WO2014204229A2 - Pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising peptide as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising, as an active ingredient, a peptide consisting of an amino acid sequence having SEQ ID NO: 1. The pharmaceutical composition according to the present invention has an advantageous effect of inhibiting the generation and activation of osteoclasts, as well as inhibiting the differentiation and activation of Th17 cells and TFH cells, which are important in the etiological process of rheumatoid arthritis, and is thus expected to be applicable to the fundamental treatment of rheumatoid arthritis.

Description

Pharmaceutical composition for the prevention or treatment of rheumatoid arthritis comprising the peptide as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating rheumatoid arthritis, and more particularly to a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising a peptide consisting of the sequence of SEQ ID NO: 1 as an active ingredient.

Rheumatoid arthritis is a systemic inflammatory disease of unknown origin that is characterized by a number of chronic disease infections, but at the same time causes many organ damages. Rheumatoid arthritis progresses chronically with repeated deterioration of remission and exacerbation, and if left untreated, leads to destruction or deformation of the joints and eventually to dysfunction of the motor. Can be life threatening. Thus, patients with rheumatoid arthritis suffer a great deal of physical and mental pain throughout their lives.

Conventional therapeutic agents for treating such rheumatoid arthritis include nonsteroidal anti-inflammatory drugs (aspirin, ibuprofen), contraindications, penicylamines, steroid hormones, and the like. However, the most improved steroid hormones have the problem of long-term side effects. Recently, the treatment of rheumatoid arthritis on the market is mainly Biologic Response Modifier, which is mainly a TNF inhibitor and IL-1 inhibitor that can inhibit the action of inflammatory cytokines TNF-α and IL-1β that are excessively produced in the pathology of rheumatoid arthritis. In addition, selective B cell inhibitors and selective costimulation modulators have been developed and used as therapeutic agents to inhibit the production of autoimmune antibodies.

However, these therapies are known to cause side effects such as pain, itching, respiratory infections, and edema, and they have a fractional effect that inhibits the action of the resulting inflammatory cytokines, which do not provide the ultimate effective treatment. there is a problem. In other words, the production of inflammatory cytokines is important in the pathogenesis of rheumatoid arthritis, but ultimately the production and maturation of osteoclasts, which are important for the regulation of bone production and activity, are important in the pathogenesis of rheumatoid arthritis. There is a limit to the fundamental treatment by not having an inhibitory effect on osteoclast generation and activation.

As such, research on the development of a composition for inhibiting osteoclast generation and activation and the treatment of rheumatoid arthritis has been made (Korea Patent Publication No. 10-2013-0049558, etc.), but it is still inadequate. .

The present invention has been made to solve the above problems in the prior art, an object of the present invention to provide a pharmaceutical composition for preventing or treating rheumatoid arthritis comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. .

In another aspect, the present invention to provide a health functional food composition for preventing or improving rheumatoid arthritis comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising as an active ingredient a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the peptide is characterized in that it binds to the formyl peptide receptor (FPR).

In another embodiment of the present invention, the peptide is characterized in that binding to formyl peptide receptor 2 (FPR2).

In another embodiment of the present invention, the pharmaceutical composition is characterized by inhibiting the production of osteoclasts.

In another embodiment of the present invention, the pharmaceutical composition is characterized by reducing immunoglobulin and / or inflammatory cytokine levels.

In another embodiment of the invention, the inflammatory cytokine is selected from the group consisting of interleukin-17 (IL-17), interleukin-21 (IL-21) and TNF-α (Tumor necrosis factor-alpha) It is done.

In another embodiment of the invention, the pharmaceutical composition is characterized in that it further comprises a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a health functional food composition for preventing or improving rheumatoid arthritis comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

In one embodiment of the present invention, the peptide is characterized in that it binds to the formyl peptide receptor (FPR).

In another embodiment of the present invention, the composition is characterized by inhibiting the production of osteoclasts.

The present invention provides a method for preventing or treating rheumatoid arthritis, comprising administering to a subject a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The present invention provides a use of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 in the prevention or treatment of rheumatoid arthritis.

A pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising the peptide consisting of the amino acid sequence of SEQ ID NO: 1 according to the present invention, osteoclasts, in addition to inhibiting the differentiation and activation of T17 cells and T _FH cells important in the pathogenesis of rheumatoid arthritis In addition to inhibiting the production and activation of FPR2 through the effect of reducing the production of IL-17, IL-21 and immunoglobulin, it is expected to be applicable to the fundamental treatment of rheumatoid arthritis.

1 is a view showing the results of measuring the hind paw thickness using a caliper (caliper) in the mice treated with and without the WKYMVm peptide in Example 1.

FIG. 2 shows the results of calculating the clinical scores added after scoring by scoring redness and swelling in each of four feet of mice treated with and without the WKYMVm peptide in Example 1; The figure which shows.

Figure 3 is a view showing the results of observing the osteoclasts produced by optical microscopy by H & E staining or TRAP staining bone tissue samples of mice treated with and WKYMVm peptide group in Example 2.

Figure 4 is a schematic diagram showing the inhibitory effect verification experiment of the osteoclast generation step of WKYMVm peptide.

Figure 5 is a view showing the osteoclast generation inhibitory results of the WKYMVm peptide in the early differentiation in Example 3.

Figure 6 is a view showing the osteoclast generation inhibitory results of the WKYMVm peptide in the differentiation stage in Example 3.

FIG. 7 shows the results of measuring interleukin-10 (IL-10) levels in mice of the WKYMVm peptide treated group and the untreated group of Example 3. FIG.

Figure 8 shows the results of confirming the expression of FPR2 in Example 4-1 (a) Th0 cells not stimulated with CD3 / CD28 (b) Th0 cells stimulated with CD3 / CD28 beads, Th17 and T _FH cells Drawing.

Figure 9 is a view showing the results confirmed by the quantitative real-time PCR to change the expression level of RORγ-t and Bcl-6 in accordance with the treatment of the WKYMVm peptide in Example 4-2.

Figure 10 shows the results confirmed by FACS analysis whether Th17 inhibits IL-17 production according to the treatment of the WKYMVm peptide in Example 4-3.

Figure 11 shows the results confirmed by FACS analysis whether the inhibition of IL-21 production of T _FH in accordance with the treatment of the WKYMVm peptide in Example 4-3.

FIG. 12 shows the results of calculating the clinical scores added after scoring by scoring redness and swelling in the CIA mouse model treated with the WKYMVm peptide and the untreated CIA mouse model in Example 5-2. FIG. Drawing.

FIG. 13 is a view showing photographs observing the CIA mouse model treated with the WKYMVm peptide and the untreated CIA mouse model in Example 5-2. FIG.

14 is a diagram showing the results confirmed by ELISA analysis of IL-17, IL-21 and TNF-α production changes in the CIA mouse model treated with WKYMVm peptide and the untreated CIA mouse model in Example 5-2 .

FIG. 15 shows the results obtained by FACS analysis of cytokine production of CD4 T cells in splenocytes of CIA mouse in Example 5-3. FIG.

FIG. 16 shows the results of confirming collagen-specific T _FH cell (CD4 ⁺ CXCR5 ⁺ PD-1 ⁺ ) population by WKYMVm peptide treatment in Example 5-3. FIG.

FIG. 17 shows the results of confirming germinal center formation using an immunohistochemical analysis in the CIA mouse model treated with the WKYMVm peptide and the untreated CIA mouse model in Example 5-3. FIG.

FIG. 18 shows the results of confirming immunoglobulin amounts according to germinal center formation in mouse serum in Example 5-3 through ELISA analysis. FIG.

FIG. 19 shows the results of calculating the clinical scores calculated after scoring by scoring redness and swelling in the CIA mouse model treated with CsH and WRW ⁴ , which are antagonists of FPR1 and FPR2, in Example 5-4. The figure shown.

20 is a diagram showing the results confirmed by FACS analysis of the production of IL-17 and IL-21 in collagen-specific CD4 T cells in Example 5-4.

