KR101827878B1 - Compositon comprising topiramate for metabolic bone disease - Google Patents

Abstract

The present invention relates to a composition comprising topiramate for promoting osteoblast differentiation. The composition for promoting osteoblast differentiation comprising topiramate according to the present invention increases the expression level of any one of Id-1, Dlx-5 and Runx-2, which are osteoblast differentiation marker genes, increases the activity of alkaline phosphatase, an osteogenic marker, and increases phosphorylation of any one of signaling proteins among Smad1, Smad5, and Smad9, thereby preventing and treating metabolic bone diseases and improving bone functions.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing and treating bone metabolic diseases including topiramate,

The present invention relates to a composition for prevention and treatment of bone metabolic diseases including topiramate.

In modern society, the number of people with senile disease continues to increase due to an increase in life expectancy. Therefore, management of geriatric diseases is important. Among them, bone diseases such as osteoporosis and degenerative arthritis are diseases affected by environmental factors and genetic factors such as diet and lifestyle. The causes of these bone disease patients are not only diverse, but also require a lot of cost and effort for treatment. In particular, the incidence of postmenopausal women is high. As a result, it has been desired to develop osteoporosis therapeutic supplements and various health functional foods by the burden of the increasing number of people.

Bone is a dynamic tissue that forms the body structure, and the process of balancing osteoclast bone absorption and osteoblast bone formation is constantly occurring. In general, adult bone resorption and formation maintain homeostasis, and bones have periodicity and reshaping. The bone remodeling is a process of replacing the old bone with a new bone after the growth has been completed. It maintains the homeostasis according to the balance of osteoblast and osteoclast activity. There are two phenomena that the osteoblast activity becomes weak or the osteoclast activity becomes strong Bile metabolic disease occurs when combined.

Methods for the prevention and treatment of bone metabolic diseases can be divided into a mechanism of decreasing the activity of osteoclasts or increasing the activity of osteoblasts.

Currently available osteoporosis medications are vitamin D, female hormone, bisphosphonate, selective estrogen receptor modulator, and calcitonin preparation. Most of them inhibit the loss of bone by regulating osteoclast activity by decreasing osteoclast activity.

Korean Patent Laid-Open No. 10-2017-0019790 discloses a composition for inhibiting osteoclast differentiation using CTRP3 as an active ingredient and a therapeutic agent for bone diseases comprising the same, and Korean Patent Publication No. 10-2015-0019179 Suggests a pharmaceutical composition for osteoclast formation and inhibition of bone resorption, including (2S) -2'-methoxycurarinone and a pharmaceutically utilizable carrier, but most of them inhibit osteoclast differentiation The present invention relates to an agent for preventing and treating osteoporosis by increasing the activity of osteoblast.

On the other hand, topiramate (2,3: 4,5-bis-o- (1-methylethylidene) β-D-fructo-pyranose sulfamate (TPM) was the first treatment for seizures, . In 2008, the drug was used as an adjunct to existing drug therapy to control intractable partial seizures by blocking the interstitial effects of antidepressant drugs. In 2008, FDA Of the drug.

TPM is known to exhibit various biochemical / pharmacological effects that can modulate a wide range of nervous system activities, including anticonvulsants, analgesics, and emotion-stabilizing properties. TPM positively affects the activity of gamma-amiobutyric acid (GABA) in GABAA receptors, and the effects of glutamate on kainate / AMPA and CA-Ⅱ and CA-Ⅳ of voltage-dependent sodium channels and carbonic anhydrase (CA) isomers Negative control over activity.

In general, the mechanisms of anticonvulsants are related to the enhancement of GABA (gamma-aminobutyric acid) activity and the inhibition of AMPA receptors and voltage-sensitive sodium and calcium channels. TPM showed weight loss effects in both humans and rats in preclinical studies for the treatment of seizures. This differs significantly from other anticonvulsants, which generally lead to weight gain.

There have been no studies related to osteoblast differentiation and bone metabolic disease prevention and treatment using antipsychotic drugs. The present inventors have studied osteoblast differentiation effect using antipsychotic drugs from Topiramate, which is an antipsychotic drug, And the possibility of prevention and treatment of bone metabolic diseases, leading to the present invention.

