KR101508041B1 - A pharmaceutical composition for preventing or treating bone metabolic disease comprising dibenzazepine - Google Patents

Abstract

The present invention relates to pharmaceutical compositions and food compositions comprising dibenzazepine. To a composition for use in the prevention or treatment of bone metabolic diseases, particularly osteoporosis, periodontal disease, fracture, or Paget's disease.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating bone metabolic diseases including dibenzazepine,

The present invention relates to pharmaceutical compositions and food compositions comprising dibenzazepine. To a composition for use in the prevention or treatment of bone metabolic diseases, particularly osteoporosis, periodontal disease, fracture, or Paget's disease.

Bone modeling and remodeling plays an important role in bone development, growth, and metabolism. The bone formation starts from the birth and continues until the young adult years, when the skeleton matures and the growth ends, forming the maximum bone mass from the 20s to the early 30s. After about 30 years, the bone is removed and the bone is replenished again. The bone formation and the bone absorption are balanced with each other. After this period, bone loss due to bone resorption can not be fully followed by bone formation, resulting in bone loss of about 0.3 ~ 0.5% per year. Especially in women, Loss.

Bone tissue constitutes the cartilage and skeletal system. It plays a role of support and muscle attachment by mechanical function, protects organism and bone marrow, and preserve calcium and phosphorus to maintain homeostasis. Bone tissue consists of various types of cells such as collagen, glycoprotein, and osteoblast, osteoclast, and osteocyte. Osteoblasts derived from bone marrow stromal cells play a major role in osteogenesis. Osteoclasts derived from hematopoietic stem cells are destroyed and are responsible for the absorption of aged bone, and osteoblasts and osteoclasts And thus remodeling of the bone is maintained. However, excessive activity of osteoclasts or depletion of osteoblasts leads to imbalance in the remodeling process of osteogenesis, resulting in the destruction of osteoclasts and osteoblasts, resulting in osteoporosis.

As a representative example of osteoporosis, osteoporosis is different in degree but it is an unavoidable symptom for the elderly, especially postmenopausal women. In developed countries, as the population ages, interest in osteoporosis and its therapeutic agent is gradually increasing. In addition, it is known that there is a market of about $ 130 billion related to bone disease treatment around the world, and since it is expected to increase further in the future, each research institute and pharmaceutical company in the world invests heavily in the development of bone disease treatments have. In Korea, the average life span is approaching 80 years, and the prevalence of osteoporosis is rapidly increasing. According to recent studies conducted by local residents, 4.5% of men and 19.8% Have been reported. This suggests that osteoporosis is a more common health problem than diabetes or cardiovascular disease and that osteoporosis is a very important health problem when estimating the cost of suffering or treatment for patients receiving fractures.

Several substances have been developed to treat osteoporosis. Estrogen, the most commonly used drug for osteoporosis, has not yet been proven its practical efficacy. It has the disadvantage of continuing to take it during its lifetime, and there is a side effect of increasing breast or uterine cancer when administered over a long period of time. Alrendronate is also less effective, has less absorption in the digestive tract, and causes inflammation in the stomach and esophageal mucosa. Calcium preparations are known to have good efficacy with fewer side effects, but they are more preventive than treatments. In addition, vitamin D preparations such as calcitonin are known, but studies on efficacy and side effects have not yet been conducted. Accordingly, there is a need for a novel therapeutic agent for metabolic bone disease, which has few side effects and is effective.

The periodontal disease is divided into gingivitis and periodontal disease according to the inflammatory state of the tooth supporting tissue. The progressive periodontitis causes tooth loss. It is reported that the periodontal disease accounts for 40% Tooth loss due to periodontitis is a serious problem.

The destruction of the periodontal ligament and alveolar bone in periodontal disease is due to the degradation of collagen, the main component of these tissues. Although the precise mechanism is not yet known, collagen degradation is known to be caused by neutral proteases secreted from host-secreted collagenase and periodontal disease-causing bacteria. Therefore, studies have been made to inhibit the progression of periodontal disease by inhibiting the activities of collagenase and protease. Treatment of periodontal disease has been based on the establishment of improved oral hygiene in patients, non-surgical or surgical calculus removal, root resorption, gingival curettage, and regeneration of periodontal tissue using neoadjuvant. However, these surgical treatment methods are difficult to effectively treat due to the hassle of going to the dentist for treatment, and they are limited to the treatment that is performed when the disease has progressed to some extent rather than the prevention of the disease. .

The use of systemic antibiotics and local controlled release agents have been used as adjunctive therapies. However, there has been reported a case in which periodontal disease bacteria, which are resistant to antibiotics recently used, There is a serious problem. In order to overcome the limitations of such surgical treatment and the problems of using antibiotics, the use of oral compositions such as dentifrices and mouthwashes containing anti-bacterial, anti-plaque agents, and the like are effective in the prevention and treatment of bacterial oral diseases such as dental caries and periodontal diseases .

