KR102137624B1 - Pharmaceutical composition for preventing or treating of bone disease containing activity inhibitor of peroxiredoxin 1 - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of bone disease, which contains an active inhibitor of peroxidedoxin 1 protein as an active ingredient. Specifically, the peroxidodoxin 1 protein of the present invention prevents differentiation of progenitor osteoclasts. By inducing and inhibiting the increase of osteoclasts induced by lipopolysaccharide and the bone loss in the animal model of bone loss, the antibody against the peroxyredoxin 1 protein inhibits the activity of the peroxyredoxin 1 protein in bone disease. It can be useful as a preventive or therapeutic agent.

Description

A pharmaceutical composition for the prevention or treatment of bone disease containing an active inhibitor of peroxidedoxin 1 protein as an active ingredient {PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING OF BONE DISEASE CONTAINING ACTIVITY INHIBITOR OF PEROXIREDOXIN 1}

The present invention relates to a pharmaceutical composition for the prevention or treatment of bone disease containing an active inhibitor of the peroxidedoxin 1 protein as an active ingredient.

Osteoporosis, a typical metabolic bone disease, is a disease in which bone tissue is reduced in lime and thinning of the bones, thereby increasing bone marrow cavity. As the symptoms progress, the bone weakens. It is easy to fracture even when impacted.

Osteoporosis has features such as decreased bone mass and abnormal microstructure. This creates an unbalanced balance between the absorption of old bones and the formation of new bones, resulting in less bone remodeling, which increases the risk of bones becoming loose, broken or broken. This bone mass is influenced by a number of factors, including genetic factors, nutrition, hormonal changes, and differences in exercise and lifestyle. The main causes of bone loss are old age, lack of exercise, low weight, smoking, low calcium diet, menopause, ovarian ablation or inflammation.

Although there are individual differences, in general, bone mass is highest at ages 14 to 18 and decreases by about 1% per year in old age. In particular, in women, bone loss continues to progress after the age of 30, and bone loss rapidly progresses due to hormonal changes in menopause. In other words, menopause increases the activity of osteoclasts by reducing the concentration of estrogen in the body, and eventually the bone mass decreases.

As such, osteoporosis differs in degree, but is an unavoidable symptom in older women, especially in postmenopausal women. In developed countries, as the population ages, interest in osteoporosis and its therapeutics is gradually increasing. In addition, there is a market of about 130 billion dollars related to the treatment of bone diseases worldwide, and it is expected to increase in the future, so global research institutions and pharmaceutical companies are investing heavily in the development of treatments for bone diseases.

In Korea, the prevalence of osteoporosis has increased rapidly in recent years as the average life span has reached 80. According to a recent study conducted on local residents, 4.5% of males and 19.8% of females were reported to be osteoporotic when standardized to the national population. This suggests that osteoporosis is a more common disease than diabetes or cardiovascular disease, and osteoporosis is a very important health problem when estimating the cost of suffering or treatment for patients suffering from fractures.

Estrogen, which is the most widely used therapeutic agent for osteoporosis developed to date, has yet to be tested for its actual efficacy and must be taken throughout life, and when administered for a long time, there is a side effect of increasing breast cancer or uterine cancer. In addition, alendronate also has a problem in that its efficacy is not clear, absorption in the digestive tract is slow, and inflammation in the gastrointestinal tract and esophageal mucosa is caused. On the other hand, calcium formulations have less side effects and excellent effect, but they are more of a preventive agent than a therapeutic agent. In addition, vitamin D preparations such as calcitonin have not been studied for efficacy and side effects. Accordingly, there is a need for a new therapeutic agent for bone disease with few side effects and excellent effect.

Osteoblasts are large multinucleated cells that destroy or absorb bone tissue that has become unnecessary during the process of bone growth in vertebrate animals and differentiate from osteoclast precursors. Osteoclast progenitor cells differentiate into mononuclear osteoclasts via proosteoclast (pOC) in the presence of M-CSF and RANKL, and form multinucleated osteoclasts through fusion. While osteoclasts form an acidic environment by binding to bone through αvβ3 integrin, etc., they secrete various collagenase and protease to cause bone resorption. Inhibition can be an effective way to treat bone disease.

On the other hand, peroxiredoxin 1 protein (peroxiredoxin 1, PRX1) is a type of peroxiredoxin protein, an antioxidant enzyme widely present in nature. PRX1 mediates the signaling process in mammalian cells by regulating the concentration of peroxides produced by cytokines. It exists in two types: the form present in the cytoplasm and the water-soluble form secreted out of the cell. Regarding bone homeostasis, when oxidative stress occurs due to estrogen deficiency, expression of PRX1 present in the cytoplasm of osteoblasts increases, thereby reducing oxidative damage (Du J et al., Sci Rep. 2016; 27; 6:35995), but the mechanism of secreted water-soluble PRX1 is unknown.