21 shows the treatment of CsH and WRW ⁴ , which are antagonists of FPR1 and FPR2, in Example 5-5, and (a) the population of CXCR5 ⁺ PD-1 ⁺ CD4 T cells was confirmed by FACS analysis. It is a figure showing the result confirmed the formation using the immunohistochemical analysis (immunohistochemical analysis).

FIG. 22 shows the results of confirming the immunoglobulin amount in mouse serum by ELISA analysis in Example 5-5. FIG.

Previously, the inventors have published a peptide ligand (Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ : SEQ ID NO: 1) that increases leukocyte cells such as monocytes, neutrophils, and the like. (Bae et al., 1999, J. Leukoc. Biol. 65, 241-248).

The present inventors have studied a new composition for solving the side effects of the currently available rheumatoid arthritis therapeutics, it was confirmed that the WKYMVm peptide is effective in rheumatoid arthritis by inhibiting the production of osteoclasts, based on the present invention It was completed.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising as an active ingredient a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

As used herein, the term "prevention" means any action that inhibits or delays the onset of rheumatoid arthritis by administration of a composition of the present invention.

As used herein, the term "treatment" refers to any action in which symptoms caused by rheumatoid arthritis are improved or beneficially altered by administration of a composition of the present invention.

The peptide, which is an active ingredient of the pharmaceutical composition according to the present invention, consists of an amino acid sequence of tryptophan-lysine-tyrosine-methionine-valine-di-methionine (WKYMVm) (SEQ ID NO: 1, hereinafter referred to as "WKYMVm peptide"), It binds to a formyl peptide receptor (FPR), more preferably formyl peptide receptor 2 (FPR2), and is effective in rheumatoid arthritis by inhibiting the production of osteoclasts.

In one embodiment of the present invention, the hind paw thickness and clinical score of the rheumatoid arthritis pathologic index of the mice induced by rheumatoid arthritis were treated by treating the WKYMVm peptide, and the hind paw thickness and It was confirmed that the clinical score was significantly relieved (see Example 1). In addition, in another embodiment of the present invention treated mice with rheumatoid arthritis WKYMVm peptide, and confirmed the production status of osteoclasts by H & E staining or TRAP staining, as compared with mice (control) not treated with WKYMVm peptide It was confirmed that the invasion of polymorphonuclear cells was significantly reduced (see Example 2).

In addition, the peptide as an active ingredient of the pharmaceutical composition of the present invention reduces the production of IL-17, IL-21 and immunoglobulin by binding to a formyl peptide receptor (FPR), preferably FPR2 to rheumatoid arthritis It works.

In another embodiment of the present invention confirmed that the inhibition of cytokine production according to the WKYMVm peptide treatment, IL-17 and IL- when treated with WKYMVm peptide than T17 cells and T _FH cells not treated with WKYMVm peptide 21 production was found to be reduced (see Example 4). Furthermore, in another embodiment of the present invention, using a collagen-induced-arthritis (CIA) mouse model to confirm whether the WKYMVm peptide inhibits rheumatoid arthritis through FPR2, IL- in a CIA mouse model treated with WKYMVm peptide. 17, IL-21, TNF-α and immunoglobulin production was confirmed to be reduced (see Example 5).

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. Pharmaceutically acceptable carriers included in the pharmaceutical compositions according to the present invention include, but are not limited to, saline, buffered saline, water, glycerol, polyethylene glycol, vegetable oils, isopropyl myristate and ethanol, and the like. Suitable formulations known to Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA) are all available.

In another aspect of the invention, the invention provides a method of treating rheumatoid arthritis by administering to a subject a peptide having the amino acid sequence of SEQ ID NO: 1. As used herein, "individual" means a subject in need of treatment for a disease, and more specifically human, or non-human primates, mice, rats, dogs, cats, horses, and cattle Means such mammals.

The preferred dosage of the pharmaceutical composition according to the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight is administered daily or every other day. It may be administered by dividing 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes. The method of administration is not limited and may be administered, for example, by oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine dural, or intra cerbroventricular injection.

