WO2018147516A1 - Composition for preventing or treating bone diseases comprising mussel protein-derived peptide as active ingredient - Google Patents

Abstract

The present invention relates to an osteogenic promotion peptide comprising sequence number 1 (PIISVYWK) or sequence number 2 (FSVVPSPK), and a composition, for preventing or treating bone diseases, comprising the peptide. The peptide promotes osteoblast differentiation and thus can be utilized for a bone disease treatment.

Description

Composition for the prevention or treatment of bone diseases, including mussel protein-derived peptide as an active ingredient

The present invention relates to a composition for preventing or treating bone diseases comprising mussel protein-derived peptide as an active ingredient.

Osteoporosis is postmenopausal osteoporosis, which is indicated by increased bone resorption due to the activation of osteoclasts due to rapid hormonal changes following menopause, and senile osteoporosis, in which osteoblasts decrease due to aging and osteoblasts decrease. Can be classified as Since osteoporosis fractures lead to severe activity limitations and are associated with a high mortality rate of about 15-35% in hip fractures, the diagnosis and treatment of osteoporosis is important before osteoporotic fractures occur (osteoporosis diagnosis). And Treatment Guidelines 2007, 2008).

Known osteoporosis treatments include bisphosphonate-based drugs, which are deposited on the mineral component of the bone and form an ATP analog that does not hydrolyze when osteoclasts phage on the bone where the bisphosphonate is deposited. It is known to reduce the bone resorption and thereby increase the bone density by causing toxicity to cells or causing osteoclast activity and apoptosis in various ways in osteoclasts. These drugs are known to be relatively safe, but in recent years, their long-term use affects bone remodeling or bone regeneration after bone fracture due to normal bone resorption and bone formation. Concerns have been raised about the impact, and in fact, there are reports of fatigue fractures in many patients. Therefore, there is an urgent need for the discovery of new mechanisms of bone metabolism associated with the development of osteoporosis and the need for development of preventive or therapeutic agents for osteoporosis.

An object of the present invention to provide a peptide for promoting bone formation comprising SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK).

Another object of the present invention to provide a composition for the prevention or treatment of bone diseases comprising the peptide.

Another object of the present invention to provide a composition for promoting osteoblast differentiation comprising the peptide.

In order to achieve the above object, the present invention provides a peptide for promoting bone formation, including SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK).

In one embodiment of the invention, the peptide may be derived from mussel protein.

In one embodiment of the invention, the peptide may be to promote differentiation of osteoblasts.

The present invention also provides a composition for the prevention or treatment of bone diseases comprising the peptide.

In one embodiment of the present invention, the bone disease may be any one selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease.

In addition, it provides a composition for promoting osteoblast differentiation comprising the peptide.

Peptide according to the present invention is a peptide for promoting bone formation comprising SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK), the peptide has an effect of promoting the differentiation of osteoblasts can be useful for the treatment of bone diseases Can be.

Figure 1 shows the results of measuring the ALP activity after the treatment of the PIISVYWK and FSVVPSPK peptide ( ^{a, b} p <0.05).

Figure 2 shows the results confirmed by Western blotting whether the protein expression involved in osteoblast differentiation after treatment with PIISVYWK and FSVVPSPK peptide.

Figure 3 shows the results confirmed by Western blotting whether the expression of MAPKs involved in osteoblast differentiation after treatment with PIISVYWK and FSVVPSPK peptide.

Figure 4 shows the results of confirming whether the mineral formation of osteoblasts after treatment with the PIISVYWK and FSVVPSPK peptide ( ^ac p <0.05).

Figure 5 shows the results of activation of the BMP signaling pathway with or without the treatment of PIISVYWK and FSVVPSPK peptide and BMP antagonist.

Figure 6 shows the results of activation of the MAPKs signaling pathway with or without the treatment of PIISVYWK and FSVVPSPK peptide and BMP antagonist.

Figure 7 shows the results of activation of the BMP signaling pathway after treatment with PIISVYWK and FSVVPSPK peptides and MAPKs inhibitors.

Figure 8 shows the results of ALP activity after treatment with PIISVYWK and FSVVPSPK peptides and MAPKs inhibitors.

FIG. 9 is a graph showing the quantification of the change in bone mineral density by micro CT after the PIISVYWK (P1), FSVVPSPK (P2) peptide, and estrogen (17β-estradiol) were administered to the osteoporosis model. will be.

The term "bone formation" of the present invention refers to the formation of bone tissue by the deposition of lime salt on the bone tissue.

The term "osteoblast" is a cell having the ability to calcify bone tissue by synthesizing and secreting bone matrix and depositing inorganic salts such as calcium and magnesium ions on a substrate. It can be seen at the site of the neonatal. In addition, bone formation in the advanced state is a cell that is buried in the bone tissue formed by itself become bone cells.