Accordingly, the present inventors, while exploring a substance that modulates the activity of osteoclasts to treat bone disease, discovered that the secreted peroxyredoxine 1 protein promotes the differentiation of osteoclasts, and its activity inhibitor is The present invention was completed by confirming that it inhibits differentiation and bone loss.

Du J et al., Sci Rep. 2016; 27; 6:35995

An object of the present invention is to provide a composition for the treatment of bone disease containing an active inhibitor of the peroxidedoxin 1 protein as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of bone disease, which contains an active inhibitor of peroxidedoxin 1 protein as an active ingredient.

In addition, the present invention provides a health functional food for improving bone disease, which contains an active inhibitor of peroxidedoxin 1 protein as an active ingredient.

In addition, the present invention comprises the steps of: 1) treating a test substance to bone cells or bone tissue; 2) measuring the secretion amount of peroxidedoxin 1 protein from the bone cells or bone tissue of step 1); And 3) provides a method for screening a substance for prevention or treatment of bone disease, comprising the step of selecting a test substance having a reduced secretion amount of the peroxidoxine 1 protein of step 2) compared to the untreated control group.

The peroxyredoxin 1 protein of the present invention induces differentiation of progenitor osteoclasts and increases the osteoclast-induced osteoclast induced by lipopolysaccharide and antibody to bone loss in animal models. By inhibiting, the activity inhibitor of the peroxidedoxin 1 protein may be useful as a preventive or therapeutic agent for bone disease.

1 is a photograph confirming the change in the amount of PRX1 secretion after RANKL treatment on progenitor osteoclasts (preosteoclast, pOC).
Figure 2 is a graph confirming whether or not promoting the differentiation of osteoclasts after treating PRX1 in progenitor osteoclasts.
3 is a photograph confirming the change in expression of NFATc1 after treating PRX1 in progenitor osteoclasts.
4 is a photograph confirming whether or not IκB is decomposed after treating PRX1 on progenitor osteoclasts.
5 is a view confirming the change in phosphorylation of p38 after treating PRX1 on progenitor osteoclasts.
6 is a graph comparing the survival rate when cultured by treating RANKL or PRX1 on mature multinuclear osteoclasts.
Figure 7 is a photograph confirming whether or not the degradation of IκB treated with PRX1 in mature multinuclear osteoclasts.
8 is a photograph confirming the change in phosphorylation of p-AKT by treating PRX1 on mature multinuclear osteoclasts.
FIG. 9 is a photograph confirming whether changes in osteoclast number and bone loss are induced by administering PRX1 to an animal model (Veh: no treatment group, PRX1: PRX1 antibody treatment group).
10 is a graph confirming the inhibition of RANKL-induced osteoclast differentiation by an antibody against PRX1 in progenitor osteoclasts (Veh: untreated group, anti-PRX1 1 μg/ml: 1 μg/ml PRX1 antibody treated group, anti-PRX1 5 μg/ml: 5 μg/ml of PRX1 antibody treated group).
FIG. 11 is a photograph visually confirming changes in the number of lipopolysaccharide-inducing osteoclasts and inducing bone loss by an antibody against PRX1 in an animal model (LPS: lipopolysaccharide treatment group, LPS+anti-Prx1: Lipopolysaccharide and PRX1 antibody treated groups).
12 is a graph confirming a change in the concentration of PRX1 present in the serum of the animal model induced bone loss through tail suspension (Control: normal mouse group, Tail: animal model group induced bone loss).
13 is a photograph of the results of analysis of the thigh bone of the animal model in which bone loss is induced through tail suspension administered with antibody against PRX1 by micro CT (Control: normal mouse group, Tail: animal model inducing bone loss) Group, Tail+anti-PRX1: animal model group induced bone loss administered with PRX1 antibody).
Figure 14a is a graph confirming changes in bone volume (BV/TV) for bone mineral density (BMD) and tissue volume of an animal model in which bone loss is induced through tail suspension, administered with antibodies against PRX1 (Control: normal) Mouse group, Tail: animal model group induced bone loss, Tail+anti-PRX1: animal model group induced bone loss administered PRX1 antibody).
14B is a graph confirming changes in the fiber line thickness (Tb.Th) and the number of fiber lines (Tb.N) in an animal model in which bone loss is induced through tail suspension by administering an antibody against PRX1 (Control: normal mouse group, Tail: animal model group induced bone loss, Tail+anti-PRX1: animal model group induced bone loss administered PRX1 antibody).
Figure 14c is a graph confirming changes in fibrous line separation (Tb.Sp) and structural model index (SMI) of animal models inducing bone loss through tail suspension, administered with antibodies against PRX1 (Control: normal mouse group, Tail : Animal model group induced bone loss, Tail+anti-PRX1: Animal model group induced bone loss administered PRX1 antibody).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of bone disease, which contains an active inhibitor of the peroxidedoxin 1 protein as an active ingredient.