As another aspect, the present invention provides a nutraceutical composition for preventing or improving rheumatoid arthritis comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. That is, the nutraceutical composition according to the present invention may be used simultaneously or separately with a medicament for the treatment of rheumatoid arthritis before or after the onset of rheumatoid arthritis in order to prevent or improve rheumatoid arthritis.

As used herein, the term "improvement" refers to any action that at least reduces the parameters associated with the condition being treated, such as the extent of symptoms.

The health functional food composition according to the present invention binds to the formyl peptide receptor (FPR) and inhibits the production of osteoclasts, and thus can be added to food supplements such as foods and beverages for the purpose of preventing or improving rheumatoid arthritis.

There is no particular limitation on the kind of food. Examples of foods to which the active ingredient may be added include drink, meat, sausage, bread, biscuit, rice cake, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups , Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, etc., and includes all the health functional foods in the conventional sense.

WKYMVm peptide as an active ingredient in the nutraceutical composition according to the present invention can be added to food as it is or used with other foods or food ingredients, and can be suitably used according to conventional methods. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement).

The composition for health drinks of the present invention is not particularly limited to other ingredients except for containing the active ingredient as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.

In addition to the above, the nutraceutical composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. These components can be used independently or in combination. The proportion of such additives may also be appropriately selected by those skilled in the art.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Validation of Rheumatoid Arthritis Inhibitory Effect of WKYMVm Peptides

In order to determine whether the WKYMVm peptide is effective for rheumatoid arthritis, the experiment was conducted as follows.

That is, 7-week-old BALB / c male mice were diluted 1/2 in PBS at 0 and 2 days, respectively. Rheumatoid arthritis was induced by intraperitoneal injection of 100 μl of K / BxN rheumatoid arthritis serum. In the experimental group, 4 mg / kg of WKYMVm peptide was intraperitoneal and subcutaneous injection six times at 12 hour intervals, respectively, and none of the control groups were treated.

Then, the hind paw thickness, which is one of the rheumatoid arthritis pathology tables, was measured using a caliper, and the results are shown in FIG. 1. On the other hand, the statistical analysis in the experiment was analyzed through Dunnett's Multiple Comparison Test (* P <0.05; ** P <0.01; *** P <0.001 (versus control group)).

In addition, the redness and swelling shown in each of the four paws of the mouse were scored and scored below to calculate a clinical score, which is one of the rheumatoid arthritis pathology tables, and the results are shown in FIG. 2. Shown in

0 = Normal

1 = Mild redness, slight swelling of ankle or wrist

2 = moderate swelling of ankle or wrist

3 = severe swelling of ankle and foot

4 = maximally inflamed

As shown in Figures 1 and 2, it was confirmed that the hind paw thickness and clinical score is significantly reduced in the mice treated with the WKYMVm peptide compared to the control group. More specifically, in the control group, swelling appeared on the 3rd day after injecting K / BxN rheumatoid arthritis serum, peaked on the 6th and 7th days, and gradually decreased, whereas the WKYMVm peptide After treatment with K / BxN rheumatoid arthritis serum, the joint damage was significantly reduced, and the hind paw thickness and clinical score were alleviated. From the above results, it was found that the WKYMVm peptide according to the present invention is effective in inhibiting rheumatoid arthritis.

Example 2. Verification of the inhibitory effect of osteoclast production of WKYMVm peptide

In order to determine whether the WKYMVm peptide is effective in inhibiting the osteoclast production was carried out as follows.

That is, in the same manner as in Example 1, 100 μl of K / BxN rheumatoid arthritis serum diluted 1/2 in PBS was intraperitoneally in 7-week old BALB / c male mice at 0 days and 2 days, respectively. Intraperitoneal injection caused rheumatoid arthritis. In the experimental group, 4 mg / kg of WKYMVm peptide was intraperitoneal and subcutaneous injection six times at 12 hour intervals, respectively, and none of the control groups were treated.

