KR20140021391A - A pharmaceutical composition for preventing or treating bone metabolic disease comprising dibenzazepine - Google Patents

Abstract

The present invention relates to a pharmaceutical composition which comprise dibenzazepine and a food composition. Specifically, the present invention relates to a composition used for preventing or treating bone metabolic diseases, particularly, osteoporosis, periodontal disease, bone fracture, and Paget's disease. [Reference numerals] (AA,FF,HH) Dibenzazepine; (BB) Saline solution + DMSO; (CC) Micro CT (upper surface); (DD) Side; (EE) New bone regeneration rate (%); (GG) Rat skull thickness (μm)

Description

A pharmaceutical composition for preventing or treating bone metabolic disease comprising dibenzazepine

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

Bone modeling and remodeling play an important role in bone development, growth and metabolism. Bone formation begins in the early stages and continues until the young adulthood, when the skeleton matures and the growth ends, forming the maximum bone mass in the 20s and early 30s. After about 30 years, the bone formation process is repeated to remove the bone and replenish it again. In this case, the bone formation and bone absorption are paired with each other to maintain balance. After this period, bone loss due to bone resorption cannot follow enough bone formation, resulting in bone loss of 0.3% to 0.5% per year, especially in women at the beginning of menopause, with significant bone loss of 2-3% per year. There is a loss.

Bone tissue is composed of cartilage and skeletal system, mechanical functions of supporting and muscle attachment, protecting the organs and bone marrow, and preserving them to maintain the homeostasis of calcium and phosphorus ions. Bone tissue consists of cell substrates, such as collagen and glycoproteins, and several types of cells, including osteoblasts, osteoclasts, and bone cells. Osteoblasts derived from bone marrow stromal cells play a major role in bone formation, osteoclasts derived from hematopoietic stem cells are destroyed, and osteoblasts and osteoclasts are responsible for the absorption of aged bone. It works in a balanced manner to maintain bone remodeling. However, excessive activity of osteoclasts or deactivation of osteoblasts causes an imbalance during the remodeling of bone formation, leading to bone disease by breaking the equilibrium between osteoclasts and osteoblasts in vivo.

As a representative example of bone disease, osteoporosis is an unavoidable symptom in elderly people, especially postmenopausal women. In developed countries, as the population ages, interest in osteoporosis and its therapeutics is gradually increasing. It is also known that there is a market of about $ 130 billion related to the treatment of bone diseases around the world, which is expected to increase further. have. In Korea, the prevalence of osteoporosis is rapidly increasing as the average life expectancy reaches 80. Recently, a study of local residents showed that 4.5% of men and 19.8% of women had osteoporosis when standardized to the national population. It has been reported to have. This suggests that osteoporosis is more common than diabetes or cardiovascular disease, and osteoporosis is a very important health problem when estimating the pain or cost of treating patients who suffer from fractures.

To date, several substances have been developed for the treatment of osteoporosis. Among them, estrogen, which is most used as a therapeutic agent for osteoporosis, has not yet been tested for its actual efficacy and has to be taken continuously for life, and has long-term side effects such as increased breast cancer or uterine cancer. Alendronate also has a problem that the effect is not clear, slow absorption in the digestive tract and inflammation of the stomach and esophagus mucosa. Calcium preparations are known to have fewer side effects and superior effects, but they are more preventive agents than therapeutic agents. Other vitamin D preparations, such as calcitonin, are known but have not been fully studied for their efficacy and side effects. Accordingly, there is a need for a new metabolic bone disease treatment agent having fewer side effects and excellent effects.

On the other hand, periodontal disease is divided into gingivitis and periodontitis according to the inflammatory state of the supportive tissue, and advanced periodontitis causes tooth loss, and there is a report that the percentage of periodontitis is 40% among the causes of tooth extraction in adults after 40 years old. Tooth loss due to periodontitis is a serious problem.

The destruction of periodontal ligament and alveolar bone in periodontal disease is due to the degradation of collagen, a major component of these tissues. The exact mechanism is not yet known, but collagen degradation is known to be due to collagenase secreted by the host and neutral protease secreted from periodontal disease-causing bacteria. Therefore, studies have been attempted to inhibit the progression of periodontal disease by inhibiting the activity of collagenase enzyme and protease. The treatment of periodontal disease has been used for the establishment of improved oral hygiene, non-surgical or surgical calculus removal, root graft, gingival sopa and regeneration of periodontal tissue. However, these surgical methods are difficult to treat effectively due to the hassle of going to the dentist for treatment, and they are limited to the treatments performed when the disease progresses rather than preventing the disease. Most of the time.

As an additional treatment, systemic antibiotics and topical sustained-release drugs have been used, but cases of periodontal disease bacteria have been reported that result in too much drug delivery to unnecessary areas resulting in side effects and resistance to recently used antibiotics. There is a serious problem. In order to supplement the limitations of the surgical treatment and the use of antibiotics, the use of oral compositions such as toothpastes and mouthwashes containing antibacterial and anti-gingival agents has been used to prevent and treat bacterial oral diseases such as dental caries and periodontal disease. The effect was reported as.