The peroxidedoxin 1 protein may include a polypeptide composed of any sequence known in the art. In one embodiment of the invention, the peroxiredoxin 1 protein may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The polypeptide may be a variant or fragment of amino acids having different sequences by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function of the protein. Amino acid exchanges in proteins or peptides that do not entirely alter the activity of the molecule are known in the art. In some cases, phosphorylation, sulfation, acrylation, glycosylation, methylation, or farnesylation can be modified. Accordingly, the present invention may include a polypeptide having an amino acid sequence substantially identical to the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and variants or fragments thereof. The substantially identical polypeptide may have homology to 80% or more of the polypeptide of the present invention, specifically 90% or more, and more specifically 95% or more.

The activity inhibitor may be any one or more selected from the group consisting of compounds, peptides, peptide mimetics, and antibodies that complementarily bind to the peroxiredoxin 1 protein.

The peptide and peptide mimetics may be to inhibit the activity of the peroxidoxin 1 protein by inhibiting the peroxidoxin 1 protein from binding to other proteins. Peptides may be variants or fragments of amino acids having different sequences by deletion, insertion, substitution, or combinations of amino acid residues, within a range that does not affect the function of the protein. Non-hydrolytic peptide mimetics can be prepared using β-turn dipeptide core, keto-methylene pseudopeptides, azepine, benzodiazepine, β-aminoalcohol or substituted gamma-lactam ring as main residues.

The antibody may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. Antibodies to a specific protein can be readily prepared using techniques well known in the art if the sequence of the protein is known. The monoclonal antibody may be prepared using hybridoma methods known in the art, or phage antibody library technology. In general, hybridoma cells that secrete monoclonal antibodies can be made by fusion of cancer cell lines with immune cells isolated from immunologically compatible host animals, such as mice injected with antigenic proteins. These two groups of cell fusions are fused using methods known in the art to which the present invention pertains, such as polyethylene glycol, and the antibody-producing cells are propagated by standard culture methods. Subcloning is performed using a limiting dilution method, and after obtaining a uniform cell population, hybridoma cells capable of producing an antibody specific for an antigen can be prepared by culturing in large quantities in vitro or in vivo. Antibodies prepared by the above method can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The polyclonal antibody may be prepared by injecting an immunogen biomarker protein or a fragment thereof into an external host according to a method known in the art. The external host can be a mammal such as a mouse, rat, sheep, or rabbit. When the immunogen is injected by intramuscular, intraperitoneal or subcutaneous injection, it may be administered with an adjuvant to increase antigenicity. Thereafter, blood may be regularly collected from an external host to obtain serum showing improved titers and specificity for antigens, and antibodies may be isolated and purified therefrom.

Antibodies herein can also include heavy or light chains. Each of the light and heavy chains may have a variable region at the N-terminus, and each variable region may alternately include four framework regions (FR) and three complementarity determining regions (CDRs). Typically, residues in the variable region are numbered according to a system designed by Kabat et al. The system is described in the US Department of Health and Human Services NIH's Sequences of Proteins of Immunological Interest (1987, Kabat).

In addition, the antibodies of the present invention may include antibody variants such as Fab, F(ab'), F(ab') _2, and Fv. The Fab has a structure having a variable region of light and heavy chains, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab') ₂ antibody is produced by cysteine residues in the hinge region of Fab' forming disulfide bonds. Fv is the smallest antibody fragment that has only the heavy chain variable region and the light chain variable region, and the recombinant technology to produce it is PCT international publication patent applications WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. The double-chain Fv (two-chain Fv) is a non-covalent bond, in which the heavy chain variable region and the light chain variable region are connected, and the single-chain Fv (single-chain Fv) is generally shared between the heavy chain variable region and the single chain variable region through a peptide linker. It can be linked by a bond or directly linked at the C-terminus to form a dimer-like structure such as a double-chain Fv. Such antibodies can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain yields Fab and cleavage with pepsin yields F(ab') ₂ ), preferably It can be produced through genetic recombination technology.

In addition, the bone disease may be a fracture, osteoprosis, rheumatoid arthritis, Paget disease or metastatic bone cancer.

In a specific embodiment of the present invention, the present inventors have confirmed that when RANKL is treated with progenitor osteoclasts, the amount of PRX1 secreted increases, and the addition of PRX1 promotes the differentiation of osteoclasts depending on its concentration (FIGS. 1 and 2). Reference). In addition, when PRX1 was treated with progenitor osteoclasts, the expression of NFATc1, the degradation of IκB, and the degree of phosphorylation of p38 increased (see FIGS. 3 to 5 ), thereby confirming the signaling pathways related to osteoclast differentiation by PRX1.

In addition, when PRX1 was administered to the animal model, the number of osteoclasts increased and bone loss was induced (see FIG. 9 ), and the differentiation of osteoclasts induced by RANKL by the antibody against PRX1 was suppressed in a concentration-dependent manner, and the animal model It was visually confirmed that inflammation-induced osteoclast differentiation and the resulting bone loss were reduced (see FIGS. 10 and 11 ).