For histological analysis, each mouse's paw with ankle joint was fixed in 10% Normal Buffered Formaldehyde (NBF) for 24-48 hours, followed by two weeks of replacement with 14% EDTA solution daily and demineralized. (decalcified). Then, washed with PBS, dehydrated with ethanol series, and embedded in paraffin to prepare a bone tissue sample. After obtaining 5μm thick sections from the prepared bone tissue samples, it was confirmed by H & E staining or TRAP staining to generate osteoclasts, and the results are shown in FIG. 3.

As shown in Figure 3, the control group was confirmed that the exudation of osteoclasts (polymorphonuclear cells) (trap positive cells) in the joint space (joint space) is significantly increased, while bone tissue of the mouse treated with WKYMVm peptide In, the invasion of polymorphonuclear cells was significantly reduced. From the above results, it was found that the WKYMVm peptide according to the present invention is effective in inhibiting the production of osteoclasts.

Example 3 Verification of the Inhibitory Effect of WKYMVm Peptides on the Production of Osteoclasts

Bone marrow cells (BMC) of mice were treated with macrophage colony-stimulating factor (M-CSF) to differentiate into macrophage, and then a combination of receptor activator of NF-ĸB Ligand (RANKL) and M-CSF While the osteoclasts were generated by the treatment, the WKYMVm peptide was treated at the early and middle stages of differentiation to verify the inhibitory effect of the osteoclast generation at each stage. A schematic diagram of the experimental procedure is shown in FIG. 4.

More specifically, after dispensing BMC cells (5x10 ³ cells / well) into 96 well plates using α-MEM medium, RANKL (100 ng / ml), M-CSF (30 ng / ml) and Various concentrations of WKYMVm peptides (0, 1, 10, 100 and 1000 nM) were treated, with media being replaced on day 3. In addition, after staining TRAP using SIGMA's kit, the number of trap positive cells was determined by optical microscopy to confirm the osteoclast generation inhibitory ability of the WKYMVm peptide at the beginning of differentiation, and the results are shown in FIG. 5. .

In addition, when preosteoclasts were generated, the α-MEM medium was replaced and RANKL (100 ng / ml), M-CSF (30 ng / ml) and various concentrations of WKYMVm peptides (0) for 1 day. , 1, 10, 100 and 1000 nM). After staining TRAP using the kit of SIGMA, the number of trap positive cells was determined by optical microscopy to confirm the osteoclast generation inhibitory ability of the WKYMVm peptide during differentiation, and the results are shown in FIG. 6. .

As shown in FIG. 5 and FIG. 6, BMC cells were differentiated into TRAP positive polymorphonuclear cells, and WKYMVm peptide was confirmed to inhibit the production of osteoclasts. In particular, at low doses (1-100 nM), although the inhibitory ability of osteoclast production was not large, it was confirmed that when 1 μM of WKYMVm peptide was treated, it significantly inhibited the production of osteoclasts.

In addition, on day 9, blood was collected in heparin via cardiac puncture, and then cytokine assay was performed to measure interleukin-10 (IL-10) level, and the results are shown in FIG. 7. It was.

As shown in FIG. 7, the IL-10 levels of the K / BxN treated mice did not increase significantly, whereas the mice treated with the WKYMVm peptide showed a significant increase in IL-10 levels.

Example 4 Confirmation of T Cell Function Degradation Through FPR2 of WKYMVm Peptide

4-1. Confirmation of FPR2 Expression in T-cell Lineage

To investigate the expression of FPR2 in mouse CD4 T cell lineage, CD4 ⁺ CD62L ^hi CD44 ^lo CD25 ^neg cells were isolated from mouse spleen and cultured in polarizing reagent. The isolated cells were treated with Th0 or IL-6 30ng / ml, TGFβ 5ng / ml, IL-23 50ng / ml, anti-IL-4 10μg / ml and anti-IFNγ 10μg / ml to differentiate into Th17, Differentiated into follicular helper T (T _FH ) cells by treatment with 30ng / ml IL-6, 10μg / ml anti-IFNγ, 10μg / ml anti-IL-4 and 20μg / ml anti-TGF-β (1D11) neutralizing Abs Thereafter, FACS analysis was performed using mouse FPR2 antibody with stimulation with CD3 / CD28 Dynabeads, and the results are shown in FIG. 8.