Fracture refers to the condition of broken bones, and it takes a long time to treat the fracture, and as the population ages, the fracture of osteoporosis patients, which is one of the pathological fractures, has increased significantly, and calcium, estrogen, and calcitonin, which are currently used to treat osteoporosis, (calcitonin), active vitamin D, and bisphosphonate preparations were used for the treatment of fractures, but the effect of fracture was expected, but it was possible to reduce the risk of fracture by inhibiting the reduction of bone density, and to bond and form the fracture site. Did not. This is because the mechanism of osteoporosis is genetic or constitutional factor, so that the negative equilibrium of bone is sustained, so that the bone has a fine skeletal quality, the bone is absorbed at normal speed, but the formation of bone is poor and bone is formed at normal speed. This is because the absorption of bone is increased, and thus, osteoporosis and fracture are completely different from the mechanism of development, and thus it cannot be inferred that fractures can be treated with an agent for treating osteoporosis.

Due to the difference in the mechanism of osteoporosis and fracture as described above, drugs that exhibit osteoporosis treatment by inhibiting bone resorption can also delay bone formation by inhibiting bone formation. Prolonged and continuous administration of incadronate disodium, a bisphosphonate, has been reported to delay fracture healing in femurs of rats (Li C et al., J. Bone Miner Res. 2001 Mar; 16 (3): 429-36), pretreatment with incadonate did not affect fracture healing at 16 weeks post-fracture, whereas continued incadronate treatment increased the bone but delayed the remodeling process during fracture healing. There are also reports (Li J et al., J. Bone Miner Res. 1999 Jun; 14 (6): 969-79). In addition, bFGF, a bone-forming biomarker known to be closely related to osteoporosis, has been reported to have no relationship with fracture healing (Xu et al., Chin. J. Traumatol. 6, 160-166, 2003).

Therefore, there is a limit for using the osteoporosis therapeutic agent to effectively promote the healing to a normal state when a general fracture occurs, and thus, there is an urgent need for the development of a drug having a unique fracture healing effect regardless of osteoporosis.

Accordingly, the present inventors have conducted studies on a composition containing dibenzazepine that promotes osteoblast differentiation and inhibits osteoclast differentiation, which are essential for bone formation, fracture recovery, and tooth growth and development, thereby preventing osteoclasts from absorbing bone. The present invention has been completed by confirming that a composition containing dibenzazepine that inhibits and simultaneously promotes osteogenicity of osteoblasts can be used for treating osteoporosis as well as fractures that require the promotion of bone.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, including dibenzazepine or a pharmaceutically acceptable salt thereof.

It is another object of the present invention to provide a method of treating bone metabolic diseases comprising administering the pharmaceutical composition to a subject.

Still another object of the present invention is to provide a food composition for preventing or ameliorating bone metabolic diseases, including dibenzazepine or a pharmaceutically acceptable salt thereof.

As one aspect for achieving the above object, the present invention relates to a composition for the prevention or treatment of bone metabolic diseases, including dibenzazepine or a pharmaceutically acceptable salt thereof.

The term "dibenzazepine" in the present invention is (S, S) -2-2 [2- (3,5-difluorophenyl) acetylamino] -N- (5-methyl-6- Oxo-6,7-dihydro-5H-dibenzo [b, d] azepine-7-yl] propionamide ((S, S) -2-2 [2- (3,5-difluorophenyl) acetylacetyl]- N- (5-methyl-6-oxo-6,7-dihydro-5H-dibenzo [b, d] azepin-7-yl] propionamide)), the molecular formula is C ₂₆ H ₂₃ F ₂ N ₃ O ₃ and molecular weight Is 525.7 (g / mol).

Dibenzazepine is known as a tricyclic antidepressant associated with imipramine, but there is no known use of osteoclasts to inhibit bone resorption and osteoblasts to promote bone formation.

In one embodiment of the present invention, dibenzazepine directly inhibits the activation of the gamma-secretase complex to reduce the activation of marker factors related to osteoclast differentiation and activation, and to activate the activation of key genes involved in osteoblast differentiation and activation. By stimulating, it has been confirmed that it can be used as a therapeutic agent for bone metabolic related diseases, especially those requiring bone regeneration, by inhibiting excessive bone reduction in vivo and inducing new bone formation and regeneration of bone fracture sites where bone density is significantly lowered. .

As an active ingredient of the pharmaceutical composition of the present invention, it may be used in the form of the dibenzazepine or a pharmaceutically acceptable salt thereof, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. 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

Lithium or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compounds salt, and then 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).

Pharmaceutically acceptable salts of the dibenzazepines, unless otherwise indicated, include almost salts of acidic or basic groups which may be present in the compound of dibenzazepine. 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 .

Dibenzazepine of the present invention also includes derivatives thereof. The term "derivative" refers to a compound prepared by chemically changing a part of dibenzazepine, for example, introducing, replacing, or deleting a functional group, to the extent that the therapeutic activity of dibenzazepine does not change. For example, it may include, but is not limited to, amitriptyline, desmethylimipramine, decipramine, nortriptyline, protriptyline, and the like. In addition, the composition of the present invention may further include a known osteoporosis, fracture treatment, periodontal disease treatment agent in addition to dibenzazepine.

The composition of the present invention is characterized in that it suppresses the bone resorption of osteoclasts and at the same time promotes the bone production of osteoblasts.

