Peptide for skeleton protection that inhibits synoviocyte and bone resorption

Abstract

To provide a peptide for skeleton protection capable of achieving a skeleton-protective effect against skeleton erosion resulting from rheumatoid arthritis.SOLUTION: By administering a peptide having a specific amino acid sequence, cortical bone erosion of calcaneus bone resulting from rheumatoid arthritis (RA) can be ameliorated and the decline of RA-induced bone mineral density can be restored. The peptide for skeleton protection comprising the specific amino acid sequence of the present invention inhibits bone resorption, and proliferation and migration of synoviocytes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a peptide comprising the amino acid sequence of SEQ ID NO: 1 for protecting the skeleton, and in particular to a peptide for inhibiting bone resorption and the proliferation and migration of synovial cells. [Background technology]

[0002] Rheumatoid arthritis (RA) is a multifactorial autoimmune disease that primarily manifests as a chronic inflammatory joint disease characterized by synovial hyperplasia leading to swelling, infiltration of activated immune cells, synovial pannus invasion, and cartilage and bone destruction, resulting in deformity and disability.

[0003] Although the pathogenesis of RA remains poorly understood, a growing body of literature indicates that fibroblast-like synoviocytes (FLS) are key components of the aggressive synovium and play a crucial role in the development and maintenance of destructive arthritis. In healthy synovium, these cells primarily control the composition of synovial fluid and maintain joint homeostasis. In RA, FLS acquire an aggressive tumor-like phenotype characterized by resistance to apoptosis and increased migration followed by cartilage invasion. FLS also produce large amounts of proinflammatory cytokines and chemokines, adhesion molecules, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs), all of which are involved in the progression of RA.

[0004] Bone erosion is a major consequence of RA progression and correlates with disease severity and functional decline. Therefore, in long-term RA patients, bone resorption (driven by osteoclasts) typically exceeds bone formation (driven by osteoblasts), leading to destruction of subchondral bone. Recently, several studies have shown that FLS is a key factor in osteoclast differentiation and bone erosion in inflammatory arthritis.

[0005] Conventional biologic disease-modifying antirheumatic drug (DMARD) therapy has proven effective in improving the severity and progression of RA. However, most patients still do not achieve sustained remission. Humira (adalimumab, an anti-TNF-α antibody for intravenous use) and Xeljanz (tofacitinib, a JAK inhibitor; oral use) are the two current blockbuster drugs in clinical practice for the treatment of RA. As the first JAK inhibitor, tofacitinib was previously the only oral targeted drug available for the treatment of RA. However, safety concerns related to its mechanism of action have arisen, including the risk of blood clots, liver enzyme abnormalities, elevated cholesterol levels, more severe infections, and cancer.

[0006] Thus, there remains an urgent need for methods for the treatment of symptoms resulting from RA. Patent document: TWI705818B Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the inadequacies of existing technologies, the present invention provides peptides for skeletal protection that can achieve a skeletal protective effect against skeletal erosion resulting from rheumatoid arthritis. [Means for solving the problem]

[0008] To achieve the above-mentioned object, the present invention provides a peptide for scaffold protection comprising the amino acid sequence of SEQ ID NO:1.

[0009] The present invention further provides a peptide for scaffold protection comprising the amino acid sequence of SEQ ID NO:1.

[0010] Preferably, the skeletal protection includes ameliorating bone erosion.

[0011] Preferably, the preservation of the skeleton includes maintaining the smoothness of the surface of the cortical skeleton, thereby creating a smooth bone contour with integrity.

[0012] Preferably, the skeletal protection includes improving or restoring bone mineral density (BMD).

[0013] According to the present invention, the skeleton includes cartilage, bones, ligaments (connecting bones), and joints, and therefore, protecting the skeleton according to the present invention includes protecting bones, cartilage, and joints.

[0014] Preferably, the peptide is provided with a pharmaceutically acceptable carrier, which includes, but is not limited to, a solvent, emulsifier, suspending agent, disintegrating agent, binder, excipient, stabilizer, diluent, gelling agent, lubricant, surfactant, or other similar or applicable carrier of the present invention.