As shown in FIG. 8, mouse FPR2 was not expressed in Th0 cells that did not stimulate CD3 / CD28 (see FIG. 8A), while Th0 cells, Th17, and T _FH stimulated with CD3 / CD28 beads. It was confirmed that the expression of mouse FPR2 in the cells (see Fig. 8 (b)).

4-2. Inhibition of RORγ-t and Bcl-6 Expression by Treatment of WKYMVm Peptide

After treating the WKYMVm peptide, which is an agonist of mouse FPR2, to Th17 and T _FH cells differentiated through Example 4-1, the expression changes of RORγ-t and Bcl-6 were analyzed by quantitative real-time PCR. The results are shown in FIG.

More specifically, total RNA was extracted from T cells using TriZol (Invitrogen), and extracted RNA was synthesized cDNA using Superscript reverse transcriptase and oligo (dT) primers. Gene expression was analyzed using the Qiagen Rotor-Gene Q System (Qiagen, Inc.) using a SYBR green real-time PCR kit. Specific information of the primers used at this time is shown in Table 1 below.

Table 1 division Forward primer (5 '→ 3') Reverse primer (5 '→ 3') RORγ-t ACC TCC ACT GCC AGC TGT GTG CTG TC (SEQ ID NO: 2) TCA TTT CTG CAC TTC TGC ATG TAG ACT GTC CC (SEQ ID NO: 3) Bcl-6 AGA CGC ACA GTG ACA AAC CAT ACA A (SEQ ID NO: 4) GCT CCA CAA ATG TTA CAG CGA TAG G (SEQ ID NO: 5)

Relative quantitation values was 2 ^- means the difference between ^△ were expressed as ^Ct, where △ Ct is conducted three times sample and endogenous β-actin Ct average value of the control group.

As shown in FIG. 9, when WKYMVm peptide was treated, the expression of RORγ-t and Bcl-6 was significantly decreased compared to cells not treated with WKYMVm peptide.

4-3. Inhibition of cytokine secretion by treatment of WKYMVm peptide

IL-17 derived from Th17 cells is known to induce various inflammatory diseases and chronic inflammatory diseases. T _FH cells produce IL-21 to stimulate B cells, thereby inducing maturation of B cells and differentiation into plasma cells. Create

Thus, in the above embodiment, it was confirmed whether FA17 analysis suppressed the production of cytokines IL-17 and IL-21 produced by Th17 and T _FH cells according to the treatment of WKYMVm peptide, and the results are shown in FIGS. 10 and FIG. 11 is shown.

As shown in FIG. 10, IL-17 production is reduced when the WKYMVm peptide is treated (see FIG. 10 (b)) than in the case of Th17 cells not treated with the WKYMVm peptide (see FIG. 10 (a)). Could confirm.

Similarly, as shown in FIG. 11, IL-21 is treated more when treated with WKYMVm peptide (see FIG. 11 (b)) than when T _FH cells not treated with WKYMVm peptide (see FIG. 11 (a)). It was confirmed that the production was reduced more than three times.

Example 5. Confirmation of Rheumatoid Arthritis Inhibition by FPR2 of WKYMVm Peptide

In order to determine whether the WKYMVm peptide inhibits rheumatoid arthritis through FPR2, the experiment was conducted in the fourth manner using a collagen-induced-arthritis (CIA) mouse model as follows.

5-1. Fabrication of a collagen-induced-arthritis mouse model

Two groups of 7-8 week old DBA / 1 male mice were inoculated intracutaneously with bovine CII (chondrex) emulsified with CFA (chondrex). After 21 days, mice were re-administered CII. Starting at day 26, one mouse group (n = 10) was treated with 100 μg of WKYMVm peptide once a day for 2 weeks, and the other mouse group (n = 10) was treated with 100 μg of PBS as a control.