Fracture is a state of fracture of the bone. Fracture treatment takes a considerable period of time. Fracture of osteoporosis patients, which is one of the pathological fractures due to aging of the population, is significantly increased. Therefore, calcium, estrogen, calcitonin The use of calcitonin, active vitamin D, and bisphosphonate preparations in the treatment of fractures was expected to reduce the risk of fractures by inhibiting bone loss, I did not. This is because the mechanism of osteoporosis is a genetic or constitutional factor, and the bone equilibrium state continues to have delicate skeletal qualities. The bone is absorbed at a normal rate, but the bone formation is slow and the bone is formed at a normal rate This is because the osteoporosis and fracture are completely different from each other, and it can not be inferred that the fracture can be treated by osteoporosis treatment.

Because of the difference in the mechanism of osteoporosis and fracture as described above, agents that inhibit bone resorption and have the effect of treating osteoporosis may also inhibit bone formation, thereby delaying the treatment of fracture. For example, Long-term continuous administration of incadronate disodium, a bisphosphonate preparation, has been reported to delay the healing of fractures of the femur of rats (Li C et al., J. Bone Miner Res. 2001 Mar; 16 (3): 429-36), pretreatment of incadronate did not affect fracture healing at 16 weeks after fracture, whereas continuous incadonate treatment increased callus, but delayed remodeling during fracture healing (Li J et al., J. Bone Miner Res. 1999 Jun; 14 (6): 969-79). In addition, bFGF, a bone-forming biomarker known to be highly related to osteoporosis, has not been associated with fracture healing (Xu et al., Chin. J. Traumatol. 6, 160 ~ 166, 2003).

Accordingly, there is a limit to the above-mentioned reason for using osteoporosis therapeutic agents in order to effectively promote healing to a normal state at the time of occurrence of general fractures, and development of medicines having unique fracture healing effects regardless of osteoporosis is desperately required.

Accordingly, the inventors of the present invention conducted a study on a composition containing dibenzepin, which inhibits osteoblast differentiation and osteoclast differentiation, which are indispensable for osteogenesis, fracture recovery, tooth growth and development, And at the same time, promotes osteoblast osteogenesis. The present invention has been accomplished by confirming that a composition containing dibenzepine can be used for treating osteoporosis as well as fractures requiring bone formation promotion.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of a bone metabolic disease comprising dibenzazepine or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for treating a bone metabolic disease comprising administering the pharmaceutical composition to a subject.

It is another object of the present invention to provide a food composition for preventing or ameliorating a metabolic disease comprising dibenzazepine or a pharmaceutically acceptable salt thereof.

In one aspect of the present invention, the present invention relates to a composition for preventing or treating bone metabolic diseases comprising dibenzazepine or a pharmaceutically acceptable salt thereof.

The term "dibenzazepine" in the present invention refers to (S, S) -2- [2- (3,5- difluorophenyl) acetylamino] Dibenzo [b, d] azepin-7-yl] propionamide ((S, S) -2- [2- (3,5- difluorophenyl) acetylamino] -N- Dihydro-5H-dibenzo [b, d] azepin-7-yl] propionamide), the molecular formula is C ₂₆ H ₂₃ F ₂ N ₃ O ₃ and the molecular weight is 525.7 (g / mol).

Although dibenzazepine is known as a tricyclic antidepressant associated with imipramine, there is no known application for osteoclast inhibition of bone resorption and promotion of osteoblast osteogenesis.

In one embodiment of the present invention, the dibenzepine directly inhibits the activation of the gamma-secretase complex to reduce the activation of the markers involved in osteoclast differentiation and activation, activate the core genes involved in osteoblast differentiation and activation, It was confirmed that the stimulation can induce new bone formation and regeneration at a site of bone destruction with a remarkable decrease in bone density and can be used as a therapeutic agent for diseases requiring bone regeneration, particularly bone regeneration-related diseases .

As the active ingredient of the pharmaceutical composition of the present invention, it can be used in the form of the above-mentioned dibenzepine or a pharmaceutically acceptable salt thereof. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful Do. Acid addition salts can be prepared in a conventional manner, for example, by dissolving the compound in an excess amount of an aqueous acid solution and precipitating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration. As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid and hydroiodic acid may be used. It is not limited.

In addition, bases can be used to make pharmaceutically acceptable metal salts. Alkal

The metalloid or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, as the metal salt, it is preferable to produce sodium, potassium or calcium salt particularly, but not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The pharmaceutically acceptable salts of the above-mentioned dibenzepine substantially include salts of acidic or basic groups which may exist in the compound of the dibenzepine, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, (Mesylate), p-toluenesulfonate (tosylate) salts, and the like, which are known in the art and which can be prepared by methods known to those skilled in the art, such as hydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate .

The dibenzepin of the present invention also includes derivatives thereof. The "derivative" means a compound prepared by chemically changing a part of the dibenzphin, for example, introducing, substituting or eliminating a functional group to the extent that the therapeutic activity of the metabolic disease of the dibenzepine remains unchanged. But are not limited to, for example, amitriptyline, desmethylimipramine, nortriptyline, protriptyline, and the like. In addition, the composition of the present invention may further include an agent for treating osteoporosis, fracture treatment, and periodontal disease other than dibenzepin.

The composition of the present invention is characterized by inhibiting osteoclast bone resorption and simultaneously promoting bone formation of osteoblasts.

The term "osteoclast" in the present invention refers to a cell derived from macrophage precursor. Osteoclast precursor cells are macrophage colony stimulating factor (M-CSF), NF -KB (RANKL), etc., to form osteoclasts and to form multinucleated osteoclasts through fusion. The osteoclasts bind to the bone through αvβ3 integrin and the like to form an acidic environment while releasing various collagenase and protease to cause bone resorption. The osteoclasts do not proliferate to completely differentiated cells, and when they reach a shelf life of about 2 weeks, they cause apotosis.