Furthermore, PRX1 antibody inhibits bone loss in animal models induced by bone loss through tail hanging (see FIG. 13), bone mineral density reduced by bone loss, bone volume for tissue volume, fiber line thickness and number of fiber lines While increasing the indices such as, decreased indices such as fibrous line separation and structural model index increased by bone loss (see FIGS. 14A-14C ).

Therefore, a substance that inhibits the activity of peroxidedoxin 1 protein may be useful for the prevention or treatment of bone disease.

The pharmaceutical composition may include 10 to 95% by weight of the peroxidedoxin 1 protein activity inhibitor according to the present invention as an active ingredient relative to the total weight of the pharmaceutical composition. In addition, the pharmaceutical composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.

The compositions of the present invention may also include carriers, diluents, excipients, or combinations of two or more of those commonly used in biological agents. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for delivering the composition in vivo, for example, Merck Index, 13th ed., Merck & Co. Inc. The compound, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or one or more of these components may be mixed. At this time, other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.

When formulating the composition, it is usually prepared using diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.

The composition of the present invention may be formulated as oral preparations or parenteral preparations. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations include at least one excipient in one or more compositions, such as starch, calcium carbonate, sucrose, It can be prepared by mixing lactose and gelatin. Also, lubricants such as magnesium stearate and talc may be added. On the other hand, the liquid preparation includes a suspension, an intravenous solution, an emulsion or a syrup, and may include excipients such as wetting agents, sweeteners, fragrances, preservatives, and the like.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspension solutions, injections such as emulsions.

As the non-aqueous solvent and suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.

The composition of the present invention may be administered orally or parenterally depending on the desired method, and parenteral administration may be applied for external application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. Can be selected.

The composition according to the invention is administered in a pharmaceutically effective amount. This may vary depending on the type of disease, severity, drug activity, sensitivity to the drug, administration time, administration route and discharge rate, treatment duration, and drugs used simultaneously. The composition of the present invention may be administered alone or in combination with other therapeutic agents. In combination administration, administration can be sequential or simultaneous.

However, for a desired effect, the amount of the active ingredient contained in the pharmaceutical composition according to the present invention may be 0.001 to 10,000 mg/kg, specifically 0.1 to 5 g/kg. The administration may be once a day, or divided into several times.

In addition, the present invention provides a health functional food for improving bone disease, which contains an active inhibitor of peroxidedoxin 1 protein as an active ingredient.

The peroxidedoxin 1 protein activity inhibitor may have the characteristics as described above. In one example, the peroxiredoxin 1 protein may be a polypeptide composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In addition, the activity inhibitor may be any one or more selected from the group consisting of compounds, peptides, peptide mimetics, and antibodies that complementarily bind to peroxiredoxin 1 protein.

The bone disease may be a fracture, osteoporosis, rheumatoid arthritis, Paget disease or metastatic bone cancer.

The peroxidedoxin 1 protein activity inhibitor of the present invention may be added as it is to foods or used with other foods or food ingredients. At this time, the content of the active ingredient to be added may be determined according to the purpose. In general, the content in the dietary supplement may be from 0.01 to 90 parts by weight of the total food weight.

In addition, there is no particular limitation on the form and type of the health functional food. The form of the health functional food to which the above substances can be added may be tablets, capsules, powders, granules, liquids and pills.

The health functional food of the present invention may contain various flavoring agents or natural carbohydrates and the like as additional components, like a normal health functional food. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, synthetic sweeteners such as saccharin and aspartame can be used.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Alcohol and the like. These ingredients can be used independently or in combination. The proportion of such additives may be selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention comprises the steps of: 1) treating a test substance to bone cells or bone tissue; 2) measuring the secretion amount of peroxidedoxin 1 protein from the bone cells or bone tissue of step 1); And 3) provides a method for screening a substance for prevention or treatment of bone disease, comprising the step of selecting a test substance having a reduced secretion amount of the peroxidoxine 1 protein of step 2) compared to the untreated control group.

The material screening method may be used to identify compounds that regulate the secretion amount of the peroxiredoxin 1 protein in vivo or ex vivo of animal models such as human bone cells or bone tissue, as well as mouse and rat, and the test substance Furnace may be selected from the group consisting of peptides, proteins, non-peptidic compounds, active compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and plasma.

The secretion of the peroxyredoxin 1 protein of step 2) is Western blotting, Enzyme-linked immnosorbent assay (ELISA) kit, Sandwich ELISA kit, Protein chip kit, Rapid kit and Multiple reaction monitoring) can be measured using any one or more methods selected from the group consisting of kits.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

<Example 1> Culture of mouse bone marrow cells

First, in order to obtain bone marrow cells of mice, ICR mice (6-9 weeks old, males) were disinfected with 70% ethanol after cervical dislocation, and the skin of the tibia portion was dissected to detach the attached muscles. The tibia was dissected and the patella was dislocated to extract the tibia. The bone marrow cells obtained by cutting both ends of the bone with a 25 G injection needle at one end and flowing α-MEM were collected in a test tube. After centrifugation, the suspension was suspended in α-MEM to add 2x Gey' solution (Sigma), and then red blood cells were removed. The cells were centrifuged again, resuspended in α-MEM containing 10% FBS, and cultured at 37° C. and 5% CO ₂ conditions to prepare mouse bone marrow cells.