As used herein, the term "prevention" means any action that inhibits or delays the development of a bone disease by the administration of the pharmaceutical composition of the present invention. Any action that improves or beneficially changes symptoms.

The route of administration of the pharmaceutical composition of the present invention may be administered via any general route as long as it can reach the target tissue (such as a bone defect site) or cell. The pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, pulmonary, rectally, intracellularly or indirectly, as desired. To this end, the pharmaceutical compositions of the present invention may be administered by any device in which the active substance may migrate to the target cell.

The pharmaceutical composition of the present invention may comprise an acceptable carrier. The pharmaceutical composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablet pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose in one or more compounds. ) And gelatin. Liquid preparations for oral administration include suspensions, solution solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition of the present invention is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It can have either formulation.

The pharmaceutical composition of the present invention is administered in a therapeutically effective amount or in a pharmaceutically effective amount. The term “therapeutically effective amount or pharmacologically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, with an effective dose level representing the type of subject and its severity, age, sex, activity of the drug , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts.

The present invention provides a method for treating bone disease, comprising administering to a subject a pharmaceutically effective amount of said bone formation promoting peptide, or a composition comprising said peptide.

The present invention also provides a method for preventing bone disease, comprising administering to a subject a pharmaceutically effective amount of the bone formation promoting peptide, or a composition comprising the peptide.

In the above method, the peptide or composition may be administered orally or parenterally when administered, and may be used in the form of a general pharmaceutical preparation. That is, the composition of the present invention may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Is prepared using. Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and the like, which may be used in the pharmaceutical composition of the present invention at least one excipient such as starch, calcium carbonate, sucrose, lactose And gelatin etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium, styrate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol and gelatin may be used. The compositions of the present invention may be via subcutaneous injection, intravenous injection or intramuscular injection during parenteral administration.

Dosage units may contain, for example, one, two, three or four times the individual dosage, or they may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose. The effective dose of the composition of the present invention is 0.0001 to 10 g / kg, preferably 0.0001 g to 5 g / kg, may be administered 1 to 6 times a day.

In the method, the subject may be a mammal, for example a human.

The method may be used alone or in combination with methods using surgery, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of bone disease.

The present invention also provides a method for promoting osteoblast differentiation using the bone formation promoting peptide.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example  1. Experimental Materials and Methods

1.1. material

Two antioxidative peptides derived from mussel protein (PIISVYWK, 1004.57 Da: SEQ ID NO: 1; FSVVPSPK, 860.09 Da: SEQ ID NO: 2) were synthesized by Fmoc solid phase peptide synthesis (SPSS) (Peptron Inc. Seoul). Other materials used for cell culture were purchased from Gibro-BRL (Gaithersburg, MD, USA). Antibodies for protein analysis (p-Smad1 / 5, Smad1 / 5/8, Dlx5, Runx2, osterix, p-ERK, p-JNK, p-p38 and β-actin) are described in Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other reagents used in this study were purchased from Sigma-Aldrich.

1.2. Cell Culture and Reagent Processing

The mouse-derived mesenchymal stem cells used in the experiment were purchased from ATCC (D1 cell, CRL-12424) and incubated in DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 5% CO ₂ and 37 ° C. . For osteoblast differentiation, differentiation medium (DMEM + 50 μg / mL ascorbic acid, 10 mM β-glycerolphosphate and 10 ^-7 M dexamethasone) was exchanged every other day.

BMP antagonist (noggin, 100 ng / mL) or MAPKs inhibitors (10 μM SB203580, 20 μM PD98059 and 10 μM SP600125) were treated with cells for 2 hours and then incubated for a specified time period with the differentiation medium containing peptides. .

1.3. Cytotoxicity Measurement

The cytotoxicity of the two peptides used was measured by MTT (3- (4,5-dimethythiazol-2-yl) -2,5diphenyltetrazolium bromide). When cells became 70-80% confluent in 24-well plates, 1 mg / mL MTT solution was added 24 or 48 hours after the peptide was treated by concentration. After incubation for 4 hours incubator, formazan produced by removing the medium was dissolved in DMSO and the absorbance was measured at 540nm.

1.4. ALP (alkaline phosphatase) activity and staining

In order to measure ALP activity, cells were cultured in 96-well plates and treated with peptide for 7 days, and then the medium was removed. The cells were washed in PBS and then digested with 25 mM sodium carbonate buffer (pH 10) containing 0.1% Triton X-100, and 100 μL of the supernatant was digested with enzyme reaction solution (1.5 mM MgCl ₂ , 3.8 mM p). 100 μL of 25 mM sodium carbonate buffer) containing -nitrophenyl phosphate was mixed and reacted at 37 ° C. for 90 minutes, and the absorbance was measured at 405 nm. ALP activity was calculated as follows.

