JP2012072095A - Osteogenesis promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound that has effective osteogenesis promotion effects, reduced adverse effects and high safety as a medicine useful for treating or preventing various bone diseases.SOLUTION: The osteogenesis promoter comprises ursodeoxycholic acid, a conjugate or a pharmacologically acceptable salt thereof as an active ingredient. The osteogenesis promoter promotes differentiation of preosteoblasts into osteoblasts. Consequently the osteogenesis promoter is useful as a prophylactic and therapeutic agent for diseases including osteoporosis, periodontal disease and fracture that require osteogenesis promotion and reduce bone weight.

Description

  The present invention relates to an osteogenesis promoter containing ursodeoxycholic acid or a conjugate thereof or a pharmacologically acceptable salt thereof as an active ingredient, and to the prevention and treatment of bone loss diseases using the same.

  The bone tissue of a living body is kept homeostatic by the balance between osteoblasts involved in bone formation and osteoclasts that mediate bone resorption. Therefore, when bone tissue is damaged by a fracture or the like, the living body regenerates the bone tissue by normalizing osteoblasts and suppressing osteoclast activity, and restores the bone tissue to a normal state. ing. However, in bone loss diseases, the balance between the two is lost due to various causes such as aging and ovarian function, resulting in a decrease in bone mass and deterioration of bone tissue. The decrease in bone mass is 20 to 30% of the total bone mass, and it becomes easy to fracture. This can lead to bedridden, body deformation, and death depending on the fracture site such as hip fracture. is there.

  Osteoporosis, which is a typical bone loss disease, is a systemic disease in which the bone microstructure is destroyed due to a decrease in bone mass or bone mineral content, bone strength is reduced, and the risk of fracture is increased. In the case of osteoporosis patients, when a long-term bed rest is required due to a fracture, inactive bone atrophy is added, and the ease of fracture progresses more rapidly. Furthermore, it is frequently accompanied by medical complications such as dementia, becoming bedridden, and becoming a serious social and economic problem. Furthermore, since osteoporosis is a common disease after middle age, it is estimated that the number of patients will increase with the aging in recent years. In Japan, there are about 12 million osteoporosis patients, 80% of whom are women.

  Bone defects due to tumor removal such as osteosarcoma or complex fractures are incomplete or require a long healing period. In addition, a fracture is a disorder that can occur across generations due to various causes, and its healing requires a relatively long period of time. In Japan, the number of fractures is estimated at about 450,000 per year. Periodontal disease is said to be 80% over 30 years of age in Japan, of which about 6 million patients with severe periodontal disease are involved in the reconstruction of alveolar bone. There is a need for safe drugs that can be used.

  As methods for preventing and treating osteoporosis and other diseases in which bone mass decreases, (1) intake of calcium and calcium absorption promoters and (2) intake of bone metabolism improving agents are known. As calcium absorption promoters, various compounds such as twinose, fructooligosaccharides, casein phosphopeptides, calcium citrate malate, and vitamin D that enhances absorption of calcium from the intestinal tract are already known. In addition, as bone metabolism improving agents, there are known substances that suppress the activity of osteoclasts, bone resorption inhibitors that suppress bone resorption via osteoclasts, and bone formation promoters that activate osteogenesis by osteoblasts. It has been. Bone resorption inhibitors include bisphosphonic acid that binds to bone matrix and suppresses osteoclast function, selective estrogen receptor modulator (SERM), bone resorption by osteoclasts, and pain relief Certain calcitonin preparations, vitamin D and the like are already known. However, administration of hormones has a risk of causing cancer (especially breast cancer, uterine cancer, etc.) and is not a safe treatment.

  In addition to this, as a substance that suppresses osteoclasts and a substance that suppresses bone resorption via osteoclasts, ipriflavone, aminoalkyl-substituted phenyl derivatives, N-fluorocyclic alkyl-substituted phenyl derivatives, organic germanium compounds, Leukocyte activating protein factor, trehalose, β-alanyl-3,4-dihydroxyphenylalanine and the like are known.

  However, for bone diseases with increased bone resorption, although bone resorption inhibitors such as the above-mentioned bisphosphonic acid preparations have been mainly used so far, the progression of osteoporosis can be delayed, but once decreased It is difficult to regenerate the bone.