5-2. Primary Identification of Rheumatoid Arthritis Inhibition via FPR2 of WKYMVm Peptide

Through Example 4, the result was that WKYMVm peptide, a FPR2 agonist, reduced the function of Th17 and T _FH through FPR2, and based on this, whether the same result was observed even in actual rheumatoid arthritis using a CIA mouse model. Confirmed.

That is, the redness and swelling shown in the foot of each group mouse of the CIA mouse model prepared by Example 5-1 were scored by the following scores daily and summed up in the rheumatoid arthritis pathology table. One clinical score was calculated and the results are shown in FIG. 12. On the other hand, the photograph of observing the foot of the mouse of each group is shown in FIG.

0: normal;

1: erythema and mild swelling confined to the ankle joint;

2: erythema and mild swelling extending from the ankle to metatarsal or metacarpal joints;

3: erythema and moderate swelling extending from the ankle to the metatarsophalangeal or metacarpophalengeal joints;

4: erythema and severe swelling extending from the ankle to the digits.

As shown in FIG. 12 and FIG. 13, it was confirmed that the CIA model treated with the WKYMVm peptide showed a significant clinical score (p <0.001) than the CIA model treated with the WKYMVm peptide.

In addition, the amounts of IL-17, IL-21 and TNF-α from mouse serum were determined by ELISA analysis, and the results are shown in FIG. 14.

As shown in FIG. 14, TNF-α, a representative cytokine of IL-17, IL-21 and rheumatoid arthritis, was markedly reduced in serum of the CIA model treated with the WKYMVm peptide, rather than the CIA model without the WKYMVm peptide. Could.

From the above results, it was found that the WKYMVm peptide according to the present invention is effective in inhibiting rheumatoid arthritis, and more specifically, the WKYMVm peptide decreases the function of Th17 and T _FH through FPR2, thereby inhibiting rheumatoid arthritis.

5-3. Secondary Identification of Rheumatoid Arthritis Inhibition via FPR2 of WKYMVm Peptide

First, cytokine production of CD4 T cells in splenocytes of CIA mice was confirmed by FACS analysis, and the results are shown in FIG. 15. As shown in FIG. 15, IL-17 and IL-21 generated in collagen-specific CD4 T cells of the CIA model were increased, but significantly decreased in the CIA model (CIA + WKYMVm) treated with the WKYMVm peptide. there was.

Next, T _FH cells are immune cells that mainly produce IL-21, and the collagen-specific T _FH cells (CD4 ⁺ CXCR ⁵⁺ PD-1 ⁺ ) population by WKYMVm peptide treatment were identified, and the results are shown in FIG. 16. Shown in As shown in FIG. 16, CD4 ⁺ CXCR ⁵⁺ PD-1 ⁺ cells were significantly reduced in the CIA model (CIA + WKYMVm) treated with the WKYMVm peptide, rather than the CIA model without the WKYMVm peptide.

Next, germinal centers (GCs) form B cell follicles during the reaction with T cell-dependent antibodies. Thus, the formation of germinal centers can be thought to produce antigen-specific antibodies. Thus, the formation of germinal centers in the CIA group and the CIA + WKYMVm group using the specific antibody of the germinal center, peanut agglutinin (PNA), was compared by immunohistochemical analysis, and the results are shown in FIG. 17. . More specifically, the mouse spleen tissue was embedded in the OCT, frozen in isopentane in Histobath to prepare a tissue block, using a cryotome sliced the tissue block to 6 μm to prepare a slide, and then produced the slide Was fixed to acetone cold. Biotin-labeled anti-mouse PNA was applied as the primary antibody, followed by avidin and biotinylated alkaline phosphatase complex reagent. On the other hand, PNA and other agents were obtained from Vector Laboratary, and slides for PNA staining were counted after staining with Hematoxlin. As shown in FIG. 17, GC formation was maintained in the CIA model not treated with the WKYMVm peptide, while GC formation was inhibited in the CIA model (CIA + WKYMVm) treated with the WKYMVm peptide.