[0015] Preferably, the peptide is provided in an enteral or parenteral dosage form. More preferably, enteral dosage forms include, but are not limited to, enteric-coated tablets, multi-layer tablets, sugar-coated tablets, sublingual tablets, chewable tablets, buccal tablets, capsules, powders, syrups, solutions, emulsions, suspensions, mucilages, magmas, fluid extracts, extracts, spirits, elixirs, tinctures, etc., or any other oral formulation or enema. More preferably, parenteral dosage forms include, but are not limited to, injections, ointments, lotions, liniments, or aerosol sprays.

[0016] The effective dose of the peptide in mice is 0.09 milligrams per kilogram per day (mg / kg / day) to 148 mg / kg / day. Preferably, the effective dose of the peptide in mice is 1.2 mg / kg / day to 125 mg / kg / day, or 0.1 mg / kg / day to 100 mg / kg / day. More preferably, the effective dose of the peptide in mice is 0.49 mg / kg / day to 99 mg / kg / day. More preferably, the effective dose of the peptide in mice is 0.5 mg / kg / day to 50 mg / kg / day. More preferably, the effective dose of the peptide in mice is 5 mg / kg / day to 30 mg / kg / day.

[0017] The effective dose of the peptide in humans is 0.007 mg / kg / day to 12 mg / kg / day. Preferably, the effective dose of the peptide in humans is 0.1 mg / kg / day to 10 mg / kg / day, or 0.008 mg / kg / day to 8.13 mg / kg / day. More preferably, the effective dose of the peptide in humans is 0.04 mg / kg / day to 8 mg / kg / day. Even more preferably, the effective dose of the peptide in humans is 0.04 mg / kg / day to 6.15 mg / kg / day. Such dosages (effective doses of peptide in humans) are determined according to the method of "Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers" published by the U.S. Food and Drug Administration in 2005, based on a 60 kg adult. In terms of dosage, the recommended daily dose per kilogram of body weight in humans ( / kg body weight / day) is equivalent to 12.3 times the dosage in mice.

[0018] Preferably, the effective dose of the peptide in humans is 0.1 mg / kg / day to 10 mg / kg / day, and more preferably, the effective dose of the peptide in humans is 0.5 mg / kg / day to 2 mg / kg / day.

[0019] Preferably, the peptides of the present invention are administered once a day to once a month, for example, once a day, twice a day, once a week, twice a week, or three times a week.

[0020] Preferably, protecting the skeleton includes avoiding cartilage damage.

[0021] Preferably, the avoidance of cartilage damage is achieved by inhibiting the proliferation of synovial cells.

[0022] Preferably, the avoidance of cartilage damage is achieved by inhibiting synovial cell migration.

[0023] Preferably, the synovial cells are fibroblast-like synovial cells, and more preferably, the synovial cells are rheumatoid arthritis fibroblast-like synovial cells.

[0024] Preferably, the skeletal protection includes joint protection.

[0025] Preferably, the skeletal protection includes inhibiting bone resorption.

[0026] Preferably, the enteral dosage form is an oral dosage form.

[0027] Preferably, the parenteral dosage form is an injectable dosage form.

[0028] According to the present invention, the term "effective amount" refers to the dosage required to induce a desired biological response, i.e., the dosage required to demonstrate a therapeutic effect. According to the present invention, the effective dosage can achieve protection of the skeleton, improvement of bone erosion, improvement of the smoothness of the cortical bone surface, improvement or restoration of bone density, prevention of cartilage invasion, and inhibition of synovial cell proliferation and migration.

[0029] To achieve the above-mentioned object, the present invention further provides a peptide for inhibiting the proliferation of synovial cells, comprising the amino acid sequence of SEQ ID NO:1.

[0030] To achieve the above-mentioned object, the present invention further provides a peptide for inhibiting synovial cell migration, comprising the amino acid sequence of SEQ ID NO:1.

[0031] To achieve the above-mentioned object, the present invention further provides a peptide comprising the amino acid sequence of SEQ ID NO: 1 for inhibiting bone resorption.