  For the above reasons, development of an osteogenesis promoter that can promote osteogenesis and increase the decreased bone mass has been promoted.

As osteogenesis promoters, there are active vitamin D ₃ and vitamin K _{2 in} addition to protein / peptide preparations such as parathyroid hormone. In recent years, as low molecular weight compounds, statin compounds also have osteogenesis promoting action. It has been reported.

  As protein preparations and peptide preparations, parathyroid hormone (PTH) preparations (teriparatide: a recombinant human PTH partial sequence, trade name “Forteo” (Eli Lilly)) were launched in 2002, and transforming growth factors In addition to BMP (bone morphogenic protein) -2, nerve growth factor (NGF), fibroblast growth factor (FGF), transforming growth factor (TGF-β) belonging to the β superfamily, prostate specific antigen (Prostate specific antigen: It is known that PSA) protein, adipocyte formation inhibitor ADIF, β-defensin and the like have a bone formation promoting action. However, these all need to be injectable preparations, and injections administered once a day are not desirable drugs from the viewpoint of patient QOL. Furthermore, when growth factors are used as osteogenesis promoters, there is a risk of side effects such as the proliferation of an unspecified large number of cells, so confirmation of safety and stability is insufficient, and osteoporosis prevention and improvement effects It seems difficult to obtain.

  For the above-mentioned reasons, a bone disease therapeutic agent having a high activity and having a low-molecular-weight osteogenesis promoting action that can be adapted to promote the repair of bone defects is strongly desired in the bone region. In recent years, statins known as HMG-CoA reductase inhibitors have also promoted BMP-2 production from osteoblasts as a low molecular weight compound having a low molecular weight osteogenesis promoting action. It has been reported that osteogenesis is promoted by enhancing the expression of osteocalcin, which is a matrix protein and an osteoblast differentiation marker (Patent Documents 1 and 2). In addition, PPAR inhibitors (Patent Document 3), phenolic amide compounds (Patent Document 4), pterocarpan (Patent Document 5) taxoid derivatives, catechins are included as drugs for activating osteogenesis by osteoblasts. Or a catechin mixture, a thiophene derivative, a phenolsulfophthalene derivative, a benzylphosphonic acid derivative, an N-quinolylanthranyl derivative, a thiazole compound, a benzothiepiene derivative, N- [2- (dimethylamino) ethyl] -4- (5-phenylpentyl) Amide compounds such as oxy) benzamide hydrochloride, phenyl-substituted hydroxycyclopentenone analogs and the like are known.

  However, these compounds having a bone formation-promoting action are not sufficiently satisfactory in their bone formation-promoting effect, and most of them are compounds that are not originally present in the living body. Therefore, the mechanism of action of these drugs is not based on physiological conditions, and is not always satisfactory from the viewpoint of effectiveness and safety after long-term use, and the use target is limited in terms of side effects. Some are done.

On the other hand, there are a plurality of drugs that are used in clinical practice as osteogenesis promoting substances that are in-vivo substances and low-molecular compounds (including derivatives thereof). Vitamin D ₃ derivatives (such as alphacalcidol) promote calcium absorption in the intestinal tract and stimulate osteoblasts, but are also believed to promote bone resorption because of their ability to promote osteoclast differentiation. Although vitamin K ₂ cannot be expected to increase bone mass, it has been reported to help improve bone quality during bone formation (make strong bones) and reduce the fracture rate. The discussion is divided.

  In the treatment of metabolic diseases, multi-drug combination therapy has become mainstream, and therefore, development of a new drug having a mechanism of action different from conventional drugs is expected. Therefore, from the viewpoint of safety, it is possible that long-term administration is possible with a substance present in the living body, more desirably a substance that has already been used for long-term administration for the purpose of preventive treatment for other diseases, From the viewpoint of the administration method, it is required to add a compound that satisfies the condition that it is a drug that can be administered orally rather than an injection to the option of an osteogenesis promoter.

  In addition, there is a need for a safe pharmaceutical that satisfies the above conditions in promoting bone formation when the alveolar bone is melted by periodontal disease bacteria or when the alveolar bone needs to be regenerated.