Korean Patent Publication No. 10-2017-0019790 (composition through inhibition of osteoclast differentiation using CTRP3 as an active ingredient and therapeutic agent for bone diseases containing the same) Korean Patent Laid-Open No. 10-2015-0019179 (a pharmaceutical composition for osteoclast formation and inhibition of bone resorption)

Disclosure of the Invention In order to solve the above problems, an object of the present invention is to provide a pharmaceutical composition for promoting osteoblast differentiation comprising topiramate.

Another object of the present invention is to provide a functional food composition for improving bone function comprising topiramate.

In order to solve the above problems, the pharmaceutical composition for promoting osteoblast differentiation comprising topiramate of the present invention may be prepared by including topiramate and a pharmaceutically acceptable salt, and the pharmaceutical composition for promoting osteoblast differentiation The composition can be used for the improvement of bone function, the treatment and prevention of bone metabolic diseases.

The term "pharmaceutically acceptable salt" of the present invention means salts prepared by conventional methods, and can be used without limitation in methods known to those skilled in the art. "Pharmaceutically acceptable salts" include, but are not limited to, salts derived from the following inorganic and organic acids and bases which are pharmacologically or physiologically acceptable. Suitable acids include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, , Benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals, such as sodium, alkaline noble metals, such as magnesium, ammonium, and the like.

The term "osteoblast" as used herein means a cuboidal cell that forms bone, and refers to a cell that undergoes osteogenesis and remodeling. The bone is composed of bone cells and bone matrix. The bone cells are again divided into osteoblasts and osteoclasts. Osteoclasts dissolve and break down existing bone components to continue bone growth and absorb bone matrix. Osteoblasts make collagen in a lattice-shaped part of osteoclast-decomposed part, and calcium, magnesium, phosphorus To create new bone matrix. The composition of the present invention can promote bone formation by promoting osteoblast differentiation.

The term "bone metabolic disease" as used herein includes, without limitation, diseases that are problematic in osteogenesis due to lack of osteoblasts and the like, for example, bone destruction by interleukin-1 (IL-1) Inflammatory diseases and the like, and can be used for osteoporosis caused by excessive osteoclast bone resorption, bone metastatic lesion, primary bone tumor, rheumatic or degenerative arthritis, periodontal disease , Inflammatory alveolar bone disorder, inflammatory bone resorption disease, Paget's disease, and the like, but are not limited to the above-mentioned bone metabolic diseases.

The term "prophylactic" in the present invention refers to any act that inhibits or delays the onset of the disease by administration of a composition comprising topiramate according to the present invention.

The term "treatment" in the present invention refers to any action that improves or alleviates the symptoms of the disease upon administration of a composition comprising topiramate according to the present invention.

The term "administering" in the present invention means introducing the composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention may be carried out through various routes of oral or parenteral routes, And can be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular or intradermal routes. A method of treatment of the invention comprises administering a composition of the present invention in a pharmaceutically effective amount. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by the practitioner within the scope of sound medical judgment. The specific therapeutically effective amount for a particular patient will depend upon a variety of factors, including the type and extent of the response to be achieved, the specific composition, including whether or not other agents are used, the age, weight, general health status, sex and diet, The route of administration and the fraction of the composition, the duration of the treatment, the drugs used or co-used with the specific composition, and the like, well known in the medical arts. Therefore, the effective amount of the composition suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned factors.

In addition, the composition of the present invention is applicable to any animal in need of treatment and prevention of bone metabolic diseases, and the animal includes not only humans and primates but also livestock such as cows, pigs, sheep, horses, dogs and cats.

The topiramate concentration in a pharmaceutical composition for promoting differentiation of osteoblast comprising Topiramate of the present invention is not particularly limited and may be any concentration that can induce osteoblast differentiation, 1 to 50 [mu] M.

According to a specific embodiment of the present invention, the topiramate increases the expression level of any one of Id-1, Dlx-5 and Runx-2, which are osteoblast differentiation marker genes, and the expression level of osteogenic markers such as alkaline phosphatase Alkaline phosphatase) activity was increased. In addition, it was confirmed that the topiramate increased phosphorylation of one or more signal transduction proteins of Smad1, Smad5, Smad9.