ALP activity (%) = (A- A ₀ ) / A ₀ × 100

A: peptide treated group, A ₀ : untreated group

After culturing the cells in a 12-well plate for ALP staining, the peptide was treated for 7 days, and then the medium was removed and washed twice in PBS. After fixing the cells for 5 minutes by treatment with 10% formalin solution, washed again in PBS, BCIP / NBT substrate solution was added to the well plate and incubated for 15 minutes at 37 ℃ and observed under a microscope.

1.5. Alizarin red S dyeing Mineralization  Measure)

To measure calcification, the final stage of osteoblast differentiation, the cells were cultured in 12-well plates and treated with peptides for 21 days. After the incubation, the cells were washed in PBS and fixed with 70% ethanol solution at 4 ° C. for 1 hour. 2% Alizarin red S (pH 4.2) solution was treated for 15 minutes at room temperature, dyed and washed 4 times with distilled water and dried completely. The mineral formation of the cells was observed under a microscope, and the absorbance was measured at 562 nm by decolorizing the Alizarin red S solution stained using 10% cetylpyridinium chloride solution for quantification. The degree of mineral formation was calculated using the following formula.

Mineralization (%) = (A- A ₀ ) / A ₀ × 100

A: peptide treated group, A ₀ : untreated group

1.6. Western blotting

After peptide treatment for 7 days, the cells were disrupted using RIPA buffer (Sigma Chemical Co.) and protein was extracted and quantified (Pierce BCA assay kit, Thermo Fisher Scientific, MA, USA). Equal amounts of protein were separated using 10% SDS-PAGE and then transferred to the PVDF membrane. After blocking with 5% skim milk, the primary antibody was treated overnight at 4 ° C., and the secondary antibody was then reacted at room temperature for 2-3 hours, followed by an enhanced chemiluminescence assay kit (Pierce Biotechnology, IL). , USA) was used to detect proteins on PVDF membranes.

1.7. Mouse Preparation and Drug Handling Protocols

All mice used in the experiment were 7-week-old C57BL / 6N mice, purchased from Envigo (USA) and undergoing a 1 week accrual period.

A total of fourteen 12-week-old female mice (OVX) were subjected to ovarian extraction surgery (OVX), one week later, to create an osteoporosis model, and one week later, SEQ ID No. 1 and 2 peptides (50 μg / 25 g mice, respectively) and PBS. 100 μL of (Gibco) was intraperitoneally administered once a day for 2 months. As a control group, 4 female mice per group were subjected to subcutaneous incision only in the Sham model of osteoporosis, and 100 μL PBS was intraperitoneally administered.

One week after ovarian extraction, a calcium deficiency diet (Envigo, USA) was provided for efficient induction of osteoporosis. Normal mice received a normal diet without surgery.

1.8. Micro CT

The femur was separated from the mouse to remove muscle, washed with physiological saline, fixed in 4% formalin for 24 hours, and bone density was measured. Microscopic tomography of the femur was measured using a micro-CT scanner (Inveon preclinical CT, Siemens Healthcare, USA) with a sample length of 1.9 cm, width 2 cm, photon energy of 80 keV, and a current of 500 μA. For bone density measurement, a volume portion of 2 mm ³ of the same site per group was measured using Siemens Inveon Software.

Example  2. Mussel protein  origin Peptide  ALP (alkaline phosphatase ) Active results

To investigate the effect of antioxidant peptides on osteoblast differentiation, we first measured the activity of ALP, a biomarker observed in early osteoblast differentiation. As a result of treatment of peptides (PIISVYWK and FSVVPSPK) by concentration for 7 days, it was confirmed that concentration-dependently increased ALP activity, and PIISVYWK increased about 270% and FSVVPSPK increased about 242% after 100 μM treatment. It was confirmed (FIG. 1).

Example 3 Results of Protein Expression Involved in Osteoblast Differentiation

Since the two peptides were very good at the 100 μM concentration in the ALP activity measurement, the following experiment was conducted by treating the 100 μM concentration. After peptide treatment for 7 days to the cell line, the protein was extracted and subjected to Western blotting. As a result, it was confirmed that two kinds of peptides increased the expression of BMP-2 / 4 (bone morphogenetic protein) involved in osteoblast differentiation (FIG. 2).