  Various bone formation markers are used in screening for candidate compounds having an osteogenesis promoting action. Osteoblasts are osteogenic cells made of mesenchymal tissue, and are differentiated from undifferentiated mesenchymal cells into precursor osteoblasts and young osteoblasts into mature osteoblasts. The bone matrix is formed from osteoblasts, which are encapsulated in the bone matrix as bone cells. Type I collagen, alkaline phosphatase (ALP), and osteonectin are produced between the precursor cell stage and the mature cell stage, and osteopontin when differentiated to young osteoblasts, bone sialoprotein (BSP) when differentiated to mature cells, Osteocalcin (BGP) begins to be produced (Non-Patent Document 1). The level and activity of these proteins produced during the differentiation process of osteoblasts can be used as an index for promoting or suppressing bone formation. Actually, intracellular alkaline phosphatase activity is used as an index for screening a candidate for an osteogenesis promoting substance using an osteoblast culture (Patent Documents 4 and 5).

  Ursodeoxycholic acid, which is an in vivo substance (hereinafter, ursodeoxycholic acid may be referred to as “UDCA”) is an animal herbal medicine that has been prized for improving digestive symptoms in Japan. Kuma bile acid discovered and identified as a medicinal main ingredient of bear gall (Yutan), which has a history of several hundred years, is widely used as a bile acid preparation for chronic hepatitis such as cholestatic liver disease. In Japan, it has been on the market since 1962 and is now popular as a generic drug. It is also widely used overseas. In Western countries, it is effective for cholestatic liver disease and chronic hepatitis. Recently, it is useful for single or combined therapy with interferon for chronic hepatitis C. Clinical utility such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), which are autoimmune diseases recognized as intractable diseases, has been reported.

  As the pharmacological action of UDCA, (1) Biliary action: Improve cholestasis by promoting chole secretion (biliary action) (Non-patent document 2), (2) Hepatocellular protective action: in the liver , By the action of substitution of endogenous hydrophobic bile acids such as chenodeoxycholic acid (CDCA) or deoxycholic acid (DCA) with strong cytotoxicity to UDCA, the relative ratio of UDCA is increased, and hepatocellular damage of hydrophobic bile acids To reduce the action (replacement effect) and to exert hepatocellular protective action (Non-patent Document 3), (3) Inflammation-inhibiting action: UDCA suppresses the production of cytokines / chemokines and suppresses infiltration of inflammatory cells into the liver. Improve function (Patent Document 6), (4) Anti-apoptotic action (Non-Patent Document 4), (5) Gallstone dissolution action: Cholesterol gallstones, cholesterol desaturation in gallbladder bile (Non-patent document 5), Cholesterol solubilization by the formation of liquid crystals (Non-patent document 6) and cholesterol absorption suppression in the intestinal tract (Non-patent document 7), (6) Promotion of pancreatic juice secretion (non-patent document) Document 8), (7) Anti-endotoxin action (Non-patent document 9), (8) Colon cancer inhibitory action (Non-patent document 10), (9) Endothelin secretion inhibitory action (Patent document 7), (10) Suddenly A therapeutic effect on thrombocytopenic purpura is known (Patent Document 8).

  In addition, it is known that UDCA has an action to improve digestion and absorption of fat and fat-soluble vitamins. Small bowel disease and indigestion after resection of the small intestine are thought to occur due to a decrease in the amount of micelles as a result of a decrease in the amount of bile acids, and administration of UDCA increases the amount of bile acids in a supplemental manner with bile acids. It is considered that dyspepsia is improved by bringing the forming ability closer to a normal state (Non-patent Document 11). Currently available calcium preparations include vitamin D and UDCA in combination. In this case, UDCA is expected to improve the digestion and absorption of fat and fat-soluble vitamins in UDCA. Vitamin D that elevates blood is emulsified with bile acids to form composite micelles and become water soluble, and is intended to promote the absorption of calcium with fat from the small intestine, and bone formation in UDCA itself It is not administered with the expectation of promoting ability.