<Example 2> Progenitor osteoclast culture

To the bone marrow cells prepared in <Example 1>, 10 ng/ml of macrophage colony stimulating factor (M-CSF) was added and cultured for one day. The suspended cells were separated, and 30 ng/ml of M-CSF was added, followed by incubation for 3 days to generate progenitor osteoclasts (Bone marrow Macrophages, BMM). After adding 200 ng/ml of RANKL (receptor activator of NF-κB ligand, osteoclast differentiation factor), the cells were further cultured for 1 day to obtain proteooclasts (pOC) produced. The obtained cells were cultured at 37° C. and 5% CO ₂ conditions.

<Experiment 1> Confirmation of PRX1 secretion in progenitor osteoclasts

Whether or not PRX1 is secreted from the progenitor osteoclasts obtained in <Example 2> was confirmed by the following method.

Specifically, 200 ng/ml RANKL and 30 ng/ml M-CSF were added to 1×10 ⁵ cells/well progenitor osteoclasts and cultured for 48 hours, followed by western blotting of secreted proteins in the medium as follows. Analyzed by lot experiment.

First, in order to obtain the protein of the recovered medium, only the supernatant was recovered by centrifugation, and the concentration of the protein was normalized to bovine serum albumin (BSA) and quantified using a protein assay kit (Bio-Rad). 20 ng of the protein was denatured by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane at 80 V for about 1 hour and 45 minutes. The PVDF membrane was blocked with PBST solution (0.05% Tween-20 in PBS) containing 5% skim milk, and anti-PRX1 (1:1000, Ab Frontier) and anti-β-actin as primary antibodies (Sigma) The antibody was reacted with each. After the reaction, the PVDF membrane was washed 5 times with PBST, reacted with a hoseradish peroxidase (HRP)-coupled secondary antibody, and then analyzed by color development with ECL Advance (Amersham. co.).

As a result, as shown in FIG. 1, when RANKL was added to progenitor osteoclasts, the amount of PRX1 was significantly increased (FIG. 1 ).

<Experimental Example 2> Confirmation of PRX1 differentiation induction in progenitor osteoclasts

Next, the effect of the addition of PRX1 on the differentiation of osteoclasts was confirmed by the following method.

Specifically, 1×10 ⁵ cells/well of progenitor osteoclasts were cultured for 2 days by adding 1 μg/ml of PRX1 (Ab Frontier) and 30 ng/ml of M-CSF. The cultured cells were fixed with 10% formalin for 10 minutes, and then resuspended for 1 minute with an ethanol-acetone mixed solution (1:1) and stained with tartrate-resistant acid phosphatase (TRAP). The stained cells were observed under a microscope and counted by determining TRAP+ cells having three or more nuclei as differentiated multinucleated (MNC) osteoclasts.

As a result, as shown in FIG. 2, osteoclast differentiation was promoted depending on the concentration of treated PRX1 (FIG. 2 ).

<Experiment 3> Confirmation of osteoclast differentiation signaling pathway by PRX1

<3-1> Confirmation of NFATc1 Expression by PRX1

Stimulation of RANKL induces the expression of NFATc1 (nuclear factor of activated T cells c1), which is known as a key transcription factor that regulates differentiation from progenitor osteoclasts to osteoclasts. NFATc1 is initially induced by intracellular calcium signal transmission by RANKL stimulation, and then activation of NF-κB and MAPK (MAP kinase), etc., again triggers autoamplification of NFATc1 to maintain activity.

Since it was confirmed in <Experimental Example 1> that PRX1 promotes osteoclast differentiation, to examine the effect of PRX1 on the expression of NFATc1, a key transcription factor that regulates osteoclast differentiation, the Western blotting experiment is as follows. Was performed.

Specifically, progenitor osteoclasts were cultured for 2 days by adding 1 μg/ml PRX1 and 30 ng/ml M-CSF. The cultured cells were recovered, lysis buffer was added, and the cells were lysed. The supernatant was recovered by centrifugation, and the concentration of the protein was normalized to BSA and quantified using a protein analysis kit (Bio-Rad). Western blotting was performed in the same manner as in <Experimental Example 1>, except that anti-NFATc1 (1:200, Santa cruz) was used as the primary antibody.

As a result, as shown in Figure 3, when treated with PRX1 progenitor osteoclast, the expression of NFATc1 increased (Fig. 3). From this, it was found that PRX1 promotes osteoclast differentiation through increased expression of NFATc1.

<3-2> Confirmation of activation of NF-κB and p38 MAPK by PRX1

Next, in order to find out more specific signaling pathways, by PRX1 The degree of activation of NF-κB and p38 MAPK was confirmed by Western blotting experiments.