In one embodiment of the present invention, the composition of the present invention was administered to a co-culture medium of bone marrow cells differentiated into osteoclasts and primary osteoblasts, followed by TRAP (tarrateresistant acid phosphate) as an osteoclast differentiation marker. As a result, TRAP activity In a concentration-dependent manner (Example 1, Fig. 1). As described above, the composition of the present invention effectively inhibits osteoclast differentiation through inhibition of TRAP activity, so that it is possible to prevent or treat bone metabolic diseases such as osteoporosis by increasing the activity of osteoclasts.

In addition, the composition of the present invention is characterized by promoting osteoblast differentiation or activity.

The term "osteoblast" in the present invention is used to increase bone density by making bone from a cell differentiated from mesenchymal stem cells. Sometimes, when the osteoblast activity is excessively increased, Resulting in bone malformation and bone metastasis.

It was confirmed that the activity of Alizarin red S, which is a differentiation marker in the early stage of osteoblast differentiation, was remarkably increased by treating the osteoblast with a composition containing dibenzepine in one embodiment of the present invention 4, Fig. 7). In addition, it was confirmed that the bone regeneration rate was significantly increased as a result of implanting the dibenzepine of the present invention into the defect site in the bone defect of the skull region (Example 5, Fig. 8). These results not only demonstrate that the composition of the present invention can induce osteoblast differentiation at the cell level, but also suggest that the bone mineral density is increased in actual animals. Thus, Thereby promoting the prevention or treatment of bone metabolic diseases such as osteoporosis.

Accordingly, the composition of the present invention can be used for preventing or treating bone metabolic diseases, and particularly, for bone related diseases caused by imbalance of osteoblasts and osteoclasts. Examples of such diseases include diseases that promote bone destruction due to pathological bone diseases such as osteoporosis, periodontal disease, fracture or Paget disease caused by excessive osteoclast bone resorption , But is not limited to the above-mentioned bone metabolic diseases.

The term " prevention or treatment of a bone metabolic disease " in the present invention includes prevention and complete or partial treatment of the above-mentioned bone metabolic diseases, particularly osteoporosis, periodontal disease, fracture and Paget's disease. It also includes reduction or amelioration of the symptoms of bone metabolic disease, alleviation of the symptoms of pain, reduction in the incidence of bone metabolic diseases or any other changes in the patient that increase the outcome of the treatment.

The above-mentioned "osteoporosis" is a state in which the lime of the bony tissue is reduced and the density of the bone is thinned and thereby the bone marrow cavity is widened. Since the bone is weakened as the symptoms progress, . The bone mass is affected by various factors such as genetic factors, nutritional intake, hormonal changes, exercise and lifestyle differences, and the causes of osteoporosis include age, lack of exercise, low birth weight, smoking, low calcium diet, Ovariectomy and the like are known. On the other hand, there are individual differences, but blacks have a lower bone resorption level than blacks, and bone mass is higher. In general, the bone mass is highest at 14-18 years old and decreases about 1% per year at old age. Especially in women, bone reduction continues after 30 years of age, and bone turnover is rapidly progressed by hormonal changes in menopause. In other words, estrogen concentration rapidly decreases in the menopausal period, and B-lymphocyte is produced as much as IL-7 (interleukin-7), and B cell precursor (pre-B Cells accumulate, which increases the amount of IL-6, which increases the activity of osteoclasts, leading to a decrease in bone mass.

The term "periodontal disease" refers to inflammatory conditions of the tooth supporting tissues caused by bacteria, which can be separated into gingivitis and periodontitis. The cause of the disease is the formation of a bacterial membrane at the oral cavity caused by a poor oral hygiene condition. Tooth surface bacteria is a bacterial mass that grows after the bacteria stick to the surface of the teeth by using a sticky substance in the needle as an adhesive. If you do not leave the bacterial membrane in the tooth, inflammation occurs, sometimes gum bleeding, bad breath, and sometimes the symptoms of gingivitis is called. As the gingivitis further progresses, the gap between the tooth and the gum becomes deeper, resulting in a periodontal pouch, where bacteria that cause periodontal disease multiply and periodontitis occurs. As periodontitis progresses, mild irritation, such as brushing, may cause bleeding from the gums and swelling, often causing acute inflammation and pain. Such inflammation lowers the function of making bone and increases the action of absorbing bone, so that the alveolar bone is gradually lowered, causing the alveolar bone to be destroyed and eventually to lose teeth.

The "fracture" refers to fracture of the bone in a state where the continuity of the bone, alveolar plate, or joint surface is abnormally broken. Causes of fractures include trauma such as traffic accidents, safety accidents caused by industrial disorders, changes in bones due to diseases such as osteoporosis, bone cancer, metabolic diseases, and repeated bones due to sports or loads. In addition, fracture status can be classified as fracture, greenstick fracture, transverse fracture, involuntary fracture, spiral fracture, fracture fracture, fractured fracture, and avulsion fracture based on fracture line (line along the bone tip generated by osteotomy) , Compression fracture, and hamstring fracture.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a perfume or the like in the case of oral administration. A wetting agent, an isotonic agent, an isotonic agent, an isotonic agent, a stabilizer and the like may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, they may be formulated in unit dosage ampoules or in multiple dosage forms have. Other, solutions, suspensions, tablets, capsules, sustained-release preparations and the like.

