KR102393730B1 - Pharmaceutical composition for preventing or treating osteoporosis comprising Follistatin-like 1 - Google Patents

Abstract

The present invention relates to a composition for bone filling and a composition for promoting osteoblast differentiation containing FSTL1 (Follistatin-like 1) as an active ingredient, wherein FSTL1 is secreted from osteoclasts to promote the migration and differentiation of bone marrow stem cells and osteoblasts. As it has been confirmed that the facilitating effect is exhibited, the composition containing the FSTL1 as an active ingredient may be provided as a composition for filling bone and as an osteoblast differentiation promoter.

Description

A composition for filling bone containing FSTL1 as an active ingredient {Pharmaceutical composition for preventing or treating osteoporosis comprising Follistatin-like 1}

The present invention relates to a composition for filling bone containing FSTL1 (Follistatin-like 1) as an active ingredient and a composition for promoting osteoblast differentiation.

Bone tissue constitutes the cartilage and skeletal system and serves as a support and muscle attachment with mechanical functions, protects living organs and bone marrow, and preserves calcium and phosphorus ions to maintain homeostasis. Such bone tissue consists of a cell matrix such as collagen and glycoprotein, and various types of cells such as osteoblasts, osteoclasts, and osteocytes. Osteoblasts derived from bone marrow stromal cells play a major role in bone formation. Osteoclasts derived from hematopoietic stem cells are responsible for resorption of destroyed senescent bone, and maintain bone remodeling through the balanced action of osteoblasts and osteoclasts.

Bone metabolic disease occurs when the balance between osteoclasts and osteoblasts is broken in vivo. Osteoporosis is an example of a bone metabolic disease. Osteoporosis refers to a disease in which osteoclast activity is increased compared to osteoblasts, and thus total bone mass is decreased and bones are easily broken even with a slight impact. In addition, there are metastatic cancer, rheumatoid arthritis, degenerative arthritis, and periodontal disease that causes destruction of alveolar bone due to bacterial infection. Bone metabolic diseases and the like result in osteoclasts being excessively activated to easily destroy bones.

The osteoclast progenitor is a hematopoietic cell of the monocyte/macrophage lineage originating in the bone marrow. Osteoclast precursors are differentiated into osteoclasts by growth factors and cytokines produced in the bone marrow. Osteoclasts are responsible for destroying or resorbing bone.

Osteoblasts originate from mesenchymal stem cells and are formed. Mineralization including calcium formed by the differentiation of osteoblasts can maintain bone strength and play a very important function in the homeostasis of calcium and hormone metabolism throughout the body. are doing Calcium formation by osteoblast differentiation is regulated by vitamin D and parathyroid hormone, and bone morphogenetic protein (BMP), Wnt, MAP kinase, calcineurin-calmodulin kinase in cells. Alkaline phosphatase (ALP) involved in osteoblast differentiation is synthesized at the initial stage of differentiation by cross-talk of various signal transduction systems such as (calcineurin-calmodulin kinase), NF-κB, and AP-1. After the mineralization, osteopontin, osteocalcin, type 1 collagen, and the like are synthesized to form bone by differentiation of osteoblasts.

However, bone in the human body maintains homeostasis through the process of formation and remodeling, and remodeling occurs in the bone multicellular unit (BMU) composed of osteoblasts and osteoclasts, and these cells are responsible for osteoblastogenesis and bone formation, respectively. It is known to play an important role in the absorption process. In this process, if the cooperative system between the two cells is damaged, various bone metabolic diseases, including osteoporosis, occur. However, there is no effective treatment for a cure so far, and prevention is emphasized.

Republic of Korea Patent Publication No. 10-2017-0058104 (published on May 26, 2017)

Conventional bisphosphonate-based drugs inhibit osteoclast activity, thereby promoting osteoblast differentiation without inducing osteoclast apoptosis, as a representative drug for treating bone metabolic diseases or osteoclast death side effects become a problem. It is intended to provide a composition for bone filling and a composition for promoting osteoblast differentiation by providing a composition for

The present invention provides a composition for filling bone containing FSTL1 as an active ingredient.