At this time, the activation of NF-κB was confirmed to the extent of decomposition of IκB, an inhibitory molecule of NF-κB, and the activation of p38 was confirmed to increase the phosphorylation to p38. The experiment was carried out in the same manner as in <Experimental Example 1>, except that anti-IκB (1:1000, Cell signaling) and anti-p-p38 (1:1000, Cell signaling) were used as antibodies.

As a result, as shown in FIGS. 4 and 5, treatment of PRX1 on progenitor osteoclasts increased the degree of degradation of IκB and phosphorylation of p38 (FIGS. 4 and 5 ). This means that the signaling pathway of each of NF-κB and p38 MAPK is activated by PRX1. Therefore, it can be seen from this that PRX1 promotes osteoclast differentiation by increasing the expression of NFATc1 through activation of NF-κB and p38 MAP kinase.

< Experimental Example 4> On PRX1 Control of survival and signaling pathway involvement of mature multinuclear osteoclasts

<4-1> Confirmation of survival control of mature multinuclear osteoclasts by PRX1

In addition to osteoclast differentiation, the survival time of mature multinuclear osteoclasts also has an important effect on bone resorption. In this regard, it is well known that RANKL promotes osteoclast survival along with its ability to promote osteoclast differentiation. Thus, the effect of PRX1 on the survival of mature multinuclear osteoclasts was confirmed as follows.

Specifically, 200 ng/ml RANKL and 30 ng/ml M-CSF were added to 1×10 ⁵ cells/well progenitor osteoclasts, and cultured for 3 days to obtain mature multinuclear osteoclasts. 30 ng/ml M-CSF (veh) in the control group, 200 ng/ml RANKL and 30 ng/ml M-CSF (RANKL) in the experimental group, or 1 μg/ml PRX1 and 30 ng/ml M-CSF (PRX1) and then cultured for 2 days. The cultured cells were fixed with 10% formalin for 10 minutes, and then resuspended for 1 minute with an ethanol-acetone mixed solution (1:1) and stained with tartrate-resistant acid phosphatase (TRAP). The stained cells were observed under a microscope and counted by determining TRAP+ cells having three or more nuclei as differentiated multinucleated (MNC) osteoclasts. The survival rate of multinucleated osteoclasts was expressed as a percentage (%) of the number of multinuclear osteoclasts of the control group or the experimental group that survived compared to the number of initial multinuclear osteoclasts.

As a result, as shown in FIG. 6, the survival rate of the mature multinuclear osteoclasts of the control group was low, but the group added with RANKL had a high survival rate of the mature multinuclear osteoclasts. In addition, the experimental group added with PRX1 showed higher survival rate of mature multinuclear osteoclasts than the group treated with RANKL.

From this, it can be seen that PRX1 further promotes the survival of mature multinuclear osteoclasts (FIG. 6 ).

<4-2> Confirmation of PRX1 signaling pathway involvement

Mechanisms of NF-κB and AKT are known as signaling pathways involved in the survival of mature multinuclear osteoclasts. It was confirmed by Western blotting experiments that the promotion of the survival of mature multinuclear osteoclasts of PRX1 identified in <Experimental Example 4-1> regulates the lower signaling pathway.

Western blotting experiment was performed in the same manner as in <Experimental Example 1>, except that anti-IκB (1:1000, Cell signaling) and anti-p-AKT (1:1000, Cell signaling) were used as the antibody. Did.

As a result, as shown in FIGS. 7 and 8, the addition of PRX1 to mature multinuclear osteoclasts increased the degradation of IκB and phosphorylation of p-AKT (FIGS. 7 and 8 ). This means that the NF-κB and AKT signaling pathways are activated. Through this, it can be seen that the secreted PRX1 promotes the survival of mature multinuclear osteoclasts through the activation process of NF-κB and AKT.

<Experimental Example 5> Identification of osteoclast differentiation effect of PRX1 in an animal model

It was confirmed by the following method whether the effect of promoting osteoclast differentiation of PRX1 confirmed in vitro appears the same in animal models.

First, 0.25 mg of PRX1 was injected subcutaneously into the cranial canal of 11-12 week old male ICR mice (Samtako), and the same amount of PBS was injected into the control group. After 6 days, mice were sacrificed by CO ₂ treatment, and the crown was collected, and then fixed in 4% paraformaldehyde. The fixed cranial canal was TRAP stained and visually observed.

As a result, as shown in FIG. 9, the number of osteoclasts, TRAP+, increased significantly and bone loss was induced in the cranial canal of mice injected with PRX1 (FIG. 9 ). From this, it was confirmed that PRX1 effectively promotes osteoclast differentiation even in an animal model.

<Experimental Example 6> Confirmation of PRX1 antibody osteoclast differentiation inhibitory effect

<6-1> Confirmation of the effect of inhibiting osteoclast differentiation in progenitor osteoclasts

It was confirmed by the following method that the differentiation of progenitor osteoclasts is inhibited by the antibody against PRX1.