Examples of suitable carriers, diluents or excipients which may be employed herein include excipients such as starch, sugar, and mannitol; Fillers and extenders such as calcium phosphate and silicic acid derivatives; Cellulose derivatives such as carboxymethylcellulose or hydroxypropylcellulose, binders such as gelatin, alginate, and polyvinylpyrrolidone; Lubricants such as talc, calcium stearate or magnesium, hydrogenated castor oil, and solid polyethylene glycols; Disintegrating agents such as povidone, sodium croscarmellose, and crospovidone; Surfactants such as polysorbate, cetyl alcohol, and glycerol monostearate, and the like.

In another aspect, the present invention relates to a method for preventing or treating a bone metabolic disease comprising administering the pharmaceutical composition to a subject.

The term "individual" as used herein includes, without limitation, mammals including rats, livestock, humans, and the like.

In addition, the pharmaceutical composition according to the present invention can be administered through various routes. Administration in the present invention means introducing a predetermined substance into a patient by any suitable method, and the administration route of the conjugate can be administered through any conventional route so long as the drug can reach the target tissue. Specifically, it may be, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrarectal, rectal. However, when orally administered, since the peptide is digested, it is preferable to formulate the oral composition so as to coat the active agent or protect it from decomposition at the top. Preferably in the form of an injection. In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell.

In the present invention, the actual dosage of the pharmaceutical composition containing the dibenzepin is determined depending on various factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease, . Preferably, the dibenzepine as an active ingredient may be administered to the mammal, including a human, once or dividedly at 1 to 20 mg / kg, more preferably 1 to 10 mg / kg per day.

In another aspect, the present invention relates to a food composition for preventing or ameliorating a metabolic disease comprising the above-mentioned dibenzepine or a pharmaceutically acceptable salt thereof. The food composition may be used as a health functional food. When the composition of the present invention is used as a food-aid additive, the above-mentioned dibenzepine may be directly added or used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).

The term "health functional food " in the present invention refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, mixing, or the like a specific ingredient contained in a food raw material, Refers to a food which is designed and processed so that the body control function such as bio-defense, regulation of biorhythm, prevention and recovery of disease and the like can be sufficiently exhibited to the living body by the above components. Recovery, and so on.

There is no limitation on the kind of health food to which the composition of the present invention can be used. In addition, the composition comprising the dibenzphin as an active ingredient of the present invention can be prepared by mixing other suitable auxiliary ingredients and known additives, which may be contained in health functional foods, according to the selection of a person skilled in the art. Examples of foods that can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component.

The pharmaceutical composition comprising the dibenzepine according to the present invention effectively inhibits osteoclast differentiation and bone resorption and at the same time promotes osteoblast differentiation and osteogenesis. Therefore, a pharmaceutical composition containing osteoporosis, periodontal disease, fracture or Paget's disease And can be usefully used for the prevention or treatment of metabolic diseases.