  As described above, there are many reports on UDCA, and in recent years a review of its pharmacological action has been reported (Non-patent Document 12), but there is no report on UDCA having a bone formation promoting action.

WO98 / 25460 JP 2002-370982 A JP 2009-173567 A JP 2005-247748 A JP 2003-155236 A JP 2007-106730 A JP 2004-35503 A JP 2008-280206 A

H. Komori, The Bone 12: 49-59, 1998 Dumont, M .; et al. : Gastroenterology, 79, 82 (1980) Tsuneo Kimura: Japanese Society of Gastroenterology, 77, 185 (1980) Hirano Shirin et al., Hepatobiliary pancreas 43 (6) 1029 (2001) Fumio Hamada et al .: Japanese Society of Gastroenterology, 75, 492 (1978) Igimi, H .; et al. : Gastroenterologia Japan, 18, 93 (1983) To Horiuchi: Biliary tract, 2,239 (1988) Yasuhiro Hara et al .: Fukuoka Medical Journal, 65, 933 (1974) A. Kobayashi et al .: Bile and pancreas, 3,755 (1982) Earnest DL. et al. Cancer Res 54: 5071-5074 (1994) Shuji Tsuchiya et al .: Ministry of Health and Welfare Specific Disease Digestion and Absorption Disorder Investigation Research Group Festi D. et al. Curr Clin Pharmacol. 2007 May; 2 (2): 155-77

  An object of the present invention is to provide a highly safe compound having an effective osteogenesis-promoting effect and having reduced side effects as a useful pharmaceutical for treating or preventing various bone diseases.

  As a result of intensive studies to solve the above-described problems, the present inventors have found that ursodeoxycholic acid has an osteoblast differentiation promoting action, and have completed the present invention.

According to the present invention, the following [1] to [10] are provided. That is,
[1] An osteogenesis promoter containing ursodeoxycholic acid or a conjugate thereof or a pharmacologically acceptable salt thereof as an active ingredient.
[2] A therapeutic and / or prophylactic agent for bone mass-lowering diseases comprising the osteogenesis promoter according to [1] above.

[3] The therapeutic and / or prophylactic agent according to the above [2], wherein the bone mass-lowering disease is primary osteoporosis.
[4] The therapeutic and / or prophylactic agent according to the above [3], wherein the primary osteoporosis is primary osteoporosis associated with aging, primary osteoporosis associated with menopause, and primary osteoporosis associated with ovariectomy.

[5] The therapeutic and / or prophylactic agent according to the above [2], wherein the bone loss disease is secondary osteoporosis.
[6] Secondary osteoporosis is associated with glucocorticoid-induced osteoporosis, hyperthyroid osteoporosis, fixation-induced osteoporosis, heparin-induced osteoporosis, immunosuppression-induced osteoporosis, osteoporosis due to renal failure, inflammatory osteoporosis, Cushing syndrome The therapeutic and / or prophylactic agent according to [5] above, which is osteoporosis and rheumatic osteoporosis.

[7] The therapeutic and / or prophylactic agent according to the above [2], wherein the bone mass lowering disease is cancer bone metastasis, hypercalcemia, Paget's disease, bone defect, and osteonecrosis.
[8] The therapeutic and / or prophylactic agent according to the above [2], wherein the bone loss disease is an alveolar bone defect, a mandibular bone defect, or a childhood sudden bone defect.

[9] Treatment of bone loss disease includes bone formation after fracture, bone formation after bone transplantation, bone formation after artificial joint surgery, bone formation after spinal fusion, bone formation after bone reconstruction, and bone The therapeutic agent according to [2] above, which is an alternative transplantation therapy.
[10] A method for treating and / or preventing a bone-lowering disease, comprising administering a therapeutically effective amount of the osteogenesis promoter according to [1] to a mammal. It is preferable to exclude humans from the target mammal.

  As will be described later, it was revealed that ursodeoxycholic acid has an activity of promoting osteoblast differentiation, that is, an action of promoting bone formation. Therefore, this bone formation promoting action of ursodeoxycholic acid is useful for the prevention and treatment of bone loss diseases. Hereinafter, the present invention will be described in detail.

It is a graph which shows the alkaline phosphatase activity of an osteoblast culture.