Next, immunoglobulin (immunoglobulin) according to germinal center formation in mouse serum was determined by ELISA analysis, and the results are shown in FIG. 18. As shown in FIG. 18, the amount of immunoglobulin was significantly reduced in the CIA model (CIA + WKYMVm) treated with the WKYMVm peptide, rather than the CIA model without the WKYMVm peptide.

5-4. Tertiary Identification of Rheumatoid Arthritis Inhibition via FPR2 of WKYMVm Peptide

First, in order to confirm whether there is a therapeutic effect of rheumatoid arthritis through the control of FPR2, clinical scores were measured according to the method of Example 5-2 using CsH and WRW ⁴ , which are antagonists of FPR1 and FPR2, and the results are shown in FIG. 19. Shown in As shown in FIG. 19, 60 days of observation showed that the CIA + WKYMVm group had a significantly lower clinical score than the CIA group (p <0.001), and the reduction effect by WKYMVm was inhibited by WRW ⁴ injection. .

Next, production of IL-17 and IL-21 in collagen-specific CD4 T cells was confirmed by FACS analysis, and the results are shown in FIG. 20. As shown in FIG. 20, the production of IL-17 and IL-21 was significantly decreased in the CIA + WKYMVm group than the CIA group, and the reduction effect by WKYMVm was inhibited by the injection of WRW ⁴ .

5-5. Fourth Identification of Rheumatoid Arthritis Inhibition via FPR2 of WKYMVm Peptide

First, after treatment with CsH and WRW ⁴ , which are antagonists of FPR1 and FPR2, population and PNA formation of CXCR5 ⁺ PD-1 ⁺ CD4 T cells were confirmed by FACS and immunohistochemical analysis. Is shown in FIG. 21. As shown in FIG. 21, the population of CXCR5 ⁺ PD-1 ⁺ CD4 T cells was decreased in the CIA + WKYMVm group than the CIA group, and the reduction effect by WKYMVm was inhibited by the injection of WRW ⁴ .

Next, the amount of immunoglobulins (immunoglobulin) in mouse serum was determined by ELISA analysis, and the results are shown in FIG. 22. As shown in Figure 22, IgG1 and IgG2a production was also reduced in the CIA + WKYMVm group than the CIA group, the WKYMVm reduction effect was confirmed that the inhibition by WRW ⁴ injection.

From the above results, it was found that the WKYMVm peptide alleviates the symptoms of rheumatoid arthritis by reducing the production of IL-17, IL-21 and immunoglobulin (immunoglobulin) through FPR2.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The peptide consisting of the amino acid sequence of SEQ ID NO: 1 according to the present invention not only inhibits the production and activation of osteoclasts, but also inhibits the production and activation of osteoclasts, as well as inhibits Th17 and T _FH cell differentiation and activation, which are important in the pathogenesis of rheumatoid arthritis. It has an effect of reducing the production of IL-21 and immunoglobulin, and is expected to be useful as an active ingredient in pharmaceutical compositions or functional food compositions for the treatment of rheumatoid arthritis.

Claims (10)

Rheumatoid arthritis prevention or treatment pharmaceutical composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. The pharmaceutical composition of claim 1, wherein the peptide binds to a formyl peptide receptor (FPR). The pharmaceutical composition of claim 2, wherein the peptide binds to formyl peptide receptor 2 (FPR2). The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits the production of osteoclasts. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition reduces immunoglobulin and / or inflammatory cytokine levels. The method of claim 5, wherein the inflammatory cytokine is selected from the group consisting of interleukin-17 (IL-17), interleukin-21 (IL-21) and TNF-α (Tumor necrosis factor-alpha), Pharmaceutical compositions. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Health functional food composition for preventing or improving rheumatoid arthritis comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. A method for preventing or treating rheumatoid arthritis, comprising administering to a subject a peptide consisting of the amino acid sequence of SEQ ID NO: 1. Use for the prevention or treatment of rheumatoid arthritis of a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

Images