[0032] The present invention demonstrates that the peptide of SEQ ID NO: 1 has the advantage of improving the cortical surface erosion of the calcaneus and restoring BMD destruction in mice with rheumatoid arthritis, thereby achieving a skeletal protective effect. The peptide of SEQ ID NO: 1 can also inhibit the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes, thereby avoiding joint and cartilage damage, and inhibiting bone resorption to achieve a skeletal protective effect. [Brief explanation of the drawings]

[0033] [Figure 1A] FIG. 1A is an image of the mouse calcaneus in Study 1, first obtained by micro-CT scanning and then segmented and analyzed by CTAn software. [Figure 1B] FIG. 1B shows enlarged images of selected groups from FIG. 1A at weeks 0, 4, and 6: the control group, the peptide of SEQ ID NO: 1 (10 mg / kg)-treated group, and the tofacitinib-treated group. [Figures 2A-2E] Figures 2A-2E show the bone mineral density of the calcaneus of mice in the control group, the tofacitinib-treated group, and the peptide of SEQ ID NO: 1 (1 mg / kg, 3 mg / kg, and 10 mg / kg)-treated group, respectively, in Study 1. * indicates p<0.05, *** indicates p<0.001, and ns indicates no significant difference. [Figure 3A] Figure 3A shows the cell proliferation rate of SW982 synoviocytes after treatment with peptide of SEQ ID NO: 1 (at different concentrations) in Study 2. ** means p<0.01 (compared to the control group), and *** means p<0.001 (compared to the control group). [Figure 3B] FIG. 3B shows the cell proliferation rate of SW982 synoviocytes treated with IL-1β in Study 2. [Figure 3C] FIG. 3C shows the inhibitory effect on cell proliferation rate of SW982 synoviocytes after combined treatment with the peptide of SEQ ID NO:1 and IL-1β in Study 2. [Figure 4A]Figure 4A shows the cell migration rate of SW982 synoviocytes after treatment with the peptide of SEQ ID NO: 1 (at different concentrations) in Study 3. *** indicates p<0.001 (compared to the control group). [Figure 4B] FIG. 4B is a cell migration image of SW982 synoviocytes after 24 hours of peptide treatment of SEQ ID NO:1 (at different concentrations) in Study 3. [Figure 5A] FIG. 5A shows the depression area resulting from coated calcium phosphate resorption in a bone resorption test. [Figure 5B] FIG. 5B shows the inhibitory effect on bone resorption of RAW264.7 cells after combined treatment with the peptide of SEQ ID NO: 1 and RANKL in a bone resorption assay. DETAILED DESCRIPTION OF THE INVENTION

[0034] The technical means adopted by the present invention to achieve the intended objects will be illustrated with reference to the drawings and the following preparation and experimental examples.

[0035] Preparation Example 1: Peptide of SEQ ID NO: 1 The peptide used in this invention is the same as that described in Taiwan Patent No. I705818. Specifically, the peptide of this invention, Glp Glu Thr Ala Val Ser Ser His Glu Gln Asp, was synthesized using a standard Fmoc method and microwave peptide synthesizer. Glp is pyroglutamic acid (Cas Number 98-79-3). Generally, Wang resin (loading 0.6 mmol / g) preloaded with D residues was weighed into a reaction vessel, and fresh dimethylformamide (DMF) (10 mL–15 mL) was added to prepare for swelling before synthesis. The swelling time was set to 3 minutes (min) in the microwave peptide synthesizer.

[0036] To liberate the N-terminal amine group, the first and subsequent Fmoc groups were removed with 5 mL of 20% piperidine in DMF by standard deprotection treatment ("DEP" step) (in the first step, the temperature was 75°C, the power was 155 W, and the temperature hold time was 15 seconds; in the second step, the temperature was 90°C, the power was 30 W, and the temperature hold time was 50 seconds).

[0037] Then, 0.5 M N'-diisopropylcarbodiimide (DIC) in DMF was added to activate the C-terminus ("ACT" step), and the desired amino acid (Fmoc-AA(protected side chain)-OH) was added in a 5-fold excess (0.2 M concentration in DMF) and 1.0 M oxime in DMF to initiate the coupling reaction ("AA" step). Standard coupling reactions were performed (Step 1: temperature 75 °C, power 170 W, temperature hold time 15 seconds; Step 2: temperature 90 °C, power 30 W, temperature hold time 230 seconds). After approximately 4 minutes of "AA" step, the Wang resin was washed with DMF solution. The DEP-ACT-AA step was repeated to assemble the peptide from the C-terminus to the N-terminus.