As used herein, the term "osteoclast" refers to a cell derived from a macrophage precursor, wherein the osteoclast progenitor cells are macrophage colony stimulating factor (M-CSF), NF. It is differentiated into osteoclasts by the receptor activator ligand of κB (RANKL) and the like and forms a multinucleated osteoclast through fusion. Osteoclasts bind to bone through αvβ3 integrin, etc., create an acidic environment and secrete various collagenases and proteases to cause bone resorption. Osteoclasts do not proliferate into fully differentiated cells and cause apoptosis at the end of about two weeks of life.

In one embodiment of the present invention, after administering the composition of the present invention to the co-culture of bone marrow cells and primary osteoblasts differentiated into osteoclasts, the resultant staining of the osteoclast differentiation marker TRAP (tarrateresistant acid phosphate), TRAP activity It was confirmed that the concentration-dependent inhibition (Example 1, Figure 1). As described above, since the composition of the present invention effectively inhibits the differentiation of osteoclasts through TRAP activity inhibition, it is possible to prevent or treat bone metabolic diseases such as osteoporosis due to increased activity of osteoclasts.

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

As used herein, the term "osteoblast" is a cell produced by differentiating from mesenchymal stem cells, and serves to increase bone density by making bone quality, and sometimes, when osteoblast activity is excessively increased, bone density increases. This can lead to bone malformations or osteoporosis.

In one embodiment of the present invention, when the composition containing dibenzazepine was treated to osteoblasts, it was confirmed that the activity of Alizarin red S, a differentiation marker, was markedly increased in the early stages of osteoblast differentiation (Example 4, FIG. 7). In addition, as a result of transplantation of the dibenzazepine of the present invention into the defective site in the experimental rat bone-deficient bone, it was confirmed that the bone regeneration rate is significantly increased (Example 5, Figure 8). This result not only demonstrates that the composition of the present invention can differentiate osteoblasts at the cellular level, but also suggests an increase in bone mineral density in real animals. It is to promote the activity to support the prevention or treatment of bone metabolic diseases such as osteoporosis.

Therefore, the composition of the present invention can be used for the prevention or treatment of osteo metabolic diseases, in particular, can be used without limitation in bone-related diseases caused by the imbalance of osteoblasts and osteoclasts. Examples of such diseases include diseases that promote bone destruction due to pathological bone diseases such as osteoprosis, periodontal disease, fracture or Paget disease caused by excessive osteoclast absorption of bone. It is not limited to the above metabolic disease.

As used herein, the term "prophylaxis or treatment of bone metabolic disease" includes the prevention and complete or partial treatment of the above osteobolic diseases, in particular, osteoporosis, periodontal disease, fractures and Paget's disease. It also includes reducing or improving symptoms of bone metabolic disease, alleviating the pain of the symptoms, reducing the incidence of bone metabolic disease, or any other change in the patient that increases the outcome of treatment.

The "osteoporosis" is a state in which bone mineralization is reduced by thinning the bone tissue, resulting in thinning of the bone marrow cavity, and thus widening of the bone marrow cavity. Easy to be Bone mass is influenced by several factors, including genetics, nutrition, hormone changes, differences in exercise and lifestyle, and the causes of osteoporosis include old age, lack of exercise, low weight, smoking, low calcium diet, menopause, Ovarian ablation is known. On the other hand, although there are individual differences, blacks have lower bone resorption levels than whites, resulting in higher bone mass, usually the highest in 14 to 18 years of age, and about 1% per year in old age. In particular, women after 30 years of bone reduction continues to progress, and by the hormonal changes, bone reduction rapidly progresses. In other words, estrogen concentration rapidly decreases at the end of menopause, in which a large amount of B-lymphocytes are generated, as in the case of IL-7 (interleukin-7), and the B cell precursor (pre-B) is added to the bone marrow. cells) accumulate, thereby increasing the amount of IL-6, increasing the activity of osteoclasts, and eventually reducing bone mass.

The "periodontal disease" refers to the inflammatory state of the tooth support tissue caused by bacteria, can be separated into gingivitis and periodontitis. The cause is caused by oral bacteria due to poor oral hygiene to form dental plaque. Toothpaste bacterium refers to a mass of bacteria that grows after the bacteria adhere to the tooth surface by using a sticky substance in the saliva as an adhesive. If left untreated, the bacterial membranes are inflamed and sometimes bleed from the gums and cause bad breath. These symptoms are called gingivitis. As the gingivitis progresses further, the gap between the teeth and the gum becomes deeper, and the periodontal sac is formed, and the periodontal disease is caused by the bacteria that cause periodontal disease. As periodontitis progresses, even weak stimuli such as brushing may bleed and swell the gums, often turning into acute inflammation, causing pain. This inflammation lowers the function of making bones, and the bone absorbing effect is increased, the alveolar bone becomes lower and lower, and the alveolar bone is destroyed and eventually loses teeth.