  Ursodeoxycholic acid (UDCA) is a known compound represented by the following formula 1, and is a kind of bile acid present in the living body.

  The osteogenesis promoting activity of UDCA was determined by adding the compound to the cultured osteoblastic cell line MC3T3-E1 at a concentration in the range of 0.1 to 100 μM and then culturing it for 4-6 days. Using alkaline phosphatase (ALP) activity, which is a cell differentiation index, as an index of bone formation (Patent Document 4), measurement is performed according to the method of Lowry et al. (J. Biol. Chem., 193, 265-273, 1951). Can do. The higher this ALP activity is compared to the control without the compound, the higher the osteogenesis promoting activity of the compound.

  Alternatively, UDCA was raised to experimental animals such as rats (including pathological model animals such as ovarioskeletal osteoporosis model) for 4 weeks or longer by administration of gastric sonde or feeding of test feed, and the animal's femur (bone trunk and / or By measuring the amount of calcium and the alkaline phosphatase activity, the bone formation promoting effect can be measured by measuring the amount of calcium and the alkaline phosphatase activity. The amount of calcium in the bone tissue was determined by washing the bone tissue with a 0.25M sucrose solution and drying at 100 ° C. for 6 hours, measuring the dry weight, adding concentrated nitric acid and decomposing at 120 ° C. for 24 hours. The amount is quantified with an atomic absorption photometer. Alkaline phosphatase activity was determined by washing bone tissue with a cold 0.25M sucrose solution, crushing in 6.5 mM barbital buffer (pH 7.4), sonicating for 60 seconds, centrifuging at 3000 rpm, and supernatant fraction. Is measured by a known method (Methods of Enzymatic Analysis, Vol. 1-2, Academic Press, New York, pp 856-860, 1965) or a commercially available ALP activity measurement kit.

  In addition to measurement of bone density, bone fracture strength, and bone morphology measurement, serum calcium concentration (calcium C-Test Wako, Wako Pure Chemicals), serum alkaline phosphatase concentration (alkaline phosphat B-test Wako, Wako Pure Chemicals), serum osteocalcin concentration (calcitonin kit, Daiichi Radioisotope Laboratories), serum parathyroid hormone concentration (Rat PTH (IRMA) kit, Japan Mediphysix), urinary calcium excretion (calcium C-test Wako, sum Any of bone metabolic markers, such as light pure drug and creatinine-Test Wako, Wako Pure Chemical), urinary hydroxypurine, pyridinoline and deoxypyridinoline excretion (clinical chemistry 21, 18-25, 1992 according to the method of Sekine et al.) By simultaneously measuring these, U It is also possible to additionally validate the osteogenesis promoting action of the CA.

  In the examples described later, ALP activity was measured when UDCA was added in an assay system using osteoblastic cell line MC3T3-E1, and the results were shown. UDCA was significantly more effective than the control without addition. It was revealed that alkaline phosphatase activity was increased in a dependent manner. From this result, it became clear that UDCA has an activity of promoting osteoblast differentiation, that is, an action of promoting bone formation.

In the present invention, ursodeoxycholic acid (UDCA)) has a bone formation-promoting action, so that bone loss diseases such as
1) Primary osteoporosis (eg, primary osteoporosis associated with aging, primary osteoporosis associated with menopause, primary osteoporosis associated with ovariectomy, etc.),
2) Secondary osteoporosis (eg, associated with glucocorticoid-induced osteoporosis, hyperthyroid osteoporosis, fixation-induced osteoporosis, heparin-induced osteoporosis, immunosuppression-induced osteoporosis, osteoporosis due to renal failure, inflammatory osteoporosis, Cushing syndrome Osteoporosis, rheumatic osteoporosis, etc.),
3) Useful for prevention and / or treatment of bone diseases such as cancer bone metastasis, hypercalcemia, Paget's disease, bone defects (alveolar bone defects, mandible bone defects, childhood sudden bone defects, etc.), osteonecrosis, etc. In addition to bone formation after bone surgery (eg, bone formation after fracture, bone formation after bone transplantation, bone formation after artificial joint surgery, bone formation after spinal fusion, other bone reconstruction It is considered useful as an agent for promoting / healing bone formation and the like, and as an alternative therapy for bone grafting.