In the experiment, 200 ng/ml RANKL and 30 ng/ml M-CSF were added to 1×10 ⁵ cells/well progenitor osteoclasts, and 1 μg/ml or 5 μg/ml PRX1 antibody (Ab Frontier) ) Was performed in the same manner as in <Experimental Example 2>, except that additionally added.

As a result, as shown in FIG. 10, the addition of RANKL promoted the differentiation of progenitor osteoclasts, but the differentiation of osteoclasts was inhibited in a concentration-dependent manner by adding an antibody against PRX1 (FIG. 10 ).

<6-2> Confirmation of the effect of suppressing osteoclast differentiation in animal models

The inhibitory effect of osteoclast differentiation of the antibody against PRX1 was confirmed by the following method using an animal model. Clinically, frequent bone loss is observed in patients with arthritis or periodontitis due to an increase in osteoclasts according to an inflammatory response, and lipopolysaccharide (LPS) is well known as a causative agent thereof. Thus, lipopolysaccharide was used to confirm the bone loss inhibitory effect of the antibody against PRX1.

First, bone loss was induced by subcutaneous injection of 0.1 mg of lipopolysaccharide once daily to the cranial canal of male ICR mice of 11 to 12 weeks. Mice inducing bone loss were injected subcutaneously into the cranial canal once daily with 10 μg/ml antibody against PRX1. The subsequent procedure was observed with the naked eye by collecting the cranial canal under the same conditions and methods as in <Experimental Example 5> and staining with TRAP.

As a result, as shown in FIG. 11, the number of osteoclasts was significantly increased in the control group and bone loss occurred, but when the antibody against PRX1 was administered, the increase in the number of osteoclasts induced by lipopolysaccharide was significantly decreased ( Fig. 11). From this, it was confirmed that the antibody of PRX1 can effectively suppress osteoclast differentiation.

< Experimental Example 7> PRX1 Antibody Bone loss Check the inhibitory effect

<7-1> Bone loss In animal models PRX1 Confirm concentration increase

Changes in the concentration of PRX1 in animal models induced bone loss through tail suspension were confirmed by the following method.

First, 8-week-old male ICR mice were kept for 2 weeks with the tail of the rat suspended so that the hind paws float about 30 degrees from the ground. At this time, it was possible to freely access the feed and drinking water. Two weeks later, blood was drawn from the heart, and the concentration of PRX1 present in the serum was measured using an ELISA kit for PRX1 (Cat#. MBS760435, Mybiosource, USA).

As a result, as shown in FIG. 12, the concentration of PRX1 present in serum was significantly increased in the animal model inducing bone loss compared to normal mice (FIG. 12 ).

<7-2> PRX1 By antibodies Bone loss Check the inhibitory effect

The bone loss-inhibiting effect of PRX1 antibody was confirmed by the following method using an animal model inducing bone loss.

Specifically, the bone loss-inducing mice prepared in Experimental Example 7-1 were administered intravenously with antibodies against PRX1 at a dose of 100 μg/kg. The administration was performed once a day, for a total of 4 times, 3 days apart. After the administration was completed, the mice were sacrificed to obtain the thigh bone, and the results of the 2D results of micro CT (micro CT) analysis are shown in FIG. 13.

As shown in FIG. 13, bone loss was suppressed by administering PRX1 antibody to mice in which bone loss was induced by tail hanging (FIG. 13 ).

<7-3> PRX1 By antibodies Goal related Confirmation of changes in expression of indicators

Changes in expression of various bone-related indicators by PRX1 antibody were confirmed using a CTAn analysis program using an animal model inducing bone loss.

Specifically, 2D or 3D micro CT data was collected using a CTAn analysis program, and shape and density were measured therefrom. First, 2D measurement was performed using a model 1172 (Model 1172, Skyscan, Belgium) micro CT scanner and analysis software at a voxel size of 9 μm. The measured images were collected at an intensity of 220 kW of energy of 35 kW and an integration time of 590 kW. The threshold value of the collected result values was set to subdivide the bone, and the same threshold value was set for all samples. Meanwhile, the 3D microstructure evaluation was performed using CTAn software release 2.5 (Skyscan, Belgium), and a volume of interset (VOI) was applied. At this time, the volume of interest was set to the smallest size including the entire sample. In addition, 3D microscopic bone structure-related indicators were measured using CTAn software.

As a result, as shown in Fig. 14, bone mineral density (BMD, g/cm 3) decreased by tail hanging, bone volume per tissue volume (BV/TV, %), Indicators such as trabecular thickness (Tb.Th, μm) and trabecular number (Tb.N, μm) were increased by PRX1 antibody (FIGS. 14A and 14B ). On the other hand, indices such as trabecular separation (Tb.Sp, μm) and structure model index (SMI) decreased by tail suspension were reduced by PRX1 antibody (FIG. 14C ). Therefore, it was found from the above that administration of PRX1 antibody significantly inhibited bone loss.