The term "fracture" refers to a fracture of a bone in a state in which continuity of a bone, a veneer, or a joint surface is abnormally broken. The causes of fractures include traumas such as traffic accidents, safety accidents caused by industrial disorders, bone changes caused by diseases such as osteoporosis, bone cancer, metabolic disorders, and repetitive bone stress caused by sports or loads. In addition, fracture status is based on fracture line (line along bone tip generated by bone cutting), crack fracture, greenstick fracture, transverse fracture, filamentous fracture, spiral fracture, segmental fracture, comminuted fracture, avulsion fracture , Compression fractures, depression fractures, and the like.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a suspending agent, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a coloring agent, a flavoring agent, and the like during oral administration. In the case of an injection, a buffer, a preservative, An analgesic agent, a solubilizer, an isotonicity agent, a stabilizer, etc. can be mixed and used, and base use, an excipient, a lubricant, a preservative etc. can be used for topical administration. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or multiple dosage forms. have. Others may be formulated into solutions, suspensions, tablets, capsules, sustained release preparations and the like.

Examples of suitable carriers, diluents or excipients that may be employed include excipients such as starch, sugars, and mannitol; Fillers and extenders such as calcium phosphate and silicic acid derivatives; Binders such as cellulose derivatives such as carboxymethyl cellulose or hydroxypropyl cellulose, gelatin, alginate, and polyvinyl pyrrolidone; Lubricants such as talc, calcium stearate or magnesium, hydrogenated castor oil, and solid polyethylene glycols; Disintegrants such as povidone, sodium croscarmellose, and crospovidone; Surfactants such as polysorbate, cetyl alcohol, 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.

As used herein, the term "individual" includes without limitation mammals, including rats, domestic animals, humans, and the like.

In addition, the pharmaceutical compositions according to the invention can be administered via several routes. Administration in the present invention means introducing any substance into the patient in any suitable manner and the route of administration of the conjugate may be administered via any general route as long as the drug can reach the target tissue. Specifically, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. However, upon oral administration, since the peptide is digested, it is desirable to formulate the oral composition to coat the active agent or to protect it from degradation in the stomach. Preferably in the form of an injection. In addition, the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.

In the present invention, the actual dosage of the pharmaceutical composition comprising dibenzazepine is the type of drug that is the active ingredient, along with several related 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. It depends on. Preferably dibenzazepine as an active ingredient may be administered to a mammal, including humans, once or divided into 1 to 20 mg / kg, more preferably 1 to 10 mg / kg during the day.

As another aspect, the present invention relates to a food composition for preventing or ameliorating bone metabolic diseases comprising the dibenzazepine 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 additive, the dibenzazepine may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The blending amount of the active ingredient may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment).

The term "health functional food" in the present invention refers to a food prepared and processed by a method of extracting, concentrating, refining, and mixing a specific ingredient as a raw material or contained in a food ingredient for the purpose of health supplement, By means of the ingredient refers to foods that are designed and processed to fully exert bioregulatory functions on the living body such as biodefense, regulation of biorhythms, prevention and recovery of diseases, and the composition for health foods prevents diseases and prevents diseases. It can perform functions related to recovery.

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 dibenzazepine of the present invention as an active ingredient can be prepared by mixing the known additives with other appropriate auxiliary ingredients that may be contained in the health functional food according to the choice of those skilled in the art. Examples of foods that can be added include meats, sausages, breads, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products, including ice cream, various soups, beverages, teas, drinks, alcoholic beverages and Vitamin complexes, and the like, can be prepared by adding the extract according to the present invention as a main ingredient juice, tea, jelly and juice.

The pharmaceutical composition comprising dibenzazepine according to the present invention effectively inhibits osteoclast differentiation and bone resorption and at the same time promotes osteoblast differentiation and bone formation, and thus bone such as osteoporosis, periodontal disease, fracture or Paget's disease. It can be usefully used for the prevention or treatment of metabolic diseases.