Figure 1 shows the inhibitory effect of dibenzfene on osteoclast formation. (Left): Co-culture conditions of bone marrow cells and primary osteoblasts containing 1α, 25-dihydroxyvitamin D3 and prostaglandin E ₂ (10 ^-6 M prostaglandin E₂) The osteoclast formed by the addition of star dibenzfine (0, 0.1, 0.5, 1.0 μM) was stained with TRACP staining method and observed with an optical microscope. (Right): The number of osteoclasts stained with TRACP staining showed a decrease in osteoclast formation (0.1, 0.5, 1.0 μM) dependent on the concentration of dibenzepin. The control group used osteoclast differentiation-induced without treatment of dibenzepin.
Figure 2 shows the effect of dibenzepine on osteoclast differentiation. (Left): osteoclasts formed by the addition of dibenzepine (0.1, 0.5, 1.0 μM) to each culture medium in macrophage culture conditions containing M-CSF (30 ng / ml) and RANKL Were stained with TRACP staining method and observed with an optical microscope. (Right): The number of osteoclasts stained with TRACP staining was expressed by the number of intracellular nuclei: 1.0 μM The number of nuclei in osteoclasts treated with dibenzephene was 3 or more (black), 10 or more Gray), and the number of nuclei in the osteoclasts over 25 (white) decreased by TRACP staining. The control group used osteoclast differentiation-induced without treatment of dibenzepin.
Fig. 3 shows the effect of dibenzepine on osteoclast actin ring formation. The morphology of actin rings stained with rhodamine phalloidin in mature osteoclast differentiation through macrophage culture was observed by confocal microscopy. In the experimental group treated with 1 μM dibenzepine, normal actin ring formation was not observed. The control group used osteoclast differentiation-induced without treatment of dibenzepin.
Figure 4 shows the effect of dibenzepine on osteoclastic bone resorption ability. (Left): Mature osteoclasts isolated by 0.2% collagenase were cultured on OAAS plates coated with carbonated calcium phosphate by addition of 0.1 μM dibenzazepine, and the bone resorption bones formed were observed under an optical microscope Respectively. The formation of bone resorption holes was significantly inhibited in the osteoclasts to which dibenzazepine was added. (Right): The area of the bone resorption cavity formed by mature osteoclasts is expressed as a ratio of the total area of the culture dish. The rate of formation of bone resorption was significantly decreased in the osteoclasts to which dibenzazepine was added. The control group used osteoclast differentiation-induced without treatment of dibenzepin.
FIG. 5 shows the inhibitory effect of dibenzepine on interleukin-1-induced mouse skull absorption. (Upper): TRACP staining (top) and micro CT images of the whole tissues of the mouse skull (bottom). Although the mouse skull induces severe bone destruction by interleukin-1, bone resorption by interleukin-1 was significantly inhibited when dibenzepine (60, 200 μg / kg) was administered in vivo in mice. Bone tissue volume (left) and bone mineral (right) per unit area of the extracted mouse skulls were shown. The volume and mineral content of bone tissue decreased by interleukin-1 was increased by dibenzepine.
Figure 6 shows the anti-resorptive enhancement effect in the mouse model of dibenzepine. (Phase): TRACPTRAP (tarrateresistant acid phosphate) staining was performed using a paraffin block section of mouse skull tissue and observed with a microscope. Interleukin-1 induces osteoclasts and severe bone destruction, but was strongly inhibited by dibenzepine. (Bottom): the number of osteoclasts per unit area formed in the same tissue. The number of osteoclasts formed by interleukin-1 was significantly reduced in a concentration-dependent manner by dibenzepin.
Figure 7 shows an osteoblast differentiation promoting effect of diben restraint pin. (Left): The cell culture conditions using osteoblast differentiation promoting medium induced calcification of osteoblasts and stained with Alizarin red S. Dibenzaphinone increased bone calcification by osteoblasts in a dose - dependent manner. (Right): The expression level of messenger RNA of osteocalcin, a mature osteoblast differentiation marker, was measured by real-time PCR. The amount of messenger RNA expression of osteocalcin was increased by concentration - gradient by dibenzfene.
Fig. 8 shows the effect of stimulating new bone formation of dibenzepine. (Phase): Bone destruction of rat skull tissue was induced, and bone regeneration pattern at that site was shown by micro CT image. The bone regeneration ability increased by BMP-2, an osteogenic factor, was also enhanced in a concentration-dependent manner by dibenzepine (0.1, 0.5 μg). (Bottom): Graph showing the ratio of newly regenerated bone mass quantitatively to bone destruction region of each group (left) and graph of skull tissue thickness (right). Treatment of dibenzepine (0.1, 0.5 μg) significantly increased new bone formation rates and skull tissue thickness.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Cell preparation

Example 1-1. Preparation of bone marrow cells and osteoblasts

Bone marrow cells and primary osteoblasts were obtained using ICR male mice (Orient Bio, Seongnam, Gyeonggi Province), 6 weeks old. Specifically, the tibia and femur of the tibia and femur at the posterior part of the 6-week-old ICR male mice (Orient Bio, Seongnam, Gyeonggi-do) were aseptically removed and then the cartilage and fatty tissue were cleanly removed from the separated bone tissue and the syringe was injected into the bone marrow To obtain bone marrow cells. The obtained cells were centrifuged at 1600 rpm for 5 minutes, treated with ACK buffer for 1 minute to remove red blood cells and other adipocytes, and then centrifuged at 1600 rpm for 5 minutes to obtain mouse bone marrow cells Respectively.

In order to obtain primary osteoblast cells, the skulls of 1 day old ICR mice (Orient Bio, Seongnam, Gyeonggi Province) were cut into appropriate sized pieces and then placed in 3x HBSS (Hank's Balanced Salt solution, Weljin, Daegu Metropolitan City) 6 cm culture dish. The cells were treated with 0.1% collagenase (Gibco BRL) and 0.2% dispase (Boehringer Mannheim) for 15 min at 37 ° C. The cells obtained from the two treatments were collected and centrifuged at 1300 rpm for 5 minutes to obtain precipitated osteoblast precursors.

Examples 1-2. Osteoclast culture

The co-culture system used to induce differentiation of osteoclasts from the bone marrow-derived cells of the mice was used, and co-culture was carried out using mouse derived bone marrow cells and osteoblasts. Specifically, the bone marrow cells and primary osteoblasts of the mice obtained in Example 1-1 were suspended in 10 ^-8 M 1α, 25-dihydroxyvitamin D 3 (Sigma) and 10 ^-6 M prostaglandins MEM supplemented with 10% FBS (fetal bovine serum), penicillin (50 units / mL) and streptomycin (50 mg / mL) under conditions treated with E ₂ (10 ^-6 M prostaglandin E ₂ minimum essential medium. Six days after coculture, mature osteoclasts were obtained.

Example 2: Effect of dibenzepine on inhibition of osteoclast differentiation

Example 2-1. Inhibition of osteoclast differentiation by dibenzepine

The bone marrow cells and osteoblasts were co-cultured in the mouse, and bone marrow cells were differentiated into mature osteoclasts with the aid of osteoblast cells. In this system, whether or not dibenzepine inhibited osteoclast differentiation was examined by Lee et al. (Journal of Biological Chemistry, 2009, 284: 13725-13734).