[0038] Finally, the peptide was removed from the solid support by treatment with 95% TFA / 2.5% HO / 2.5% TIPS in an ice-water bath, and the reaction was then allowed to warm to room temperature for 2 hours. After filtration, the filtrate was collected and added with cold ether for precipitation, followed by centrifugation to remove the supernatant. The precipitate was then washed with ether. This process was repeated 3 to 6 times. The final precipitate was collected and lyophilized to obtain the final product, a white, flocculent-like product. The purity of the final product was determined by high-performance liquid chromatography, and the molecular weight and sequence were analyzed by LC-MS / MS, yielding the peptide of SEQ ID NO:1.

[0039] Test 1: Animal testing This animal experiment was performed according to the adjuvant-induced arthritis (AIA) model designed by Academia Sinica, Taipei, Taiwan. Specifically, arthritis induction was performed according to the method described in Gauldie, SD, McQueen, DS, Clarke, CJ, & Chessell, IPA, "Robust model of adjuvant-induced chronic unilateral arthritis in two mouse strains." J Neurosci Methods 139, 281-291 (2004). Briefly, this animal experiment was performed on 30 C57BL / 6 mice, 8 weeks old and weighing approximately 20-25 grams, purchased from the National Laboratory Animal Center in Taiwan. Using a 30-gauge needle attached to a 100 μl Hamilton syringe, 5 μg of Complete Freund's Adjuvant (CFA) (F5881, Sigma, USA) was injected into the right ankle joint of each subject mouse once a week for four consecutive injections. The first injection was performed on week 0 (day 0). Mice were anesthetized and maintained with a 2% isoflurane supply during the injection. The AIA model can induce long-term inflammation and swelling of one ankle joint in C57BL / 6 mice, simulating the long-term chronic pain experienced by patients with rheumatoid arthritis.

[0040] After CFA induction, mice were weighed weekly and scored using the rheumatoid arthritis score estimation (data not shown) according to Hsieh, WS, et al. (2017). TDAG8, TRPV1, and ASIC3 involved in establishing hyperalgesic priming in experimental rheumatoid arthritis. Scientific Reports, 7(1), 8870. All mice showed persistent rheumatoid arthritis by week 4 (day 28). Mice were assigned to five groups: (1) control group, (2) P1 group (given peptide of SEQ ID NO:1 at 1 mg / kg / day), (3) P3 group (given peptide of SEQ ID NO:1 at 3 mg / kg / day), (4) P10 group (given peptide of SEQ ID NO:1 at 10 mg / kg / day), and (5) T group (given tofacitinib at 12.4 mg / kg / day). The animals were grouped based on their rheumatoid arthritis scores using a random sampling method. From week 4 (day 28) to week 6 (day 42), the drug administration was as follows: the target mice received the following solutions orally (by gavage) at 5 μL / g body weight (bw) once daily: (1) peptide of SEQ ID NO: 1 (1 mg / kg bw, 3 mg / kg bw, or 10 mg / kg bw dissolved in water) in groups P1, P3, and P10, respectively; (2) tofacitinib (12.4 mg / kg bw dissolved in 0.5% carboxymethylcellulose aqueous solution) in group T; and (3) water in the control group. The target mice were sacrificed on day 45.

[0041] A. Micro-computed tomography (μCT) scanning Each mouse was anesthetized (2.5% isoflurane, 1.0 L / min air) and maintained under anesthetic gas throughout the scan. The mouse was placed in a carbon fiber half-cylinder bed, with its right hind leg positioned in a cylindrical polystyrene foam holder, inside a micro-CT scanner (Skyscan 1076). The mouse's foot was fixed to the centerline of the scanner to prevent inappropriate movement.

[0042] The microCT scanner (SkyScan-1076; Bruker MicroCT, Belgium) settings were as follows: isotropic voxel size: 9 μm, tube voltage: 49 kV, current: 167 μA, Al filter: 0.5 mm, and 180° scan with a 0.6° rotation interval (720 projections) and 3 frame averaging. MicroCT projections were then backprojected and reconstructed using NRecon software (NReconServer 64bit, Bruker MicroCT, Belgium), volume-rendered, and visualized in 3D using CTVox software (Bruker MicroCT, Belgium). Images of the calcaneus were segmented and analyzed using CTAn software (Bruker MicroCT, Belgium).