Figure 1 shows the inhibitory effect of dibenzazepine on osteoclast formation. (Left): Concentrations in co-culture of bone marrow cells and primary osteoblasts containing 1α, 25-dihydroxyvitamin D₃ and prostaglandin E ₂ (10 ^-6 M prostaglandin E₂) Osteoclasts formed by the addition of star dibenzazepine (0, 0.1, 0.5, 1.0 μM) were stained by TRACP staining and observed under an optical microscope. (Right): The number of osteoclasts stained by TRACP staining showed that osteoclast formation was reduced depending on the concentration of dibenzazepine (0.1, 0.5, 1.0 μM). The control group used osteoclasts induced differentiation without treatment with dibenzazepine.
Figure 2 shows the cell fusion effect during the osteoclast differentiation process of dibenzazepine. (Left): osteoclasts formed by addition of dibenzazepine (0.1, 0.5, 1.0 μM) at each concentration to macrophage culture conditions containing M-CSF (30 ng / ml) and RANKL (100 ng / ml) Was stained by TRACP staining and observed with an optical microscope. (Right): The number of osteoclasts stained by TRACP staining is indicated by the number of intracellular nuclei: The number of osteoclasts treated with 1.0 μM dibenzazefen was 3 or more (black), 10 or more ( Gray), the number of nuclei in more than 25 (white) osteoclasts showed reduced TRACP staining formation. The control group used osteoclasts induced differentiation without treatment with dibenzazepine.
Figure 3 shows the osteoclast actin ring formation effect of dibenzazepine. In the course of mature osteoclast differentiation through macrophage culture, the actining formation stained by rhodamine phalloidin was observed by confocal microscopy. In the experimental group treated with 1 μM dibenzazepine did not form a normal actin ring. The control group used osteoclasts induced differentiation without treatment with dibenzazepine.
Figure 4 shows the effect of dibenzazepine mature osteoclast bone resorption capacity. (Left): Osteatomic cells isolated from 0.2% collagenase were cultured by adding 0.1 μM dibenzazepine to OAAS plates coated with carbonated calcium phosphate, followed by optical microscopy. The result is observed. The formation of bone resorption holes was markedly inhibited in osteoclasts to which dibenzazepine was added. (Right): The area of bone absorption hole formed by mature osteoclasts is shown as a ratio with respect to the total area of a culture dish. In the osteoclasts to which dibenzazepine was added, the rate of formation of bone resorption holes was significantly reduced. The control group used osteoclasts induced differentiation without treatment with dibenzazepine.
Figure 5 shows the inhibitory effect of dibenzazepine on mouse cranial uptake induced by interleukin-1. (Top): TRACP staining (top) and micro CT picture (bottom) of mouse skull whole tissue. Mouse skulls induce severe bone breakage by interleukin-1, but significantly inhibited bone uptake by interleukin-1 when dibenzazepine (60, 200 μg / kg) was administered in vivo. Bone tissue volume (left) and bone mineral (right) per fixed area of the extracted mouse skull were shown. Bone tissue volume and minerals decreased by interleukin-1 were increased by dibenzazepine.
Figure 6 shows the anti-resorptive improvement effect in the mouse model of dibenzazepine. (Phase): Paraffin block section of mouse skull tissue was performed using a tarrate resistant acid phosphate (TRACPTRAP) stain and observed under a microscope. Interleukin-1 induced osteoclasts and severe bone breakage but was strongly inhibited by dibenzazepine. (Bottom): The number of osteoclasts per unit area formed in the same tissue is shown. The number of osteoclasts formed by interleukin-1 was significantly reduced, depending on the concentration of dibenzazepine.
Figure 7 shows an osteoblast differentiation promoting effect of diben restraint pin. (Left): Calcification of osteoblasts was induced by cell culture conditions using osteoblast differentiation promoting medium and stained with Alizarin red S. Dibenzazepine increased bone calcification by osteoblasts in a concentration dependent manner. (Right): The amount of messenger RNA expression of osteocalcin, a mature osteoblast differentiation marker, was measured by real-time PCR. Dibenzazepine increased the concentration of messenger RNA expression of osteocalcin.
Figure 8 shows the new bone production promoting effect of dibenzazepine. (Top): Induced bone breakage of rat skull tissue and the bone regeneration pattern occurring in the site by micro CT image. The bone regeneration ability increased by BMP-2, a bone formation factor, was also concentration-dependently enhanced by dibenzazepine (0.1, 0.5 μg). (Bottom): Graph showing quantitatively the percentage of newly regenerated bone volume at each site of bone break (left) and graph showing the thickness of skull tissue (right). Treatment with dibenzazepine (0.1, 0.5 μg) significantly increased new bone formation and thickness of skull tissue.

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. Bone Marrow and Osteoblast Preparation

Bone marrow cells and primary osteoblasts were obtained using 6-week-old ICR male mice (Oriental Bio, Seongnam, Gyeonggi-do). Specifically, aseptically isolate the tibia and femur of the hind limbs of 6-week-old ICR male mice (Oriental Bio, Seongnam, Gyeonggi-do), remove cartilage and adipose tissue from the separated bone tissue, and inject a syringe into the bone marrow. The bone marrow cells were obtained. The obtained cells were precipitated by centrifugation at 1600 rpm for 5 minutes, and treated with ACK buffer for 1 minute to remove red blood cells and other adipocytes, and then precipitated by centrifugation at 1600 rpm for 5 minutes to obtain mouse bone marrow cells. It was.

In addition, ICR mice (Orient Bio, Seongnam, Gyeonggi) skulls were isolated and cut into pieces of appropriate size to obtain primary osteoblasts.They contained 3x HBSS (Hank's Balanced Salt solution, Weljin, Daegu Metropolitan City). Collected 6 cm culture dish. 0.1% collagen degrading enzyme (collagenase, Gibco BRL) and 0.2% dispase (Boehringer Mannheim) were added and treated 5 times at 37 ° C. for 15 minutes. The cells obtained from the two treatments were collected to obtain osteoblast precursors precipitated by centrifugation at 1300 rpm for 5 minutes.

Examples 1-2. Osteoclast culture

In order to induce differentiation of osteoclasts from mouse bone marrow-derived cells, a commonly used co-culture system was used, and co-culture was performed 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 treated with 10 ^-8 M 1α, 25-dihydroxyvitamin D₃ (Sigma) and 10 ^-6 M prostaglandin. Alpha-MEM containing 10% FBS (fetal bovine serum), penicillin (50 units / mL) and streptomycin (50 mg / mL) under E ₂ (10 ^-6 M prostaglandin E₂ (Sigma)) treatment co-cultivation using a minimum essential medium). Six days after coculture mature osteoclasts were obtained.