In order to examine the effect of dibenzepine on the inhibition of osteoclast differentiation, dibenzepine was treated (0.1, 0.5, 1 μM) for each concentration in the mouse-derived bone marrow cells and osteoblast- The cells were cultured for 6 days and differentiated into osteoclasts. At this time, the control group used differentiation-induced osteoclasts without treatment with dibenzepine. The differentiation-induced osteoclasts were fixed with 10% formalin for 10 min. After formalin was removed, 0.1% tryptone X-100 (Triton X-100) was treated for 5 minutes, then tryptone X-100 solution was removed and stained with TRACP (tartrate-resistant acid phosphatase) for 10 minutes. TRACP staining was performed using a leukocyte acid phosphatase kit (Sigma). The TRACP staining solution was removed, washed twice with distilled water, and dried. Then, TRACP positive osteoclasts were analyzed under an optical microscope (X100).

As shown in FIG. 1, in the dibenzepin-treated experimental group, the number of multicellular osteoclasts, which are TRACP-positive, was significantly decreased in a concentration-dependent manner compared with the control group not treated with dibenzfene. As a result, the pharmaceutical composition for the prevention and treatment of bone metabolic diseases including dibenzepine showed that 10 ^-8 M 1 ?, 25-dihydroxyvitamin D? And 25-dihydroxyvitamin D? ^{6 -} M prostaglandin E ₂ (10 ^-6 M prostaglandin E ₂ ).

Example 2-2. Inhibition of osteoclast fusion of dibenzepine

Since osteoclasts are fused with hematopoietic progenitor cells, the nucleus can be differentiated into an average of 10 to 20 giant polynuclear cells and can absorb bone. Thus, the ability of osteoclast fusions to fuse is a function of osteoclast function It is important.

In this example, the effect of dibenzepine on osteoclast fusion inhibition was measured using monocyte progenitor cells derived from bone marrow cells of mouse hematopoietic lineage. Specifically, a system that differentiates mature osteoclasts in the presence of M-CSF (macrophage colony-stimulating factor) and RANKL (Receptor activator of NF-κB ligand) by culturing macrophage-derived monocyte precursor cells of mouse hematopoietic cell line And the effects of dibenzepin on the osteoclast fusogenic activity were investigated as follows.

Bone marrow cells of the mice were obtained as described in Example 1-1, and the cells were suspended in 10% alpha-MEM and M-CSF (30 ng / ml) was added. After culturing in a 100 cm culture dish for 3 days Respectively. After 3 days, 1 μM of dibenzepine was added to the culture medium by adding M-CSF (30 ng / ml) and RANKL (100 ng / ml) to the culture medium and incubated for 4 days to induce differentiation into osteoclasts Respectively. At this time, the control group used differentiation-induced osteoclasts without treatment with dibenzepine. The differentiation-induced osteoclasts were stained with TRACP (Tartrate-resistant acid phosphataseform), and TRACP-positive osteoclasts were observed under an optical microscope (X100). The number of osteoclasts in which the number of nuclei in TRACP-positive osteoclasts is 3, 10 or 25 or more is shown in Fig.

As shown in FIG. 2, the dibenzepine shrinks the mature osteoclast cell membrane to a somewhat smaller size than the normal osteoclast, but does not affect the osteoclast formation of TRACP-positive polynuclear cells and the nuclear number of such cells I did. As a result, dibenzepin did not affect cell fusion, an important process for mature multinuclear osteoclast formation.

Examples 2-3. Inhibition of osteoclast actin ring formation by dibenzfentin

The cytoskeletal process of osteoclasts involves attaching to the bone in order to distinguish between the extracellular matrix and the space for absorbing the actual bone, and at the same time, the actin of the osteoclast is organized into one huge ring. The formation of rings is an important marker of the ability of osteoclasts to absorb bone.

In this example, the effect of dibenzepine on the osteoclast actin ring formation was analyzed using monocytic progenitor cells derived from bone marrow cells of the mouse hematopoietic lineage. Specifically, we established a system to differentiate osteoclasts into mature osteoclasts in the presence of M-CSF and RANKL by culturing macrophages, the bone marrow-derived mononuclear precursor cells of mouse hematopoietic lineage, and the effect of dibenzepine on the actin ring formation of osteoclasts Were measured as follows.

To investigate the effect of dibenzfene on osteoclast actin ring formation, macrophages were treated with 1 μM dibenzepine while cultured in the presence of M-CSF and RANKL. To distinguish the actin ring formation pattern of osteoclasts differentiated from macrophages by the above culture composition, immunocyte staining was performed with an antibody to actin-forming factor Rhodamine-Phalloidin. The cultured osteoclasts were washed with PBS, fixed with 2% paraformaldehyde, and blocked with nonspecific binding with bovine serum albumin. Since actin formation marker is Rhodamine-was observed with ^{ProLong ⓡ Gold antifade reagent with DAPI (} Invitrogen, San Diego, CA) to produce a specimen in a confocal microscope (X100) were reacted by the addition of palroyi Dean antibody.

As shown in FIG. 3, in the experimental group treated with dibenzepin compared with the control group not treated with dibenzfene, rhodamine-paloidin staining of the perivitial actin was not normally performed. In addition, it was confirmed that the dibenzepine inhibited normal actin ring formation and the relationship between the cell shape contracted by the dibenzepine and the cell membrane in Fig.