[0043] B. Statistical analysis Statistical analysis and graphing were performed using GraphPad Prism 9.3.1 (GraphPad Software, USA). Data are expressed as mean ± standard deviation. The level of statistical significance was set at 5% (P < 0.05) for all analyses. Data analysis of indices derived from μCT images of the mouse calcaneus was performed. For example, bone mineral density data was analyzed using one-way RM ANOVA (one-way repeated measures analysis of variance) with Tukey's post-hoc test.

[0044] C. Preliminary Safety Profile To evaluate the safety of the peptide of SEQ ID NO:1, the mice were weighed six times at weekly intervals. All data were converted into percentage weight changes using the weight at week 0 (before AIA induction) as the denominator. No significant differences in weight changes were observed after one or two weeks of treatment (i.e., week 5 or week 6) between mice treated with the peptide of SEQ ID NO:1 or mice treated with tofacitinib (P > 0.05 compared with week 4 or the control group) (data not shown). Observation of mice treated with the peptide of SEQ ID NO:1 revealed no signs of irritation, loss of vitality, or any changes in skin or hair. Therefore, the peptide of SEQ ID NO:1 was deemed safe for mice.

[0045] The observation time frames for μCT scanning were set before AIA induction (week 0), at the end of induction (week 4), and after one or two weeks of treatment (i.e., weeks 5 and 6). Images were then segmented and analyzed using CTAn software, as shown in Figure 1A. For clearer comparison, images from weeks 0, 4, and 6 were enlarged, as shown in Figure 1B. Figures 1A and 1B clearly show that at week 4 (at the end of induction, before treatment), the integrity of the calcaneal surface was compromised, as indicated by the irregularities in the cortical bone (dashed circle; week 4 vs. week 0). As shown in Figure 1A, treatment with the peptide of SEQ ID NO: 1 at 1 mg / kg or 3 mg / kg did not demonstrate any benefit in restoring bone contour. In contrast, as shown in Figure 1B, mice treated with 10 mg / kg of the peptide of SEQ ID NO: 1 for two weeks had fewer sharp protrusions and a smoother cortical surface on the calcaneus (week 6 vs. week 4, dashed circle). It should be noted that tofacitinib treatment did not reduce the degree of surface roughness or distortion of bone contour, and even worsened bone destruction in some areas (solid circle, week 6 vs. week 4). Therefore, treatment with 10 mg / kg of the peptide of SEQ ID NO: 1 could improve bone destruction, demonstrating a better therapeutic effect than the conventional drug tofacitinib.

[0046] Bone mineral density (BMD, unit: g / cm 3 The results of the evaluation are shown in Figures 2A-2E. As expected, BMD significantly decreased at week 4, and this phenomenon persisted until week 6 (P<0.001 compared with week 0, Figure 2A). Furthermore, in group T, neither 1-week nor 2-week treatment with tofacitinib reversed the BMD loss (P>0.05, weeks 5 / 6 vs. week 4, Figure 2B). Furthermore, in groups P1 and P3, mice treated with 1 mg / kg or 3 mg / kg of the peptide of SEQ ID NO:1 showed no significant changes in BMD after 1 or 2 weeks of treatment (P>0.05, weeks 5 / 6 vs. week 4, Figures 2C and 2D). In the P10 group (given 10 mg / kg of the peptide of SEQ ID NO:1), no increase in BMD was observed after 1 week of treatment, but a significant increase in BMD was observed after 2 weeks of treatment (P<0.05, week 6 vs. week 4; Figure 2E). This indicates that a relatively high dose (10 mg / kg) of the peptide of SEQ ID NO:1 has a high therapeutic effect on bone protection in inflammatory arthritis. Therefore, treatment with 10 mg / kg of the peptide of SEQ ID NO:1 can ameliorate bone destruction and restore BMD in AIA mice, demonstrating a better therapeutic effect than that of tofacitinib.