Example 2 Effect of Dibenzazepine on Inhibition of Osteoclast Differentiation

Example 2-1. Dibenzazepine Inhibits Osteoclast Differentiation

Co-culture of mouse bone marrow cells and osteoblasts to form a system in which bone marrow cells are differentiated into mature osteoclasts with the support of osteoblasts, and whether dibenzazepine inhibits the differentiation of osteoclasts in this system (Journal of Biological The experiment was performed by the method reported by Chemistry, 2009, 284: 13725-13734.

To examine the effect of dibenzazepine on osteoclast differentiation, dibenzazepine was treated (0.1, 0.5, 1 μM) at different concentrations in the co-culture of mouse-derived bone marrow cells and osteoblasts in Example 1-2. The cells were cultured for 6 days to induce differentiation into osteoclasts. The control group used osteoclasts induced differentiation without treatment with dibenzazepine. In order to fix differentiated osteoclasts, the cells were treated with 10% formalin for 10 minutes. Formalin was removed, treated with 0.1% Trypton X-100 (Triton X-100) for 5 minutes, then the Tryptone X-100 solution was removed and stained with TRACP (tartrate-resistant acid phosphatase) for 10 minutes. TRACP staining was performed using the leukocyte acid phosphatase kit (LEUKOCYTE ACID PHOSPHATASE KIT, Sigma). The TRACP staining solution was removed, washed twice with distilled water, dried, and analyzed for TRACP-positive multinucleated osteoclasts under an optical microscope (X100).

As shown in FIG. 1, in the experimental group treated with dibenzazepine, the number of osteoclasts of multinucleated cells that were TRACP positive was significantly reduced in comparison with the control group treated with dibenzazepine. As a result, the pharmaceutical composition for the prevention and treatment of bone metabolic diseases comprising dibenzazepine is 10 ^-8 M 1α, 25-dihydroxyvitamin D (25-dihydroxyvitamin D₃) and 10 under coexistence conditions of bone marrow cells and osteoblasts. ^-6 M prostasin Rogaland Dean ^{_{E 2 (10 -6 M prostaglandin E₂}} ) strongly inhibited the formation of osteoclasts generated by.

Example 2-2. Dibenzazepine Inhibits Osteoclast Fusion

Osteoclasts are capable of absorbing bones by fusion of hematopoietic stem cell mononuclear progenitor cells into an average of 10 to 20 giant multinucleated cells, which is why osteoclast fusion is very important in osteoclast function. It is important.

In this example, the effect of dibenzazepine on osteoclast fusion inhibition was measured using macrophages, which are mononuclear progenitor cells derived from hematopoietic stem cells. Specifically, macrophages, which are bone marrow-derived mononuclear progenitor cells of mouse hematopoietic cells, are cultured to differentiate into mature osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). In order to determine the effect of the dibenzazepine on the fusion ability of the osteoclasts was carried out as follows.

As described in Example 1-1, mice were obtained with bone marrow cells, suspended with 10% alpha-MEM, added M-CSF (30 ng / ml), and cultured in a 100 cm culture dish for 3 days. It was. After 3 days, M-CSF (30 ng / ml) and RANKL (100 ng / ml) were added to the culture medium and treated with 1μM dibenzazepine to the culture composition, followed by incubation for 4 days to induce differentiation into osteoclasts. It was. The control group used osteoclasts induced differentiation without treatment with dibenzazepine. Differentiation-induced osteoclasts were stained with TRACP (Tartrate-resistant acid phosphataseform) staining to observe TRACP-positive osteoclasts under an optical microscope (X100). In addition, 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. 2.

As shown in FIG. 2, dibenzazepine slightly contracted the cell membrane morphology of mature osteoclasts, but exhibited a slightly smaller cell morphology than normal osteoclasts, but did not affect the formation of TRACP-positive multinucleated osteoclasts and the number of intracellular nuclei. Did. As a result, dibenzazepine did not appear to affect cell fusion, an important process for the formation of mature multinucleated osteoclasts.

Examples 2-3. Dibenzazepine Inhibits Osteoclast Formation

The osteoskeletal cell skeletal process involves attaching bone to the extracellular matrix and the space that actually absorbs the bone, while at the same time organizing the actin of the osteoclast into one large ring. The formation of rings is an important marker for the ability of osteoclasts to absorb bone.

In this example, the effects of dibenzazepine on osteoclast actin ring formation were analyzed using macrophages, which are mononuclear progenitor cells derived from hematopoietic stem cells. Specifically, a system for differentiating mature osteoclasts in the presence of M-CSF and RANKL by culturing macrophages, bone marrow-derived mononuclear progenitor cells of mouse hematopoietic cells, and effect of dibenzazepine on actin ring formation of osteoclasts Was measured as follows.

To investigate the effect of dibenzazepine on osteoclast actining formation, macrophages were treated with 1 μM dibenzazepine in the presence of M-CSF and RANKL. In order to distinguish the actining formation of osteoclasts differentiated from macrophages by the culture composition, immunocytostaining was performed with Rhodamine-Phalloidin antibody, an actin-forming marker. The cultured osteoclasts were washed with PBS, fixed with 2% paraformaldehyde, and blocked with nonspecific binding with bovine serum albumin. After reacting with the addition of actin-forming marker rhodamine-palloidine antibody was prepared with a ProLong ^® Gold antifade reagent with DAPI (Invitrogen, San Diego, CA) and observed by confocal microscopy (X100).