Examples 2-4. Inhibition of bone resorption by dibenzepine via bone resorption experiment

As shown in Example 1-2, when bone marrow cells and osteoblast cells were co-cultured, osteoclast cells were differentiated into mature osteoclasts with the aid of osteoblast cells, and mature osteoclasts were cultured through collagenase In order to investigate the effect of dibenzfyne on bone resorption after separation, the following experiment was carried out.

In order to induce the differentiation of mature osteoclasts, bone marrow cells (1 x 10 ⁷ ) and primary osteoblasts (1 x 10 ⁶ ) were inoculated into a collagen coated culture dish, and 10 ^-8 M 1α, 25-dihydroxyvitamin D 3 And 10 ^-6 M prostagalin E ₂ were added for 6 to 7 days. The cultured cells were treated with 0.2% collagenase (Invitrogen) at 37 ° C for 10 minutes, and then re-inoculated into a culture dish for induction of osteoclast differentiation. After incubation for 4 hours, osteoclasts and other bone marrow cells floating in the culture medium and mature osteoclasts attached to the bottom of the culture dish were obtained.

For bone resorption experiments, 1 μM dibenzepine was re-inoculated onto OAAS plates (Osteogenic Core Technologies Inc., Korea) coated with carbonated calcium phosphate with osteoclasts and left for 12 hours. After 24 hours, the cells were removed, and a total absorbance pit formed by the osteoclasts was photographed. The pro-plus program (Pro-Plus program, version 4.0, Media Cybernetics).

As a result, as shown in FIG. 4, the pharmaceutical composition containing dibenzaphin was significantly reduced the pore formation on the OAAS plate, thereby showing a substantial effect of inhibiting bone resorption by dibenzepine.

Example 3: Experimental study on inhibition of bone resorption by dibenzfentan

Example 3-1. Interleukin-1 (IL-1) Induced Inhibition of Mouse Skull Disruption by Dibenzepin

To investigate the effect of dibenzepine on the inhibition of mouse bone loss, the effect of interleukin-1-induced in vivo on the loss of mouse skull was examined by Ha et al. (Journal of Immunology, 2006, 176: 111-117) Experiments were conducted in the reported manner. Specifically, 5-7 ml of collagen (Cellmatrix type IA, Wako co., Japan) was poured into Petri dish (60 x 15 mm) and lyophilized. After lyophilized collagen sponge was divided into appropriate sizes for implantation, IL-1 (interleukin-1, Peprotech, London, UK) was diluted in physiological saline to induce local bone loss in the mice The mice were soaked in collagen sponge at 2 ㎍ per mouse. The mouse scalp was incised and the collagen sponge treated with IL-1 solution was contacted to the surface of the skull. The mice were transplanted into 6 mice (5-week-old male, ICR strain) Suture was maintained for 7 days and sacrificed to obtain the entire skull tissue. In the experimental group, dibenzazepine was administered to the mice one day before the start of the operation, in which DMSO (50 μl) and physiological saline (50 μl) were mixed at a dose of 60 and 200 μg / And injected intraperitoneally. As a control (vehicle-treated group or negative control group), a solution in which dibenzepin-treated DMSO (50 μl) and physiological saline (50 μl) were mixed was administered by intraperitoneal injection. Seven days later, mice were sacrificed and the whole tissues of the extracted mouse skulls were washed with physiological saline for 3-4 times and then fixed in 4% paraformaldehyde solution for 24 hours. Tumor-resistant acidic phosphatase (TRACP) staining and micro-computed tomography scan (SMX-90CT, Shimadzu, Japan) were performed to determine the bone resorption pattern of the fixed mouse skull. The obtained result is shown in Fig. 5A (Volume Graphics, VG studio Max 1.2.1). The bone mineral content (mg) and the volume (mm ³ ) of bone tissue were measured using the TRI 3D-BON (RATOC system Engineering Co., Tokyo, Japan) Respectively.

As a result, as shown in FIG. 5, the vehicle-treated group used as a negative control group induced severe destruction of the skull tissue as shown in a three-dimensional image due to progressive bone loss by interleukin-1, and TRACP positive The site was increased. In addition, in these skull tissues, bone volume and bone-mineral content were remarkably reduced, but the dibenzepine of the present invention inhibited the bone loss of the skull and showed almost no destruction of the skull tissue, The tissue volume and total bone-mineral content were increased in a concentration-dependent manner compared to the vehicle-treated group. Therefore, it was confirmed that the inhibitory effect of dibenzepine on bone resorption was confirmed by the mouse skull disappearance model.

Example 3-2. Histological analysis of the effect of dibenzepine on the loss of mouse skull

In order to histologically examine the effect of the above-mentioned dibenzepine on inhibition of mouse bone resorption, the mouse skull tissue after micro CT scans was washed and fixed in Example 3-1, and demineralized for 2-3 weeks And a paraffin block was prepared. For histological analysis, paraffin blocks were cut into 5-6 mm thickness using a rotary microtome with a razor blade and attached on a laboratory glass plate. Then, in vivo osteoclast formation, which is directly related to bone loss, To identify the loss pattern, TRACP (tartrate-resistant acid phosphatase) staining was performed. The total area of TRACP positive osteoclast formation was plotted using the mage J program.