  Ursodeoxycholic acid conjugates include taurine conjugates and glycine conjugates. The conjugate of ursodeoxycholic acid can be obtained by a known method. Further, the pharmacologically acceptable salt of ursodeoxycholic acid is not limited, but alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt, magnesium salt and barium salt, trimethylamine, Examples thereof include salts with organic bases such as triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine and dicyclohexylamine.

  The type of pharmaceutical additive used for preparing the pharmaceutical composition is not particularly limited, and it is possible to select an appropriate pharmaceutical additive depending on the form of various pharmaceutical compositions. The pharmaceutical additive may be either solid or liquid, and for example, a solid carrier or a liquid carrier can be used. As an example of the solid carrier, a normal gelatin type capsule can be used. Also, for example, the active ingredient can be tableted with or without one or more pharmaceutical additives, or can be prepared and packaged as a powder. These capsules, tablets, and powders can generally contain 5 to 95% by weight, preferably 5 to 90% by weight, of the active ingredient relative to the total weight of the preparation, and the dosage unit form is 5 to 500 mg. Preferably it contains 25 to 250 mg of active ingredient. As the liquid carrier, water, oils of animal or vegetable origin such as petroleum, peanut oil, soybean oil, mineral oil, sesame oil or synthetic oils are used. In general, physiological saline, dextrol or similar sucrose solution, glycols such as ethylene glycol, propylene glycol, polyethylene glycol and the like are preferable as the liquid carrier, and in the case of an injection solution using physiological saline, it is usually 0.00. It can be prepared to contain 5 to 20%, preferably 1 to 10% by weight of the active ingredient.

The agent of the present invention can also be used in combination with other therapeutic agents for bone diseases. Examples of drugs to be combined include calcitonin preparations (eg, eel calcitonin, salmon calcitonin, porcine calcitonin, abicatonin, etc.), vitamin D ₃ types (eg, 1α-hydroxyvitamin D ₃ , 1α, 25-dihydroxyvitamin D ₃ , Floccitriol, cecalciferol, alpha-cascirol, eldecalcitol, etc.), sex hormone-related compounds (eg, tibolone, estrogen, estradiol, osaterone, mifebriston, etc.), selective estrogen receptor modulators (eg, raloxifene, bazedoxifene) , Loxifene, droloxifene, olmeroxifene, tamoxifen, etc.), prostaglandins (eg, A1, E1, E2), bisphosphonic acids (eg, simadronate, etidronate, clodro Over DOO, pamidronate, neridronate, alendronate, olpadronate, ibandronate, tiludronate, risedronate, minodronate, incadronate, zoledronate, etc.), ipriflavone acids, fluorine compounds (e.g., sodium fluoride, etc.), vitamin K _2, bone Formation inducer (BMP), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), transforming growth factor (TGF-β), insulin-like growth factor-1 and 2 (IGF-1, -2) Parathyroid hormones (PTH) (eg, PTH (1-34), PTH (1-84), PTH (1-36), etc.), HMG-CoA reductase inhibitors (eg, pitavastatin, atorvastatin, simvastatin, Cerivastatin, Pravastatin, Fluvastatin, Robusta Down, lovastatin and the like).

  The administration period of the drug of the present invention is basically a period in which bone lesions, disorders, and defects are observed clinically, and can be continued even after recovery according to the judgment of the clinician depending on the etiology and pathology. . Furthermore, when the onset of secondary osteoporosis is expected as in adrenal steroid therapy, the agent of the present invention can be administered prophylactically.

  The dosage when the preventive and / or therapeutic agent for bone-related diseases of the present invention is used as a pharmaceutical agent varies depending on the type of disease, administration method, disease state, patient age, etc. It is 30 to 3000 mg orally, preferably 150 to 1200 mg orally, and 5 to 400 mg, preferably 50 to 200 mg intravenously, and can be administered divided into 1 to 6 times, preferably 1 to 3 times. When the additives for preparation used for preparing conjugates, salts, and pharmaceutical compositions are changed from existing ursodeoxychol preparations, they can be administered according to the above weights, and the active ingredient of the carrier used can be released slowly. What is necessary is just to change a compounding quantity suitably with property.

  Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to these examples.

Evaluation of bone formation promoting effect of ursodeoxycholic acid The effect of ursodeoxycholic acid on intracellular alkaline phosphatase activity in osteoblastic cell lines was measured as follows.

Seed in 1 ml of alpha MEM (Flow laboratories) medium (10% calf neonatal serum; ICN Biomedicals included) prepared in 24-well plate at 1 × 10 ⁵ cells / ml of osteoblastic cell line MC3T3-E1 cells. The cells were cultured at 37 ° C. and 5% CO ₂ for 48 hours. After the medium was removed, the same medium was newly added, and ursodeoxycholic acid was further added to the medium at a concentration of 0.1 to 100 μM. This was cultured in a carbon dioxide culture apparatus, and alkaline phosphatase activity of cultured cells 48 hours after the addition of the drug was measured by the following method.

  150 μl of a cell lysate (Dual-Glo (registered trademark) Luciferase Reagent, Promega) was added to the medium containing the above cells, allowed to stand at room temperature for 10 minutes, transferred to a 1.5 ml microtube, 4 ° C., 15, Centrifugation was performed at 000 rpm for 5 minutes. Transfer 40 μl of the supernatant to a 96-well microplate, mix 160 μl of chemiluminescence reagent Lumi-Phos Plus (manufactured by Lumigen) for alkaline phosphatase activity measurement, shield from light, and incubate at 37 ° C. for 20 minutes, then luminometer Luminoskan The amount of luminescence for 5 seconds was measured using RS (manufactured by Labsystems). FIG. 1 shows the activity of cells to which ursodeoxycholic acid was added, using ursodeoxycholic acid-free cells as a control. Statistical processing was performed by Student's t-test. As ursodeoxycholic acid used above, ursodeoxycholic acid / sodium salt manufactured by CALBIOCHEM was used. This revealed that ursodeoxycholic acid increased alkaline phosphatase activity in a dose-dependent manner.

  All publications and patent documents cited herein are hereby incorporated by reference in their entirety. While specific embodiments of the invention have been described herein for purposes of illustration, it will be apparent to those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.

Claims (10)

  An osteogenesis promoter containing ursodeoxycholic acid or a conjugate thereof or a pharmacologically acceptable salt thereof as an active ingredient.   A therapeutic and / or prophylactic agent for bone loss diseases comprising the osteogenesis promoter according to claim 1.   The therapeutic and / or prophylactic agent according to claim 2, wherein the bone loss disease is primary osteoporosis.   The therapeutic and / or preventive agent according to claim 3, wherein the primary osteoporosis is primary osteoporosis associated with aging, primary osteoporosis associated with menopause, or primary osteoporosis associated with ovariectomy.   The therapeutic and / or prophylactic agent according to claim 2, wherein the bone loss disease is secondary osteoporosis.   Secondary osteoporosis is glucocorticoid-induced osteoporosis, hyperthyroid osteoporosis, fixation-induced osteoporosis, heparin-induced osteoporosis, immunosuppression-induced osteoporosis, osteoporosis due to renal failure, inflammatory osteoporosis, osteoporosis associated with Cushing syndrome, or The therapeutic and / or prophylactic agent according to claim 5, which is rheumatic osteoporosis.   The therapeutic and / or prophylactic agent according to claim 2, wherein the bone loss disease is cancer bone metastasis, hypercalcemia, Paget's disease, bone defect, or osteonecrosis.   The therapeutic and / or prophylactic agent according to claim 2, wherein the bone loss disease is an alveolar bone defect, a mandible bone defect, or a childhood sudden bone defect.   Treatment of bone loss disease is bone formation after fracture, bone formation after bone transplantation, bone formation after artificial joint surgery, bone formation after spinal fusion, bone formation after bone reconstruction, or bone graft replacement therapy The therapeutic agent according to claim 2, wherein   A method for treating and / or preventing a bone-lowering disease, comprising administering a therapeutically effective amount of the osteogenesis promoter according to claim 1 to a mammal (excluding humans).

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