In addition, BMP-2 / 4 stimulates the phosphorylation of Smad1 / 5 in downstream signaling systems to promote intracellular signaling, while phosphorylated Smad1 / 5 is important for producing specific proteins involved in osteoblast differentiation and formation. The transcriptional regulators, Dlx-5, Runx-2 and Osterix, which play a role, were activated. As a result, the two types of peptides increased the expression of BMP-2 / 4 so that the downstream signaling agent Smad1 / 5 and the transcriptional regulators Dlx-5, Runx-2 and Osterix all increased the expression or activity, It was confirmed to promote differentiation of osteoblasts (FIG. 2). It was also confirmed that the expression of type I collagen, a biomarker for osteoblast differentiation, was strongly increased (FIG. 2).

In addition, mitogen-activated protein kinases (MAPKs) are known to play an important role in osteoblast differentiation, and the present inventors conducted experiments to determine whether two types of peptides have an effect of activating MARKs. As a result, the phosphorylation of MARKs p-p38, p-ERK and p-JNK was increased to confirm osteoblast differentiation (Fig. 3).

Example 4 Mineral Formation of Osteoblasts by Peptides

Mineral formation was observed by Alizarin red S staining after 21 days of treatment of two peptides (PIISVYWK and FSVVPSPK). As a result, it was confirmed that two kinds of peptides strongly promoted the mineral formation of osteoblasts. Specifically, PIISVYWK was found to increase the mineral formation of 302%, FSVVPSPK 282% compared to the control (Fig. 4).

Example  5. BMP antagonists (noggin) and MAPKs  Effect of Inhibitor Treatment on BMP Signaling Pathway and MAPKs Phosphorylation

In order to identify osteoblast differentiation by activation of BMP signaling pathway and MAPKs phosphorylation pathway, two peptides were first treated with noggin, an antagonist of BMP, and then related protein expression was confirmed by Western blotting. As a result, it was confirmed that in the group treated with noggin and peptide, the expression of BMP-2 / 4 and downstream signal transducers was significantly reduced compared to the group treated with peptide alone (FIG. 5).

In general, activation of BMPs is known to be associated with increased phosphorylation of MAPKs. However, in the present invention, the treatment of BMP antagonists does not change the phosphorylation of MAPKs (p-p38, p-ERK and p-JNK) by peptides. It was confirmed (Fig. 6).

On the other hand, MAPKs inhibitors SB203585 (p38 inhibitors), PD98059 (ERK inhibitors) and SP600125 (JNK inhibitors) were treated with BMP-2 / 4, phosphorylated Smad1 / 5 and the electronic regulator, Runx- 2 increased the expression of BMP-2 / 4, phosphorylated Smad1 / 5 and Runx-2, an electronic regulator, was inhibited in the group treated with the MAPKs inhibitor (FIG. 7).

In order to confirm the effect of MAPKs inhibitors on osteoblast differentiation, the expression of ALP, an early biomarker for osteoblast differentiation, was confirmed by staining. As a result, the expression of ALP was increased in the cells treated with the two peptides and stained very strongly. However, PD98059 (ERK inhibitor) and SP600125 (JNK inhibitor) among MAPKs inhibitors significantly reduced the expression of ALP. It was confirmed that (Fig. 8).

Therefore, the two peptides are involved in signaling pathways that affect the phosphorylation of ERK and JNK, and by activating BMPs signaling pathways, they can promote osteoblast differentiation, which is effective in the treatment of bone diseases, especially osteoporosis. It was confirmed that there is.

Example 6. Effect of Peptides on Bone Formation in Osteoporosis Model

The effects of two PIISVYWK (P1) and FSVVPSPK (P2) peptides on bone formation in osteoporosis model induced by ovarian extraction and calcium deficiency diet were measured. A micro CT photograph and a graph of bone mineral density change results are shown in FIG. 9. PIISVYWK (P1) and FSVVPSPK (P2) peptides in osteoporosis-induced models showed significantly higher bone mineral density than mice injected with PBS alone, and 17β-estradiol (Est, estrogen) used as a positive control. It showed a better degree of bone formation than). Both peptides showed higher or similar bone density than normal mice.

Claims (6)

1. Peptides for promoting bone formation comprising SEQ ID NO: 1 (PIISVYWK) or SEQ ID NO: 2 (FSVVPSPK).
2. The method of claim 1,

   The peptide is a peptide, characterized in that derived from mussel protein.
3. The method of claim 1,

   The peptide is characterized in that to promote the differentiation of osteoblasts.
4. A composition for preventing or treating bone diseases comprising the peptide of any one of claims 1 to 3.
5. The method of claim 4, wherein

   The bone disease is a composition characterized in that any one selected from the group consisting of osteoporosis, osteoarthritis, rheumatoid arthritis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease.
6. Osteoblast differentiation promoting composition comprising the peptide of any one of claims 1 to 3.

Images