As a result, as shown in FIG. 6, the vehicle-treated group used as a negative control group was found to induce osteoclast formation, which leads to an irregular shape of the skull tissue surface, On the other hand, the compound significantly decreased the formation of osteoclast-induced osteoclasts and confirmed the stabilization of the skull tissue surface.

Example 4: Effect of dibenzepine on the promotion of osteoblast differentiation

Osteoblast cells are originated from mesenchymal stem cells and develop into mature osteoblast cells that undergo differentiation process to form bones. Mineralization, including calcium, produced by osteoblast differentiation not only maintains bone strength, Of the hormone metabolism and calcium homeostasis is also very important function. The most urgent problem in studying bone related diseases is not only the inhibition of osteoclast activation but also the promotion of osteoblast activation and the ability to form new bone tissue. This is due to the fact that there is already a significant progression of significant bone loss as seen in most bone related disease patients.

In this embodiment, a system is developed to differentiate into mature osteoblastic cells capable of producing substantial bone nodules by culturing precursor cells of primary osteoblasts of mice. In this system, dibenzepine is used to promote osteoblast differentiation The experiment was carried out in the following way.

In order to differentiate the osteoblast precursor obtained in Example 1-1 into osteoblasts, osteogenic media (alpha-MEM + 10 mM beta-glycerophosphate (sigma) + 50 μg / mL ascorbate-2-phosphate Sigma) + 30 ng / mL BMP-2 (Daewoong Pharm., Korea)). Dibenzepin was treated with each concentration (0.1, 0.5 μM) for 10 days, and induced to differentiate into osteoblasts. On day 10 after culture, the cells were treated with 0.2% Alizarin Red S solution (pH 4.3; Sigma) dye to stain calcified osteoblasts. In addition, the expression level of messenger RNA in osteocalcin, a marker of differentiation of mature osteoblasts, was analyzed by real-time PCR. At this time, the osteoblast differentiation induction treatment was performed without treatment of dibenzepine.

As a result, as shown in FIG. 7, osteoblast differentiation was promoted in a culture dish treated with dibenzepin, along with a medium for promoting osteoblast differentiation, compared to a control group not treated with dibenzfene, The number of stained bone nodules was significantly increased. In addition, the messenger RNA expression of osteocalcin, a marker of osteoblast differentiation, was increased by dibenzepine, and it was confirmed that the osteoblast activation ability of dibenzepin was increased according to the concentration of dibenzepin.

Example 5: Animal experiment promoting bone formation of dibenzfene

To investigate the effect of the present dibenzepine on new bone formation, the effect of dibenzepine on bone regeneration ability in the in vivo cranial deficient region was investigated by Kim et al. (Journal of Bone and Mineral Research, 2011, 26 : 2161-2173). ≪ / RTI >

A 8 mm diameter circular bone defect was formed in the skull of approximately 250 grams of male Sprague-Dawley rats (Orient Bio, Korea), 2 months old, and a collagen sponge containing dibenzepine (Bioland, Korea) After transplantation, periosteum and skin were double sutured. At this time, as a negative control, collagen sponge containing Dibenzidine-dissolved DMSO and physiological saline solution in the same volume was injected, and collagen sponge containing BMP-2 (2 μg) was injected as a positive control. After 3 weeks, the rats were sacrificed and the skull removed. The extracted skull was rinsed with physiological saline, micro CT was performed, and a three-dimensional image was obtained. The results are shown in FIG. In addition, the 3D image file is graphically displayed by measuring the new bone regeneration rate and the thickness of the skull tissue of each group using the TRI 3D-BON program.

As a result, as shown in Fig. 8, compared with the vehicle-treated group used as the negative control group, the dibenzepin-treated group showed a higher score than the empty space created by the bone defect on the skull tissue surface as the positive control group BMP- It was found that the space filled with bones was formed by the formation of three dimensional images through micro CT and the bone regeneration rate and thickness of skull tissues were significantly increased by dibenzepine.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (6)

(S, S) -2- [2- (3,5-difluorophenyl) acetylamino] -N- (5-methyl-6-oxo-6,7-dihydro-5H -Dibenzo [b, d] azepin-7-yl] propionamide) or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is a pharmaceutical composition for preventing or treating bone metabolic diseases,
Wherein the bone metabolic disease is a disease selected from the group consisting of osteoporosis, periodontal disease, fracture, and Paget's disease.
The pharmaceutical composition according to claim 1, wherein the dibenzepine inhibits bone resorption of osteoclasts and at the same time promotes bone formation of osteoblasts.
The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
The pharmaceutical composition according to claim 1, wherein the composition is formulated in the form of an oral preparation selected from the group consisting of granules, tablets, pills, capsules, suspensions, emulsions, syrups and aerosols, .
(S, S) -2- [2- (3,5-difluorophenyl) acetylamino] -N- (5-methyl-6-oxo-6,7-dihydro-5H - dibenzo [b, d] azepin-7-yl] propionamide) or a pharmaceutically acceptable salt thereof as a pharmaceutical composition for preventing or improving bone metabolic diseases,
Wherein said bone metabolic disease is a disease selected from the group consisting of osteoporosis, periodontal disease, fracture, and Paget's disease.
6. The food composition of claim 5, further comprising a pharmaceutically acceptable food-aid additive.

Images