The present invention provides a composition for promoting osteoblast differentiation containing FSTL1 as an active ingredient.

In addition, the present invention comprises the steps of treating a candidate substance to bone marrow stem cells; confirming the expression level of osteoblast differentiation markers in bone marrow stem cells treated with the candidate material; And it provides an osteoblast differentiation promoting agent screening method comprising the step of comparing the increased level of expression of the osteoblast differentiation marker with a control.

According to the present invention, as it has been confirmed that FSTL1 is secreted from osteoclasts to promote the migration and differentiation of bone marrow stem cells and osteoblasts, the composition containing FSTL1 as an active ingredient is a composition for bone filling and osteoblasts. It may serve as a differentiation promoter.

1 is a conceptual diagram of FSTL1 secreted from osteoclasts to inhibit the differentiation and activity of osteoclasts and promote the differentiation of osteoblasts.
Figure 2 is the result of confirming the proliferation and differentiation effect of osteoblasts by FSTL1, Figure 2A is the result of MTT assay analysis confirming the proliferation-promoting effect of osteoblasts by FSTL1, Figure 2B is Alizarin Red staining confirming the osteomineralization promoting effect It is the result.
Figure 3 is a Western blot result confirming the expression change of bone differentiation markers by FSTL1, FSTL1 was treated during the differentiation process of MC3T3-E1 cells, and proteins were extracted from the cells on days 8, 15, and 28 to increase the expression of Runx2 and osteocalcin. This is the confirmed result.
4 is a result confirming the ability to induce bone marrow differentiation in vitro using FSTL1, FIG. 4A is an MTT assay analysis result confirming the proliferation of bone marrow stem cells by FSTL1, and FIG. These are the results of Alizarin Red staining confirming the promotion of differentiation into osteoblasts.
Figure 5 is a real time PCR (Real time PCR) analysis result confirming the expression change of the bone differentiation marker gene, ALP (Alkaline phosphatase), Osx (Osterix, Transcription factor Sp7), FGF23 (Fibroblast growth factor 23) in the group treated with FSTL1 ), DMP1 (Dentin matrix acidic phosphoprotein 1), Phex (Phosphate Regulating Endopeptidase Homolog X-Linked), and osteocalcin (Osteocalcin) are confirmed to increase the expression.

Hereinafter, the present invention will be described in more detail.

In general, when a part of bone tissue is missing or needs reinforcement in a specific site in the living body, bone is transplanted in that area, and the transplanted bone induces the generation of new bone in the transplant site, and the transplanted bone itself is partially or As a whole, it goes through a process of slowly decomposing. Bone to be transplanted in vivo is a mixture of a part of bone tissue extracted in vivo and artificial bone made artificially such as ceramics, which is called a bone filler or a bone substitute. The inventors of the present invention confirmed that FSTL1 promotes differentiation of bone marrow stem cells and osteoblasts, and thus completed the present invention to provide FSTL1 as a composition for bone filling for more effectively inducing new bone regeneration of bone fillers.

The present invention may provide a composition for filling bone containing FSTL1 as an active ingredient.

The FSTL1 may promote differentiation of bone marrow stem cells or osteoblasts.

The bone-filling composition may be included in a material selected from the group consisting of artificial bone, artificial joint, bone fixative, bone substitute, bone repair material, bone fusion agent, and bone graft material.

The present invention can provide a composition for promoting osteoblast differentiation containing FSTL1 as an active ingredient.

The composition for promoting differentiation may increase the expression of alkaline phosphatase (ALP) and osteocalcin, which are markers of differentiation of osteoblasts.

In addition, the composition of the present invention may be provided as a pharmaceutical composition for promoting osteoblasts by including one or more pharmaceutically or food acceptable auxiliary ingredients in addition to the FSTL1 active ingredient. When preparing the composition of the present invention, a pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. Other conventional additives such as agents, buffers, and bacteriostats may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art, or a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Aston PA.

