An equal amount of DMSO was added to the control group.

  RANKL was added 0.1 mL to all 96 wells (final concentration of RANKL 50 ng / mL) and combretastatin D-4 solution was added to the wells (combretastatin D-4 final concentration was 25, 50, 100 μM). An equal amount of DMSO not containing combretastatin D-4 was added to wells serving as control groups.

After culturing at 37 ° C. for 3 days in the presence of 5% CO ₂ , the culture solution in each well of the 96-well microplate is discarded, and this plate is washed with a phosphate buffered saline solution (PBS) and then 10% formalin. Each well was filled with -PBS (hereinafter sometimes referred to as "about 3.7% formaldehyde-PBS"), and the cells were fixed at room temperature for 15 minutes. Furthermore, it was fixed at room temperature for 1 minute using an ethanol-acetone solution (1: 1). After fixing, the fixing solution was discarded and dried at room temperature.

  After drying, the state of intracellular tartrate-resistant acid phosphatase in each well was confirmed by TRAP staining. The TRAP staining solution is obtained by dissolving 5 mg of Naphtol AS-MX phosphate (manufactured by SIGMA, catalog number N-4875) as a substrate in 0.5 ml of N, N-dimethylformamide, and further using fast red violet LB salt (manufactured by SIGMA) as a dye. , Catalog number F-1625) dissolved in 50 ml of 50 mM sodium tartrate / 0.1 M sodium acetate buffer (pH 5.0) was used in combination. That is, as in the case of the TRAP assay, the medium was removed after culturing. After washing the cell layer with PBS, the cells were fixed with about 3.7% formaldehyde-PBS for 15 minutes at room temperature. Subsequently, it was further fixed at room temperature for 1 minute using an ethanol-acetone solution (1: 1). After fixing, the fixing solution was discarded and dried at room temperature. After drying, a TRAP staining solution was added and stained at room temperature for 20-30 minutes. After staining, the staining solution was discarded, washed with running water, dried, and then observed with a microscope.

(3) Results When RAW264 cells, a mouse macrophage cell line, are cultured in the presence of RANKL, an osteoclast differentiation-inducing factor, staining using the activity of tartrate-resistant acid phosphatase (TRAP), a marker enzyme for osteoclasts. Induced many osteoclasts that stained red, and many multinucleated osteoclasts due to cell fusion were also observed. When combretastatin D-4 was added, multinucleated / mononuclear osteoclasts decreased (Fig. 7). This indicates that combretastatin D-4 suppresses osteoclast differentiation.

<Example 4>
Inhibition of osteoclast differentiation (co-culture of bone marrow cells and osteoblast cell line)
(1) Material
4-week-old male ddy mice were purchased from Chubu Scientific Materials Co., Ltd. A 26G needle and a 2.5 ml syringe were purchased from Terumo. The same α-MEM as in Example 1 was used. The osteoblast cell line UAMS-32 was provided by Dr. Masamichi Takami, School of Dentistry, Showa University. Vitamin D ₃ and PGE ₂ were purchased from Wako Pure Chemical Industries, Ltd..

(2) Osteoclast differentiation by co-culture
The femurs of ddy mice were collected, and bone marrow cells were taken out by injecting α-MEM into the femur using a 26G injection needle and a 2.5 ml syringe. Seed bone marrow cells at 2X10 ⁵ cells / well, UAMS-32 cells at 1X10 ⁴ cells / well, in each well of a 96-well microplate. Test samples at each concentration, vitamin D ₃ (1 μΜ) and PGE ₂ ( 1 μΜ) (α-MEM, 37 ° C., 5% CO ₂ incubator for 5 days.