The pharmaceutical composition for promoting osteoblast differentiation comprising topiramate of the present invention may further comprise an appropriate carrier, excipient or diluent according to a conventional method. As used herein, the term "carrier" refers to a carrier, excipient or diluent that does not significantly stimulate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition for promoting osteoblast differentiation comprising topiramate according to the present invention may be formulated into oral compositions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, A suppository, or a sterile injectable solution.

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

In another aspect, the present invention provides a functional food composition for improving bone function comprising topiramate.

The functional food composition containing Topiramate of the present invention can be used in a variety of foods and beverages for the improvement of bone function, the treatment and prevention of bone metabolic diseases, and the like. Specific examples thereof include various foods such as extracts, concentrates, beverages, gums, tea, vitamin complexes and health supplements. They can be used in form of pills, powders, granules, infusions, tablets, capsules or beverages But are not limited to.

As described above, according to the composition for promoting osteoblast differentiation comprising topiramate according to the present invention, osteoblast differentiation markers and ALP activity are enhanced to increase osteoblast differentiation, thereby preventing or treating bone metabolic diseases, There is an effect of improving.

FIG. 1 is a graph showing cytotoxicity according to the concentration of TPM in MC3T3-E1 cells. FIG.
FIG. 2 shows experimental results showing the expression of TPM-induced osteoblast differentiation marker gene.
FIG. 3 shows experimental results showing the activity of ALP by TPM.
Figure 4 shows Western blot results showing protein expression by TPM.

Specific features and advantages of the present invention will be described in detail below with reference to the accompanying drawings. The detailed description of the functions and configurations of the present invention will be omitted if it is determined that the gist of the present invention may be unnecessarily blurred.

1. Materials and Methods

1-1. Cell culture

The MC3T3-E1 cells, which are derived from mouse calvaria, were purchased from ATCC CRL-2593 (American Type Culture Collection, Manassas, VA, USA). (100 μg / mL penicillin-100 μg / mL streptomycin (GibcoBRL)) supplemented with 10% FBS (Atlas Biologicals, Fort Collins, CO, USA) and α-MEM (GibcoBRL, Grandisland, NY, USA) And incubated at 37 ° C in a 5% CO ₂ incubator.

1-2. Cytotoxicity test

MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) assay was performed to determine the cytotoxicity of TPM to osteoblast. MC3T3-E1 cells were inoculated into 48 well plates at a density of 1 × 10 ⁴ cells / well, cultured for 24 hours, treated with TPM at 1, 20, 50, 100, and 200 μM for 24 hours. The cells were treated with 0.05 mg / mL MTT (Sigma Aldrich, St. Louis, Mo., USA) for 1 hour and then the medium was removed. Insoluble formazan crystals were dissolved in DMSO (Duchefa, Haarlem, The Netherlands) And the absorbance was measured at 540 nm.

1-3. Gene expression assay

Total RNA was extracted and isolated from MC3T3-E1 cells and C3H10T1 / 2 cells using Tri-solution (Bioscience technology, Gyeongsan, Korea). Complementary DNA (cDNA) was synthesized using 3 μg RNA with TOP script ™ RTDryMIX (Enzynomics, Daejeon, Korea). A mixture of 19 μL of a primer and 1 μL of cDNA was subjected to a polymerase chain reaction (PCR) cycle 30 times with Emerald Amp GT PCR Master Mix (TaKaRa Bio Inc., Tokyo, Japan). The PCR reaction conditions were 95 ° C for 10 minutes, 95 ° C for 30 seconds, annealing temperature for 30 seconds, 72 ° C for 30 seconds, and 72 ° C for 5 minutes. qPCR was performed 40 cycles of polymerase chain reaction (PCR) with 19 μL of a mixture containing AmpiGene ™ qPCR Green Mix Hi-ROX (Enzo Life Sciences, Farmingdale, NY, USA) and primer and 1 μL of cDNA. The primer information used in the polymerase reaction is shown in Table 1 Respectively.