Formulations of the composition of the present invention may be granules, powders, granules, coated tablets, tablets, capsules, decoctions, extracts, suppositories, syrups, juices, suspensions, emulsions, sustained-release preparations of the active compound, etc. . The dosage or intake of the composition of the present invention is preferably 1.5 mg/kg/day when administered orally, and the type of dosage form and the patient's age, weight, general health, sex and diet, administration time, administration route, and composition It can be adjusted according to various factors including secretion rate, duration of treatment, and concomitant drugs.

In addition, the present invention comprises the steps of treating a candidate substance to bone marrow stem cells; confirming the expression level of osteoblast differentiation markers in bone marrow stem cells treated with the candidate material; And it may provide an osteoblast differentiation promoting agent screening method comprising the step of comparing the increased level of expression of the osteoblast differentiation marker with a control.

The osteoblast differentiation marker may be one or two or more selected from the group consisting of alkaline phosphatase (ALP), Osx, FGF23, DMP1, Phex and osteocalcin.

The expression level of the osteoblast differentiation marker is reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry (immunohistochemistry), microarray (microarray), western blotting (western blotting), and may be measured by any one or more selected from the group consisting of flow cytometry (FACS).

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Reagents

Alizarin red S, alkaline phosphatase (ALP) staining kit, ascorbic acid, benzoyl peroxide, DMSO (Dimethyl sulfoxide), ethanol, fast red violet LB, glycerol, naptol As-Mx phosphate, N,N-dimethylformamide (N,N-dimethylformamide), paraformaldehyde (PFA), picric acid solution ), sodium acetate, sodium carbonate, and β-glycerophosphate were purchased from Sigma (St. Louis, MO, U.S.A). Acetone, formaldehyde solution, sodium acetate trihydrate, and sodium tartrate dihydrate were purchased from JUNSEI (Nihonbashi-honcho, Chuo-ku, Tokyo).

α-Minimum Essential Medium (α-MEM) Hyclone and permount were purchased from Thermo scientific (Rockford, IL, U.S.A). Fetal bovine serum (FBS) is GIBCO (Grand Island, NY, U.S.A), Human Macrophage-Colony Stimulation Factor (M-CSF) is PEPROTECH, INC. (Rocky Hill, NJ, USA), Methyl methacrylate (MMA) for Merck KGaA (Darmstadt, Germany), N,N-DIMETHYL-p-TOLUIDINE for MP Biomedicals (Solon-Ohio, BC, U.S.A), oligo(dT) is Promega (Madison, WI, USA), penicillin/streptomycin is Lonza (Rockland, ME, USA), erythrocyte lysate is BioLegend (San Diego, CA, U.S.A), Receptor Activator for Nuclear Factor κB Ligand (RANKL) was purchased from R&D systems (Minneapolis, MN, U.S.A), SuperScript Ⅱ Reverse Transcriptase was Intron (iNtRON Biotechnology, Gyeinggi-do, Korea), and SYBR green master mixture was purchased from ABI (Carlsbad, CA, U.S.A). did

2. Experimental equipment

ViiA7 (Applied Biosystems, CA, U.S.A) was used for real-time PCR, Nano drop 2000 was used for DNA and RNA quantification, and Thermo scientific (Rockford, IL, U.S.A) was used for the incubator. Protein quantification and MTT were performed using an ELISA reader (TECAN, Mannedorf, Switzerland).

3. Cell Culture

MC3T3-E1 (mouse calvaria osteoblast cell line) cells were cultured using α-MEM containing 10% (v/v) FBS, penicillin (100 units/ml) and streptomycin (100 units/ml) at 37°C, 5 % (v/v) CO ₂ Cultured in a constant temperature and humidity incubator. Bone marrow stem cells were obtained from femur and tibia bone marrow of 8-week-old male mice, and 10% (v/v) FBS, penicillin (100 units/ml) and streptomycin (100 units/ml) were used in DMEM at 37°C, 5% (v/v) CO ₂ Cultured in a constant temperature and humidity incubator. In the osteoblast differentiation experiment, osteoblasts or bone marrow stem cells were inoculated in a 48-well culture dish so that it was 5 × 10 ⁴ /well, and when the culture dish was 100% filled, ascorbic acid (50 μg/ml) and β-glycerol Phosphate (β-glycerophosphate, 10 mM) was added to induce osteoblastization. During the differentiation period, the differentiation medium was changed every 3 days, and staining was performed on the 28th day of differentiation.