[0047] In vitro testing

[0048] Test 2: Cell viability test Because primary rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) have a limited lifespan, the fibroblast-like synoviocyte (FLS) cell line SW982 synoviocytes (hereafter referred to as SW982 cells) is well accepted as a useful in vitro model for conducting experiments on the pathogenesis of RA. Briefly, SW982 cells were seeded into 96-well plates at a cell density of 6,000 cells per well. After treatment, the effect of each treatment group on SW982 cell viability was tested using a cell proliferation detection kit (Cell Counting Kit-8, CCK8; Elabscience, Catalog No: E-CK-A361) according to the manufacturer's instructions. The treatment groups of interest were as follows: (1) Different concentrations of the peptide of SEQ ID NO: 1: SW982 cells were treated with the peptide of SEQ ID NO: 1 for 24 hours at 100 μg / ml. -6 , 10 -7 , 10 -8 , 10 -9 (2) IL-1β stimulation group: SW982 cells were stimulated with medium containing 1 ng / mL of IL-1β for 24 hours to simulate inflammatory synovial cells in rheumatoid arthritis; (3) combination treatment group: SW982 cells were stimulated with the peptide of SEQ ID NO: 1 for 72 hours at 10 μM. -9 SW982 cells were treated with IL-1β at 1 ng / mL in medium containing 1 μM IL-1β. All SW982 cells in the treatment groups were cultured in the absence of carbon dioxide. As a control, SW982 cells were cultured in L15 medium for 24 hours. In this study, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-8), included in the CCK-8 kit, reacted with the dehydrogenase enzymes of viable cells, resulting in a color change from pink to orange in all treatment groups containing WST-8. The dehydrogenase activity was determined by detecting absorbance at 450 nm, and the number of viable cells was calculated.

[0049] The results are shown in Figures 3A, 3B, and 3C. As shown in Figure 3, the peptide of SEQ ID NO: 1 -9 ~10 -6Concentrations in the μM range effectively inhibited the proliferation of SW982 cells. As shown in Figure 3B, treatment with IL-1β (1 ng / mL) for 24 hours dramatically enhanced the proliferation of SW982 cells, which is consistent with the fact that inflammatory factors in the microenvironment (as in RA) promote the proliferation of synovial fibroblasts. Of note, as shown in Figure 3C, (10 -9 The results show that the peptide of SEQ ID NO: 1 (at 1 μM) can significantly inhibit IL-1β-induced proliferation of SW982 cells.

[0050] Test 3: Measurement of SW982 cell migration Fibroblast-like synoviocytes are known to secrete inflammatory cytokines, chemokines, and matrix metalloproteinases (MMPs), inducing cell migration and invasion that damage joints and cartilage. Next, we investigated the effect of the peptide of SEQ ID NO: 1 on SW982 cell migration using a conventional wound healing experiment. Specifically, Culture-Insert 2 Well (ibidi), which contains two cell culture reservoirs separated by a 500 μm wall, was placed on the surface of a cell culture plate. Each reservoir was filled with L15 medium and 4 x 10 cells to allow cell growth within the designated area. 4 After proper cell attachment of all SW982 cells, the Culture-Insert 2 Well was removed to create a cell-free gap of approximately 500 μm. The cell monolayer was washed with PBS buffer to remove detached cells and debris. SW982 cells were incubated in L15 medium with peptide of SEQ ID NO: 1 (10 -5 , 10 -6 , 10 -7Cells were cultured for 24 hours in the absence of carbon dioxide with either 0.1 μM of the peptide or L15 medium alone (as a control). Direct microscopic visualization was used to create a reference point at the bottom of each field, and cell migration and wound healing rates were analyzed by measuring the remaining cell-free area in images of the same field taken immediately after 24 hours of treatment with the peptide of SEQ ID NO:1. Wound healing / cell migration kinetics was analyzed using Image J (National Institutes of Health, NIH) software. Specifically, the area of ​​the cell-free area was calculated using Image J. The formula for calculating migration rate is as follows:

[0051] (Size of cell-free area before peptide treatment - Size of cell-free area after peptide treatment) ÷ Size of cell-free area before peptide treatment × 100%

[0052] The results are shown in Figures 4A and 4B. As shown in Figure 4A, the peptide of SEQ ID NO: 1 (10 -7 ~10 -5 Treatment with 10 μM of the peptide of SEQ ID NO:1 for 24 hours significantly reduced the lateral migration of SW982 cells. Cell images are shown in Figure 4B. It should be noted that, as shown in Figure 4B, -5 μM) is a high concentration (10 -7 Although it may exert stronger inhibition at concentrations lower than 1 μM, the results shown in Figure 4A did not confirm statistical significance.