After culturing, the cells were fixed with 10% formalin solution and re-fixed with 100% ethanol. After the dish was dried, it was stained with tartrate-resistant acid phosphatase (TRAP), which is an osteoclast marker enzyme. The same method as in Example 3 was followed for TRAP staining.
(3) Results When mouse bone marrow cells and osteoblast cell line UAMS-32 cells are cultured in the presence of Vitamin D ₃ and PGE ₂ , osteoclasts that are stained red by TRAP staining are induced, and further due to cell fusion. Many multinucleated osteoclasts are also observed. When combretastatin D-4 was added, multinucleated / mononuclear osteoclasts decreased (Fig. 8). This indicates that combretastatin D-4 also suppresses osteoblast-induced osteoclast differentiation closer to the living body.

<Example 5>
Bone resorption inhibitory effect (co-culture of bone marrow cells and osteoblast cell line)
(1) Material
4-week-old male ddy mice were purchased from Chubu Scientific Materials Co., Ltd. A 26G needle and a 2.5 ml syringe were purchased from Terumo. The same α-MEM as in Example 1 was used. The osteoblast cell line UAMS-32 was provided by Dr. Masamichi Takami, School of Dentistry, Showa University. Vitamin D ₃ and PGE ₂ were purchased from Wako Pure Chemical Industries, Ltd.. Toluidine blue was purchased from SIGMA. The ivory slice was prepared by cutting the ivory to a thickness of about 0.7 mm, making a 4 mm diameter slice from this ivory sheet using a punch, sterilizing with ethanol and drying.

(2) Osteoclast differentiation by co-culture
The femurs of ddy mice were collected, and bone marrow cells were taken out by injecting α-MEM into the femur using a 26G injection needle and a 2.5 ml syringe. Seed bone marrow cells at 2X10 ⁵ cells / well, UAMS-32 cells at 1X10 ⁴ cells / well, in each well of a 96-well microplate. Test samples at each concentration, vitamin D ₃ (1 μΜ) and PGE ₂ ( 1 μΜ) (α-MEM, 37 ° C., 5% CO ₂ incubator for 5 days.

  In addition, after culturing these cells on the ivory section under the same conditions as described above, the part (bone resorption pit, hereinafter sometimes referred to as “pit”) that is melted by osteoclasts on the ivory section Whether or not it was formed was stained with 1% toluidine blue staining solution.

(3) Results When mouse bone marrow and osteoblastic cell line UAMS-32 cells are cultured on ivory slices in the presence of Vitamin D ₃ and PGE ₂ , the bone surface melts and has a hole, which becomes purple by toluidine blue staining. Numerous bone resorption pits are formed. When combretastatin D-4 was added, this bone resorption pit formation was suppressed (FIG. 9). This indicates that combretastatin D-4 suppressed osteoclast differentiation and suppressed bone resorption.

<Example 6>
Osteoclast differentiation inhibitory action (RAW264 cells)
The materials and test methods were the same as in Example 3.

  Osteoclast differentiation inducing factor (RANKL) was dissolved in 10% FBS-MEM to prepare a 100 ng / mL solution. In addition, D-4, Iso-D-4, Me-D-4 and Me-Iso-D-4 were each dissolved in DMSO at a concentration of 100 mM to prepare a stock solution at −20 ° C. Stored. When performing treatment with D-4, Iso-D-4, Me-D-4, and Me-Iso-D-4, dilute the stock solution to prepare a solution having a concentration 100 times the treatment concentration. Was added to each well (2 μL) so that the final concentration was less than 1%. The concentrations of D-4, Iso-D-4, Me-D-4 and Me-Iso-D-4 in each well were all 15, 50 and 150 μM. An equal amount of DMSO was added to the control group (CON).

  Add RANKL to all 96 wells (0.1 mL each) (RANKL final concentration 50 ng / mL) and add D-4, Iso-D-4, Me-D-4 and Me-Iso-D-4 solutions to the wells (The final concentrations of D-4, Iso-D-4, Me-D-4, and Me-Iso-D-4 were all 15, 50, and 150 μM). To the control wells, DM-4 containing no D-4, Iso-D-4, Me-D-4 and Me-Iso-D-4 was added.