4. Cell proliferation assay

Cell proliferation was evaluated by MTT assay. For MTT assay, MC3T3-E1 cells and bone marrow stem cells were dispensed in a 96-well multi-plate by 5 × 10 ³ cells/well, and 24 hours later, FSTL1 was treated at 0, 10 and 100 ng/ml concentrations and cultured for 24 hours. Then, 50 μl of MTT solution (5 mg/ml) was put into each well and incubated for 4 hours. The solution was completely removed, and 150 μl/well of DMSO was added to dissolve formazan crystals, and then the absorbance was measured at a wavelength of 570 nm using an ELISA reader.

5. Cell migration assay (Migration assay)

In order to observe the mobility of stem cells, a cell migration analysis experiment using a Transwell was performed. Bone marrow stem cells were seeded into the up-well at 1×10 ⁵ cells/well, and 24 hours later, the down-well part was treated with 0, 10 and 100 ng/ml of FSTL1. After 48 hours of treatment with FSTL1, the bone marrow stem cells that passed through the transwell membrane were stained using crystal violet to evaluate the mobility of the bone marrow stem cells.

6. Alkaline phosphatase (ALP) staining

MC3T3-E1 and bone marrow stem cells were seeded at 5×10 ⁴ cells/well in a 48-well multi plate, replaced with differentiation induction medium 2 days later, and FSTL1 was treated at 0, 10, and 100 ng/ml concentrations for 7 days. cultured. After incubation, the cells were washed 3 times with PBS, and fixed with a fixative [citrate or citrate acid (5 ml) + acetone (13 ml) + formaldehyde solution (1.6 ml)] for 45 seconds. Washed twice with PBS, added alkaline-dye mixture, blocked light at room temperature, and reacted for 30 minutes. After removing the solution, it was washed 3 times for 5 minutes with PBS and observed under a microscope.

7. Alizarin red S staining

MC3T3-E1 and bone marrow stem cells were dispensed in a 48-well multi-plate at 5×10 ⁴ cells/well, and 2 days later, while replacing the differentiation induction medium, FSTL1 was treated at concentrations of 0, 10 and 100 ng/ml for 21 days and Cultured for 14 days. After incubation, the cells were washed 3 times with PBS, and fixed at room temperature for 10 minutes using 4% formaldehyde as a fixing solution. Then, it was washed three times with tertiary distilled water, and alizarin red solution (pH 4.2) was added thereto and dyed at room temperature for 10 minutes. It was washed several times with PBS and observed under a microscope.

8. Western blotting

Western blotting was performed to confirm the protein expression change of osteoblast differentiation markers. By inducing differentiation of osteoblasts, proteins were extracted on the 8th, 15th and 28th days of differentiation, and the protein was quantified by the Bradford method, and 20 μg of the protein was loaded on 8% and 15% acrylamide gels. After blocking for 1 hour using 5% skim milk, washed 3 times for 5 minutes each with TBS-T (0.1% tween 20/Tris-buffered saline), the primary antibody was reacted at room temperature for 1 hour and 30 minutes. After washing 3 times with TBST, the secondary antibody was reacted at room temperature for 1 hour, and the color was developed using ECL solution to confirm the protein expression level of the osteoblast differentiation marker.

<Example 1> FSTL1 Confirmation of proliferation and differentiation of osteoblasts by administration

To confirm the effect of FSTL1 (Follistatin-like 1) secreted from mature osteoclasts on the proliferation and differentiation of osteoblasts, cell proliferation analysis using osteocytes (MC3T3-E1) and Alizarin red S staining was performed.

As a result, as shown in FIG. 2 , it was confirmed that the proliferation of MC3T3-E1 was increased and differentiation was promoted by FSTL1.