As shown in FIG. 3, the experimental group treated with dibenzazepine compared to the control group not treated with dibenzazepine did not normally perform rhodamine-palloidine staining of actin around the cell membrane. In addition, it was confirmed that dibenzazepine inhibits normal actin ring formation and the cell morphology of the cell membrane contracted by dibenzazepine in FIG. 2.

Examples 2-4. Inhibition of Bone Absorption of Dibenzazepine through Bone Absorption Experiment

As in Example 1-2, the bone marrow cells and osteoblasts were co-cultured with the bone marrow cells to support osteoblasts, and then applied to a system that differentiates into mature osteoclasts. To determine the effect of dibenzazepine on bone resorption after the separation, the experiment was performed as follows.

To induce differentiation of mature osteoclasts, bone marrow cells (1 x 10 ⁷ ) and primary osteoblasts (1 x 10 ⁶ ) are inoculated in a collagen-coated culture dish, and 10 ^-8 M 1α, 25-dihydroxyvitamin D₃ And 10 ^-6 M prostaglandin E ₂ was incubated for 6-7 days. The cultured cells were treated with 0.2% collagenase (collagenase, Invitrogen) for 10 minutes at 37 ° C, and then reinoculated into a culture dish for osteoclast differentiation induction experiments. 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 plate were obtained.

For bone uptake experiments, 1 μM dibenzazepine was reinoculated with osteoclasts onto OAAS plates coated with carbonated calcium phosphate (Osteogenic Core Technologies Inc., South Korea) and left for 12 hours. Thereafter, the cells were cultured in the presence of RANKL (100 ng / ml), and after 24 hours, the cells were removed, and the whole pit pictures formed by the osteoclasts were taken and a Pro-Plus program (version) was taken. 4.0, Media Cybernetics).

As a result, as shown in FIG. 4, the pharmaceutical composition containing dibenzazepine significantly reduced the formation of absorbing pit generated in the OAAS plate, thereby exhibiting a substantial bone retardation effect by dibenzazepine.

Example 3 Inhibition of Bone Absorption of Dibenzazepine Animal Experiment

Example 3-1. Inhibitory Activity of Dibenzazepine Induced Interleukin-1 (IL-1) in Mice

To determine the effect of dibenzazepine on the inhibition of bone loss in mice, the effect of intraleukin-1-induced mouse cranial loss in vivo by Ha et al. (Journal of Immunology, 2006, 176: 111-117) The experiment was carried out by the reported method. Specifically, 5-7 ml of collagen (Cellmatrix type IA, Wako co., Japan) was poured into a Petri dish (60 × 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 mice. 2 μg of each mouse was soaked in the collagen sponge. Incision of the scalp of the mouse was made so that the collagen sponge treated with IL-1 solution was brought into contact with the skull surface, and transplanted into 6 mice (5 weeks old, male, and ICR strain), respectively, in the experimental and control groups. Sutures were grown for 7 days and sacrificed to give the entire skull tissue. At this time, dibenzazepine was mixed with DMSO (50 μl) and physiological saline (50 μl) at a dose of 60 and 200 (μg / kg), once every two days, from the day before the operation to the day before the sacrifice of the mice. Dissolved in and administered by intraperitoneal injection. As a control group (vehicle treatment group or negative control group), a solution containing DMSO (50 μl) and physiological saline (50 μl) treated with dibenzazepine was administered by intraperitoneal injection. After 7 days, the mice were sacrificed and the whole tissue of the extracted mouse skull was washed 3-4 times with physiological saline and fixed in 4% paraformaldehyde solution for 24 hours. Immobilized mouse skulls were subjected to tartrate-resistant acidic phosphatase (TRACP) staining and micro-computed tomography scan (SMX-90CT, Shimadzu, Japan). The results are shown in FIG. 5A (Volume Graphics, VG studio Max 1.2.1). In addition, the three-dimensional image file was measured using the TRI 3D-BON (RATOC system Engineering Co. Tokyo, Japan) program to measure the bone mineral content (mg mineral content) and the volume of bone tissue (mm ³ ) Figure 5 Indicated on

As a result, as shown in Figure 5, the vehicle treated group used as a negative control actively progressed bone loss by interleukin-1, induced severe breakage of the skull tissue as shown in the three-dimensional image and TRACP positive in the same site The site was increased. In addition, although the bone tissue volume and bone-mineral amount were significantly reduced in these skull tissues, the dibenzazepine of the present invention inhibited the bone loss of the skull and showed little damage to the skull tissues through 3D images. It was confirmed that the tissue volume and the overall bone-mineral amount were increased in a concentration-dependent manner of dibenzazepine compared to the vehicle treated group. Therefore, the bone resorption inhibitory activity of the dibenzazepine identified at the cellular level was confirmed through the mouse cranial disappearance model.