After culturing at 37 ° C. for 3 days in the presence of 5% CO ₂ , tartrate-resistant acid phosphatase (TRAP) activity was measured as follows. After discarding the culture solution in each well of the 96-well microplate and washing this plate with phosphate buffered saline solution (PBS), each well was filled with approximately 3.7% formaldehyde-PBS, and the cells were fixed at room temperature for 15 minutes. . Furthermore, it fixed for 1 minute at room temperature using the ethanol-acetone solution (1: 1). After fixing, the fixing solution was discarded and dried at room temperature. After drying, measurement of the tartrate-resistant acid phosphatase activity of the cells in each well (hereinafter sometimes referred to as “TRAP assay”) and staining were performed.

  Measurement of TRAP activity in cultured cells was performed using 50 mM citrate buffer (pH 4.6) containing 3.7 mM p-nitrophenol-disodium hydrogen phosphate and 10 mM tartaric acid. 0.1 mL of this buffer was added to each well and reacted at 37 ° C. for 30 minutes. After 30 minutes, the reaction was stopped by adding 0.1 mL of 0.1 M NaOH, and the released p-nitrophenol was measured at a measurement wavelength of 405 nm using a spectrophotometer (manufactured by Dainippon Pharmaceutical Co., Ltd.). .

  The results of the osteoclast early differentiation inhibition test were expressed as a relative value (%) relative to the control group when the TRAP activity of the control group (control) was 100.

(3) Results
Mouse macrophages for D-4 and its isomer, Iso-D-4, as well as Me-D-4 and Me-Iso-D-4, which are methylated derivatives of phenolic hydroxyl groups of these compounds. The effect on osteoclast differentiation in RAW264 cells, a cell line, was evaluated by measuring the activity of tartrate-resistant acid phosphatase (TRAP), a marker enzyme of osteoclasts. As a result, TRAP activity was reduced by any compound (FIG. 10). This indicates that combretastatin D-4 and its derivatives suppress osteoclast differentiation.

<Example 7>
Tooth germ growth promoting action (1) Materials Dulbecco's phosphate buffered saline (hereinafter sometimes referred to as “D-PBS”) was purchased from Dainippon Pharmaceutical Co., Ltd. Affi-Gel agarose beads (Affigel heparin catalog number: 15306173) were purchased from BioRad. Five-day-old C57BL / 6 mice were purchased from Sankyo Lab Service Co., Ltd. Moreover, as a host mouse | mouth, C57BL / 6 mouse | mouth (female) 10-15 weeks old purchased from Sankyo Lab Service Co., Ltd. was used as a group.

(2) Preparation of D-4 impregnated agarose beads
Agarose beads impregnated with D-4 (hereinafter referred to as “D-4 impregnated beads”) were prepared as follows. D-4 was diluted with D-PBS to a concentration of 100 μM (final concentration). Affi-Gel agarose beads were washed with PBS. The washed agarose beads were placed in a 1.5 ml square bottom Eppendorf tube, and the D-4 solution diluted as described above was added in an amount of 100 μl. Agarose beads were impregnated with D-4 by incubating at room temperature for 30 minutes or more. The obtained D-4 impregnated agarose beads were used for implantation in the following dental pulp. Further, as a negative control, agarose beads prepared in the same manner as D-4 impregnated beads (hereinafter referred to as “D-PBS impregnated beads”) were used except that D-PBS containing no D-4 was impregnated. .

(3) Implantation in dental pulp Five-day-old C57BL / 6 mice immediately before the formation of root-periodontal tissue were decapitated after ether anesthesia. After removing the lower jaw, the lower first molar was removed together with the dental follicle. In the sample administration group, D-4 impregnated agarose beads were implanted in the pulp of the extracted tooth (mandibular first molar). In the control group, D-PBS-impregnated beads were implanted in the pulp of the extracted tooth (mandibular first molar).