<110> Sookmyung University-Industry Collaboration Foundation <120> PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING OF BONE DISEASE CONTAINING ACTIVITY INHIBITOR OF PEROXIREDOXIN 1 <130> 2017P-12-042 <150> KR 10-2017-0016175 <151> 2017-02-06 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> human <400> 1 Met Ser Ser Gly Asn Ala Lys Ile Gly His Pro Ala Pro Asn Phe Lys 1 5 10 15 Ala Thr Ala Val Met Pro Asp Gly Gln Phe Lys Asp Ile Ser Leu Ser 20 25 30 Asp Tyr Lys Gly Lys Tyr Val Val Phe Phe Phe Tyr Pro Leu Asp Phe 35 40 45 Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Ala Glu 50 55 60 Glu Phe Lys Lys Leu Asn Cys Gln Val Ile Gly Ala Ser Val Asp Ser 65 70 75 80 His Phe Cys His Leu Ala Trp Val Asn Thr Pro Lys Lys Gln Gly Gly 85 90 95 Leu Gly Pro Met Asn Ile Pro Leu Val Ser Asp Pro Lys Arg Thr Ile 100 105 110 Ala Gln Asp Tyr Gly Val Leu Lys Ala Asp Glu Gly Ile Ser Phe Arg 115 120 125 Gly Leu Phe Ile Ile Asp Asp Lys Gly Ile Leu Arg Gln Ile Thr Val 130 135 140 Asn Asp Leu Pro Val Gly Arg Ser Val Asp Glu Thr Leu Arg Leu Val 145 150 155 160 Gln Ala Phe Gln Phe Thr Asp Lys His Gly Glu Val Cys Pro Ala Gly 165 170 175 Trp Lys Pro Gly Ser Asp Thr Ile Lys Pro Asp Val Gln Lys Ser Lys 180 185 190 Glu Tyr Phe Ser Lys Gln Lys 195 <210> 2 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> mouse <400> 2 Met Ser Ser Gly Asn Ala Lys Ile Gly Tyr Pro Ala Pro Asn Phe Lys 1 5 10 15 Ala Thr Ala Val Met Pro Asp Gly Gln Phe Lys Asp Ile Ser Leu Ser 20 25 30 Glu Tyr Lys Gly Lys Tyr Val Val Phe Phe Phe Tyr Pro Leu Asp Phe 35 40 45 Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Ala Asp 50 55 60 Glu Phe Lys Lys Leu Asn Cys Gln Val Ile Gly Ala Ser Val Asp Ser 65 70 75 80 His Phe Cys His Leu Ala Trp Ile Asn Thr Pro Lys Lys Gln Gly Gly 85 90 95 Leu Gly Pro Met Asn Ile Pro Leu Ile Ser Asp Pro Lys Arg Thr Ile 100 105 110 Ala Gln Asp Tyr Gly Val Leu Lys Ala Asp Glu Gly Ile Ser Phe Arg 115 120 125 Gly Leu Phe Ile Ile Asp Asp Lys Gly Ile Leu Arg Gln Ile Thr Ile 130 135 140 Asn Asp Leu Pro Val Gly Arg Ser Val Asp Glu Ile Ile Arg Leu Val 145 150 155 160 Gln Ala Phe Gln Phe Thr Asp Lys His Gly Glu Val Cys Pro Ala Gly 165 170 175 Trp Lys Pro Gly Ser Asp Thr Ile Lys Pro Asp Val Asn Lys Ser Lys 180 185 190 Glu Tyr Phe Ser Lys Gln Lys 195

Claims (8)

Fractures containing as an active ingredient an antibody that complementarily binds to the peroxidedoxin 1 protein, a peroxidoxine 1 protein, osteoporosis, rheumatoid arthritis, paget disease ) Or metastatic bone cancer (metastatic bone cancer) is a pharmaceutical composition for the prevention or treatment of bone disease.
The method of claim 1, wherein the peroxiredoxin 1 protein is SEQ ID NO: 1 or SEQ ID NO: A pharmaceutical composition for preventing or treating bone disease, which is an amino acid sequence described as 2.
delete delete Fractures containing as an active ingredient an antibody that complementarily binds to the peroxidedoxin 1 protein, a peroxidoxine 1 protein, osteoporosis, rheumatoid arthritis, paget disease ) Or metastatic bone cancer (metastatic bone cancer) is a health functional food for the improvement of bone disease.
The method of claim 5, wherein the peroxiredoxin 1 protein is SEQ ID NO: 1 or SEQ ID NO: A health functional food for improving bone disease, which is the amino acid sequence described in 2.
delete 1) treating the separated osteoclast with a test substance;
2) measuring the secretion amount of peroxidedoxin 1 protein from the osteoclast of step 1); And
3) Fracture, osteoporosis, rheumatoid arthritis, Paget's disease, including the step of selecting a test substance having a reduced secretion amount of the peroxidoxine 1 protein of step 2) compared to an untreated control group (Paget disease) or metastatic bone cancer (metastatic bone cancer) for the prevention or treatment of bone diseases screening materials.

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