In addition, in order to confirm the changes in the osteoblast differentiation markers in osteoblasts by FSTL1 administration, MC3T3-E1 cells were treated with recombinant FSTL1 and then Western blot was performed to determine the osteoblast differentiation marker protein during the osteoblast differentiation process. Expression changes were confirmed.

As a result, as shown in FIG. 3 , in the group treated with FSTL1 (100ng/ml), increases in Runx2 and osteocalcin were observed.

<Example 2> Confirmation of bone marrow stem cell proliferation, migration and osteoblast differentiation by FSTL1 administration

To determine the effect of FSTL1 on the proliferation, migration and differentiation of bone marrow stem cells, bone marrow stem cells were isolated from the femur and tibia of an 8-week-old male mouse and used in the experiment.

The proliferation of bone marrow stem cells was confirmed by performing MTT assay, and alkaline phosphatase (ALP) staining was performed to confirm the differentiation effect. In addition, in order to observe the mobility of stem cells, a cell migration assay using a transwell was performed.

As a result, it was confirmed that the proliferation of bone marrow stem cells by treatment with FSTL1 did not change as shown in FIG. 4, but the migration of bone marrow stem cells and differentiation into osteoblasts were promoted.

<Example 4> Confirmation of changes in the expression of bone marrow stem cells of bone marrow differentiation marker genes by FSTL1 administration

Changes in gene expression of osteogenic markers were confirmed during the differentiation process of osteoblasts.

Real-time PCR was performed to confirm changes in gene expression of osteoblast differentiation markers. Bone marrow stem cells were treated with FSTL1 (100ng/ml) to induce differentiation into osteoblasts, and on the 1st, 8th, and 13th days of differentiation, total RNA was extracted using the RNA extraction kit (Easy-blue), and 2 μg of total RNA was extracted. It was synthesized into cDNA using reverse transcriptase. Real-time PCR was performed by mixing 0.5 μl of the synthesized cDNA with the 2× SYBR green PCR master mixture (5 μl) and specific primers (0.2 μl). Primers used for real-time PCR are listed in Table 1. Primers were designed using Primer Express software (ABI).

As a result, in the group treated with FSTL1 (100ng/ml) as shown in FIG. 5, alkaline phosphatase (ALP), Osx (Osterix, Transcription factor Sp7), FGF23 (Fibroblast growth factor 23), DMP1 (Dentin matrix acidic phosphoprotein 1) ), it was confirmed that the expression levels of Phex (Phosphate Regulating Endopeptidase Homolog X-Linked) and osteocalcin were increased.

oligo name sequence mALP-F TCC CAC GTT TTC ACA TTC GG mALP-R CCC GTT ACC ATA TAG GAT GGC C mOsx F AGA GGT TCA CTC GCT CTG ACG A mOsx R TTG CTC AAG TGG TCG CTT CTG mOsteocalcin-F TTC TGC TCA CTC TGC TGA CCC T mOsteocalcin-R CCT GCT TGG ACA TGA AGG CTT mFgF23-F CCT GAG GAA TGA TTC GCC AA mFgF23-R GAG AGT TCG AGC GTT TGC ATT T mDMP1-F GGT TTT GAC CTT GTG GGA AA mDMP1-R CAT ATT GGG ATG CGA TTC CT mPHEX F TGA TTC CAC ATG AGA ACC GAA C mPHEX R TGA TAT AGC CCA GCC AGT CAA A

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

delete delete delete A reagent composition for inducing or promoting osteoblast differentiation of bone marrow stem cells containing FSTL1 as an active ingredient. delete treating the candidate substance to bone marrow stem cells in vitro;
One or more expression levels of FGF23 (Fibroblast growth factor 23), DMP1 (Dentin matrix acidic phosphoprotein 1) and Phex (Phosphate Regulating Endopeptidase Homolog X-Linked), which are osteoblast differentiation markers, in bone marrow stem cells treated with the candidate material checking; and
An osteoblast differentiation promoting agent screening method comprising the step of comparing the expression level of the osteoblast differentiation marker with a control. delete

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