Example 3-2. Histological Analysis of Inhibition of Mouse Skull Loss of Dibenzazepine

In order to determine histologically the effect of the dibenzazepine on the inhibition of bone loss in mice, the microcranial tomography of the mouse cranial tissues in Example 3-1 was washed with water, fixed and demineralized for 2-3 weeks. And a paraffin block was produced. For histological analysis, paraffin blocks were cut 5-6 mm thick using a razor-attached rotary microtome, attached onto an experimental glass plate, and in vivo osteoclast formation and bone resulting from bone loss. TRACP (tartrate-resistant acidic phosphatase) staining was performed to investigate the loss pattern. The total area of TRACP positive osteoclast formation was shown graphically using the mage J program.

As a result, as shown in FIG. 6, the vehicle treated group used as a negative control group actively promoted bone loss by interleukin-1, resulting in irregular morphology of the skull tissue surface and promoting osteoclast formation leading to bone loss. On the other hand, the compound was found to significantly reduce the formation of osteoclasts causing bone loss and at the same time stabilize the skull tissue surface.

Example 4 Effect of Dibenzazepine on Accelerating Osteoblast Differentiation

Osteoblasts originate from mesenchymal stem cells and develop into mature osteoblasts that form bones through differentiation. Mineralization, including calcium, produced by osteoblast differentiation not only maintains bone strength, but also the entire body. Is also very important for hormone metabolism and calcium homeostasis. The most urgent problem in studying bone-related diseases is not only to inhibit the activation of osteoclasts but also to enhance the ability to form new bone tissue by inducing the promotion of osteoblast activation. This is because severe bone loss is already advanced, as can be seen in the characteristics of most bone-related patients.

In this example, a system is formed in which the primary osteoblasts of the mouse are cultured to differentiate into mature osteoblasts that can produce a substantial bone nodule. In this system, dibenzazepine promotes osteoblast differentiation. The experiment was conducted in the following way.

Osteoblast differentiation promoting medium (osteogenic media, alpha-MEM + 10 mM beta-glycerophosphate (sigma) + 50 μg / mL ascorbate-2-phosphate (1) in order to differentiate the osteoblast precursors obtained in Example 1-1 into osteoblasts sigma) + 30 ng / mL BMP-2 (Daewoong Pharmaceutical, South Korea)). Dibenzazepine was treated in the culture solution at each concentration (0.1, 0.5 μM) and then cultured for 10 days to induce differentiation into osteoblasts. On day 10 of the culture, the calcified osteoblasts were stained by treating the cells with a 0.2% Alizarin Red S solution (pH 4.3; Sigma) stain. In addition, the expression level of messenger RNA of osteocalcin, a marker for differentiation of mature osteoblasts, was analyzed by real-time PCR. At this time, the control group used osteoblasts induced differentiation without treatment with dibenzazepine.

As a result, as shown in FIG. 7, osteoblast differentiation was promoted in the culture dish treated with dibenzazepine with osteoblast differentiation promoting medium and osteoblast differentiation was increased compared to the control without dibenzazepine, and the bone calcification was increased to alizarin red S. Stained bone nodule was significantly increased. In addition, messenger RNA expression of osteocalcin, a osteoblast differentiation marker, was increased by dibenzazepine, and thus the osteoblast activating ability of dibenzazepine was increased according to the concentration of dibenzazepine.

Example 5: Bone Formation Promoting Animal Experiment of Dibenzazepine

To investigate the effect of dibenzazepine of the present invention on new bone formation, the effects of dibenzazepine on bone regeneration in the skull defect area in vivo were described by Kim et al. (Journal of Bone and Mineral Research, 2011, 26). (2161-2173), the experiment was carried out by the method reported.

Approximately 250 grams of male Sprague-Dawley rats (Orient Bio, South Korea), 2 months old, form a circular bone defect with a diameter of 8 mm and a collagen sponge containing dibenzazepine (Bioland, South Korea). After implantation, periosteum and skin were double sealed. At this time, a collagen sponge containing only DMSO and physiological saline mixed solution in which dibenzazepine was dissolved was used as a negative control group, and a collagen sponge containing BMP-2 (2 µg) as a positive control group. Three weeks later the rats were sacrificed and the skulls removed. The extracted skull was washed with physiological saline and then micro computed tomography was performed. The three-dimensional image was obtained and the results are shown in FIG. 8. In addition, the three-dimensional image file by using a TRI 3D-BON program is shown in a graph measuring the new bone regeneration rate and the thickness of the skull tissue for each group.

As a result, as shown in Figure 8, compared to the vehicle-treated group used as a negative control, the dibenzazepine treatment group is a new bone than the empty space caused by bone defects on the skull tissue surface, as a result of the positive control group BMP-2 The formation of more bone-filled space by formation was shown in three-dimensional images through micro computed tomography, and it was found that bone regeneration rate and thickness of skull tissue were significantly increased by dibenzazepine.

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)

As a pharmaceutical composition for the prevention or treatment of bone metabolic diseases comprising dibenzazepine or a pharmaceutically acceptable salt thereof,
The bone metabolic disease is a pharmaceutical composition 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 dibenzazepine 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 of claim 1, wherein the composition is formulated in the form of an oral dosage form selected from the group consisting of granules, tablets, pills, capsules, suspensions, emulsions, syrups and aerosols, or sterile injectable solutions. .
As a plant composition for the prevention or amelioration of bone metabolic diseases comprising dibenzazepine or a pharmaceutically acceptable salt thereof,
The bone metabolic disease is a food composition 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 food acceptable food additive.

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