1-4. Alkaline 포스화제 (ALP) staining

In order to measure the activity of ALP expressed on osteoblast differentiation, MC3T3-E1 cells were seeded at 24 × 10 ⁴ cells / well on 24 weII plates and treated with 50 μM TPM every other day for 4 days. After 4 days, the culture medium of the plate was removed and fixed with 4% formaldehyde for 5 minutes. After washing three times with PBS, BCIP / NBT (Sigma aldrich) was treated with rocker. After the reaction, the plate was washed with distilled water and the stained plate was scanned.

1-5. Western blot analysis

The culture medium of MC3T3-E1 cells cultured in a 60 mm dish was removed and washed twice with PBS. RIPA buffer (Thermo Scientific) containing protease inhibitor (Roche) and phosphatase inhibitor (Roche) was added to the cells and incubated for 5 minutes in ice. The dish was transferred to a microcentrifuge tube, centrifuged at 13,000 rpm for 10 min at 4 ° C, and the supernatant was transferred to a new microcentrifuge tube. The extracted proteins were quantified by the Bradford method. The same amount of protein was added to the sample buffer, boiled at 100 ° C for 5 minutes, and incubated for 10 minutes at 100 V in SDS polyacrylamide gel electrophoresis (SDS-PAGE) 10% gel and for 1 hour 30 minutes at 120 V. Mini-PROTEAN ^® Tetra CeII (Bio-Rad). The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane at 100 V for 1 hour using a transfer device. Then, ponceau (Sigma) was stained. After confirmation, it was washed with Tris-buffered saline with Tween 20 (TBST) buffer. After blocking the membrane with 5% skim milk (Sigma), the primary antibody was reacted at 4 ° C overnight or at room temperature for 2 hours. After washing four times for 10 minutes with TBST, the secondary antibody was reacted at room temperature for 2 hours. After washing four times with TBST for 10 min each, WesternBright ECL (Advansta Inc.) reagent 1 and reagent 2 were mixed at a ratio of 1: 1 and the membrane was treated with Fusion solo (Vilber Lourmat, Eberhardze IIl, Germany) The primary antibodies used in the experiments were Runx2 (Santa Cruz Biotechnology, Inc.), p-Smad1 / 5/9 (Abcam), t-Smad1 (abcam), and β- actin (Santa Cruz Biotechnology, Inc.) Secondary antibodies are goat anti-rabbit (Santa Cruz Biotechnology, Inc.), goat anti-mouse (Santa Cruz Biotechnology, Inc.), rabbit anti-goat (Santa Cruz Biotechnology, Inc.).

1-6. Statistical processing

The results obtained in this study were expressed as standard deviations. Statistical analysis was performed using Student's t-test. Significant differences were found between the control and TPM treatment groups when * P <0.05, ** P <0.01, *** P < .

2. Experimental results

2-1. Cytotoxicity test

MC3T3-E1 cells are osteoblast cell lines derived from mouse calvaria and have metabolic characteristics such as osteoblast proliferation, differentiation and calcification, and are used in studies related to bone formation. The toxicity of TPM was confirmed in MC3T3-E1 cells by MTT assay.

FIG. 1 is a graph showing cytotoxicity according to the concentration of TPM in MC3T3-E1 cells. FIG.

At the concentration of 1, 20, 50, 100, 200 μM, no cytotoxicity was observed at the concentrations of 1, 20, and 50 μM, but toxicity was observed at the concentrations of 100 μM. Therefore, the experiment was carried out at a concentration of 50 μM in the subsequent experiments.

2-2. TPM  Gene expression

TPM confirmed mRNA levels of marker genes of osteoblast differentiation in MC3T3-E1 cells. MC3T3-E1 cells were treated with 50 μM of TPM and cultured for 4 days. Expression of each gene was confirmed by RT-PCR method.

Fig. 2 shows the expression of the osteoblast differentiation marker gene induced by topiramate.

Expression of the marker genes Id1, Dlx5, and Runx2 in osteoblast differentiation was increased. Id1 showed up to 2 days, Dlx5 and Runx2 showed up to 4 days time-dependent increase in mRNA expression.