[0053] From the above, the present invention has confirmed that the peptide of SEQ ID NO: 1 can actually improve the cortical surface erosion of the calcaneus in mice with RA and restore BMD destruction in mice with RA, thereby achieving a skeletal protection effect. The peptide of SEQ ID NO: 1 can also inhibit the proliferation and migration of SW982 synovial cells, thereby avoiding joint and cartilage damage.

[0054] Test 4: Bone resorption test In this study, RANKL (receptor activator of nuclear factor-κB ligand) was used as an inducer of osteoclast differentiation to detect whether the peptide of SEQ ID NO: 1 could inhibit the osteoclast resorption function induced by RANKL in vitro. This study included three groups: a control group, a RANKL group, and a treatment group.

[0055] First, RAW264.7 cells were plated onto calcium phosphate (CaP)-coated 96-well plates at 2 × 10 cells per well. 3 Cells were seeded at a density of 1000 μg / ml. RAW264.7 is a macrophage cell line, and RAW264.7 cells can differentiate into osteoclasts. Cells in all groups were cultured for 7 days according to the manufacturer's instructions for the bone resorption assay kit (Cosmo Bio). The differences between the control group, RANKL group, and treatment group are as follows:

[0056] (1) Control group: neither RANKL nor the peptide of SEQ ID NO: 1 was administered to RAW264.7 cells during the 7-day culture period. (2) RANKL group: RANKL at a concentration of 100 ng / mL was administered to RAW264.7 cells on day 1 and cultured for 7 days. (3) Treatment group: On day 1, RAW264.7 cells were simultaneously treated with RANKL at a concentration of 100 ng / mL and the peptide of SEQ ID NO: 1 at a concentration of 100 μM, and then cultured for 7 days.

[0057] After a 7-day culture period, all wells in all groups were washed with 0.2–0.5 ml of 5% sodium hypochlorite to remove cells attached to the surface. All plates were further washed with water and dried. The area within each well in all groups was photographed under a light microscope at 100x magnification, as shown in Figure 5A. The depression area resulting from osteoclast absorption of the coated calcium phosphate was measured and calculated using image analysis software (Image J). The depression area results, representing the mean ± SD of three independent samples, are shown in Figure 5B. *** indicates p<0.001 relative to the RANKL group.

[0058] Figure 5B shows that the depression area in the RANKL group was 1-fold, which was significantly higher than that in the treatment group (approximately 0.5-fold). Therefore, it was confirmed that the peptide of SEQ ID NO: 1 can also inhibit calcium phosphate absorption by osteoclasts (bone resorption), thereby preventing damage to joints and cartilage.

[0059] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any respect. Although the present invention has been described in connection with certain preferred embodiments, it should be understood that the present invention should not be unduly limited to those specific embodiments. It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the present invention. Indeed, various modifications apparent to those skilled in the art are intended to be encompassed within the scope of the following claims.

Claims

1. A peptide for backbone protection, comprising the amino acid sequence of SEQ ID NO:

1.

2. 10. The peptide of claim 1, wherein the skeletal protection comprises ameliorating bone erosion.

3. The peptide of claim 1 , wherein the protection of the skeleton includes improving the smoothness of the surface of cortical bone.

4. 10. The peptide of claim 1, wherein the skeletal protection comprises improving or restoring bone density.

5. The peptide according to any one of claims 1 to 4, wherein the effective amount of the peptide is in the range of 0.1 mg / kg / day to 10 mg / kg / day.

6. 2. The peptide of claim 1, wherein the protection of the skeleton comprises avoiding cartilage damage.

7. The peptide of claim 6, wherein the avoidance of cartilage damage is achieved by inhibiting synovial cell proliferation.

8. The peptide of claim 6, wherein the avoidance of cartilage damage is achieved by inhibiting synovial cell migration.

9. The peptide according to claim 7 or 8, wherein the synoviocytes are fibroblast-like synoviocytes.

10. The peptide of claim 1 , wherein the skeletal protection comprises inhibiting bone resorption.

11. 10. The peptide of claim 1, wherein the skeletal protection includes joint protection.

12. A peptide for inhibiting the proliferation of synovial cells, comprising the amino acid sequence of SEQ ID NO:

1.

13. A peptide for inhibiting synovial cell migration, comprising the amino acid sequence of SEQ ID NO:

1.

14. A peptide for inhibiting bone resorption, comprising the amino acid sequence of SEQ ID NO: 1.

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