(4) Transplant culture
The extracted teeth embedded with D-4 impregnated beads and the extracted teeth embedded with D-PBS impregnated beads as a negative control were transplanted and cultured under the kidney capsule of the host mouse. The host mice were lit for 12 hours and kept at room temperature of 25 ° C., and were allowed to freely take water and feed for 3 weeks. Three weeks after the transplantation of the above extracted teeth, the host mice were sacrificed by cervical dislocation, the kidneys were extracted, and the extracted teeth (transplanted teeth) after culture of each group were collected. Periodontal periodontal tissue formation (elongation), periodontal ligament, and bone in the collected transplanted teeth after observation were observed under a stereomicroscope and imaged with μCT. The observation results of the transplanted teeth in each group after the transplantation culture are shown in FIG.

(5) Results After collecting the first molars of 5-day-old mice, implanting agarose beads infiltrated with the compound, transplanting them under the kidney capsule of the host mice, raising them for 3 weeks, and then taking μCT images, The effect on growth was investigated. As a result, it was shown that in the transplanted teeth embedded with D-4 impregnated agarose beads, the tooth growth was remarkably advanced and the root elongation was promoted compared to the negative control (Fig. 11). . This indicates that D-4 promotes the growth and formation of teeth and alveolar bone.

<Summary>
Since D-4 and its derivatives promote early and late differentiation of osteoblasts and enhance mineralization, it is considered that D-4 and its derivatives have an effect of promoting osteogenesis. Moreover, the growth promotion effect | action of the tooth | gear and the alveolar bone was recognized. On the other hand, differentiation of osteoclasts that perform bone resorption was suppressed, and bone resorption suppression action was also observed. Therefore, it is useful for the prevention and treatment of osteopenia, rheumatoid arthritis, periodontal disease, Paget's disease, bone metastasis of cancer, etc. It is. It is also possible to induce and promote bone formation and bone regeneration in hard tissue (bone and teeth) reduction and defect sites due to fractures, accidents, surgery, and other factors. Furthermore, it can be used for prevention and regeneration of destruction of alveolar bone and periodontal tissue in periodontal disease. It is also useful for the production of these preventive and therapeutic cells.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Claims (8)

A pharmaceutical composition for preventing and / or treating osteopenic diseases, comprising a compound represented by the following chemical formula (I):
(In formula (I),
R1 is one functional group or 2 to 4 functional groups that are the same or different from each other, and is a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group,
(R2 and R3 are a hydrogen atom or a ketone group, and either R2 or R3 is a ketone group) 2. The pharmaceutical composition according to claim 1, wherein the compound represented by the chemical formula (I) is a compound selected from the group of the following chemical formulas (II) to (V).
A pharmaceutical preparation for regulating bone metabolism, comprising a compound represented by the following chemical formula (I):
(In formula (I),
R1 is one functional group or 2 to 4 functional groups that are the same or different from each other, and is a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group,
(R2 and R3 are a hydrogen atom or a ketone group, and either R2 or R3 is a ketone group)   A therapeutic agent for osteopenia, comprising the pharmaceutical preparation for bone metabolism regulation according to claim 3.   A biocompatible material containing the compound according to claim 1 or 2.   A functional food for preventing or ameliorating osteopenic diseases, comprising the compound according to claim 1 or 2.   A health food for preventing or ameliorating osteopenia, comprising the compound according to claim 1 or 2. A method for preventing and / or treating osteopenic disease, the method comprising:
A method comprising administering to a subject suffering from the osteopenic disease a pharmaceutical composition containing a compound represented by the following chemical formula (I) or a pharmaceutical preparation for regulating bone metabolism.
(In formula (I),
R1 is one functional group or 2 to 4 functional groups that are the same or different from each other, and is a functional group selected from the group consisting of a hydrogen atom, a hydroxyl group, and a methoxy group,
(R2 and R3 are a hydrogen atom or a ketone group, and either R2 or R3 is a ketone group)