This indicates that TPM induces osteoblast differentiation by increasing the expression of the marker gene in osteoblast differentiation.

2-3. TPM  ALP activity by

Based on the increase in osteoblast marker gene expression by TPM, MC3T3-E1 cells were treated with 50 μM TPM to differentiate into osteoblasts, and the degree of osteoblast differentiation was confirmed by ALP staining.

Figure 3 shows the results of experiments showing ALP activity by TPM.

ALP activity was increased in the TPM-treated group compared to the control group without any treatment, and ALP activity was observed in the TPM-treated cells compared with the cells treated with ascorbic acid and β-glycerophosphate , Respectively. Therefore, it is suggested that TPM promotes osteoblast differentiation by increasing ALP activity in osteoblast differentiation.

2-4. TPM  Protein expression by

In order to confirm the osteoblast differentiation at the protein level, TPM increases the expression of the marker gene in the osteoblast differentiation and increases ALP activity. In order to confirm the osteoblast differentiation at the protein level, the expression of Runx2, a key transcription factor of osteoblast differentiation, 5/9 phosphorylation was confirmed by Western blotting.

Figure 4 shows protein expression by TPM.

The protein expression of Runx2 was increased in cells treated with ascorbic acid and β-glycerophosphate to induce osteoblast differentiation, and also increased in TPM-treated cells, and increased with ascorbic acid, β-glycerophosphate and TPM (Fig. 4A). In addition, phosphorylation of Smad1 / 5/9 was TPM-treated alone, confirming a time-dependent increase up to 2 hours (Fig. 4B). The results suggest that TPM not only increases protein expression of Runx2, a key transcription factor for osteoblast differentiation, but also promotes osteoblast differentiation by increasing the phosphorylation of Smad1 / 5/9, an important signaling protein in the BMP signaling pathway it means.

These results suggest that TPM promotes the phosphorylation of Smad1 / 5/9, a signaling protein in the BMP signaling pathway, and increases the expression of Id1, Dlx5, and Runx2, the marker genes of osteoblast differentiation. Among them, The protein expression of Runx2, a key transcription factor, was also increased. In addition, it was confirmed that the activity of ALP was increased.

To date, most studies on TPM are antipsychotic drugs such as epilepsy, seizures, and depression, and research has been conducted on weight loss after approval of FDA as an obesity inhibitor, but research on bone has not been conducted As a result of the present invention, TPM, which is an antipsychotic drug, may be related to bone related diseases such as osteoporosis and osteopenia, and may be an example showing that antipsychotic drugs may affect bone health , And it is expected to provide a basis for application to bone related diseases using antipsychotic drugs through continuous research in the future.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken as limiting the scope of the present invention. The present invention can be variously modified or modified. The scope of the invention should, therefore, be construed in light of the claims set forth to cover many of such variations.

Claims (8)

A pharmaceutical composition for the prevention and treatment of bone metabolic diseases including topiramate.
The method according to claim 1,
Wherein the topiramate concentration is 1 to 50 [mu] M
A pharmaceutical composition for the prevention and treatment of bone metabolic diseases.
The method according to claim 1,
The topiramate
Wherein the expression level of the osteoblast differentiation marker gene is increased
A pharmaceutical composition for the prevention and treatment of bone metabolic diseases.
The method of claim 3,
The osteoblast differentiation marker gene
Id-1, Dlx-5, and Runx-2.
A pharmaceutical composition for the prevention and treatment of bone metabolic diseases.
The method according to claim 1,
The topiramate
Characterized in that the bone formation marker is increased in activity
A pharmaceutical composition for the prevention and treatment of bone metabolic diseases.
6. The method of claim 5,
The bone formation marker
Characterized in that it is an alkaline phosphatase
A pharmaceutical composition for the prevention and treatment of bone metabolic diseases.
The method according to claim 1,
The topiramate
Smad1, &lt; / RTI &gt; Smad5, Smad9, &lt; RTI ID = 0.0 &gt;
A pharmaceutical composition for the prevention and treatment of bone metabolic diseases.
A functional food composition for improving bone function comprising topiramate.

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