Pharmaceutical composition for preventing or treating behcet's disease and rheumatoid arthritis comprising a peptide or a mixture thereof as an active ingredient

Abstract

The present invention relates to a composition for preventing or treating Behcet's disease and rheumatoid arthritis, the composition comprising a peptide or a mixture thereof as an active ingredient. More specifically, it was found that intraperitoneal injection of P1 to P5 peptides or a mixture thereof or application as a skin external preparation to a mouse animal model having Behcet's disease caused by an autoimmune response can improve skin ulceration and skin inflammation in the Behcet's disease mouse model, and an improvement effect on arthritis of a rheumatoid arthritis animal model was also observed. Accordingly, a composition comprising P1 to P5 peptides or a mixture thereof as an active ingredient can be provided as a medicament for Behcet's disease and rheumatoid arthritis.

Description

Technical Field

[0001] The present disclosure relates to compositions comprising peptides or mixtures thereof as active ingredients and their medical uses. Background Art

[0002] The human immune system plays a role in protecting the body from foreign antigens, and due to its self-tolerance, it does not attack its own tissues. However, when the immune system's self-tolerance is broken, a condition known as autoimmunity occurs, causing immune cells to produce antibodies that recognize proteins normally expressed by their own genes as targets, thereby attacking and destroying normal tissues by eliciting a T cell response. When specific symptoms appear, it is called an autoimmune disease.

[0003] Rheumatoid arthritis is a chronic disease caused by inflamed synovium around the joints, with damage to the joints and disorder as the inflammation spreads to the surrounding cartilage and bone. Inflammation occurs in the synovial membrane of the joints and immune cells in the blood accumulate, causing an increase in the volume of joint fluid and swelling of the joints, causing pain. This type of rheumatoid arthritis is a disease mainly caused by an abnormal autoimmune response, and abnormal immune function triggers inflammation. The inflammatory response in the joints mainly involves T cells, B cells and macrophages, and it is known that TNF-α, M1 macrophages that secrete IL-1β and Th17 cells that secrete IL-17 secreted from these cells can aggravate rheumatoid arthritis.

[0004] Currently, TNF inhibitors (infliximab, etanercept, etc.), IL-1 inhibitors (anakinra, canakinumab), steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), cytokine inhibitors (Acemira), signal transduction inhibitors (JAK3 inhibitors) or TNF-α related antibody treatments are mainly used to treat autoimmune diseases including rheumatoid arthritis. However, these treatments can cause side effects (such as itching and respiratory tract infections), and because they focus on alleviating pain by suppressing inflammation, they can hardly be a fundamental treatment method.

[0005] Meanwhile, Behçet's disease is a rare, intractable inflammatory disease with symptoms of recurrent aphthous ulcers, uveitis, skin inflammation, and ulcers in the mouth and reproductive organs. Clinical symptoms include not only skin ulcers but also symptoms related to the joints, central nervous system, stomach, kidneys, genitourinary system, lungs, cardiovascular system, and digestive system (e.g., intestinal bleeding and intestinal perforation), as well as severe chronic inflammation with symptoms on multiple levels (e.g., superior vena cava syndrome and aortic regurgitation). These symptoms are associated with systemic vasculitis, which is the main pathophysiological feature of Behçet's disease.

[0006] While the exact cause of Behçet's disease remains unknown, autoimmunity and autoinflammatory responses are the main causes. Studies have reported that the cytokine interleukin-17 is associated with the development of inflammatory symptoms because it is highly expressed in the plasma of Behçet's disease patients. Currently, new biological drugs (such as TNF-α antibodies) are being used to treat Behçet's disease. However, these drugs are expensive, side effects (such as tuberculosis infection) have been reported, and their repeated use may reduce therapeutic efficacy due to drug resistance. Summary of the Invention

[0007] Technical problem to be solved by the invention

[0008] An object of the present disclosure is to provide a composition comprising a peptide or a mixture thereof as an active ingredient as a composition for preventing or treating Behcet's disease or rheumatoid arthritis induced by an autoimmune response.

[0009] Technical means to solve technical problems

[0010] Exemplary embodiments of the present disclosure provide a pharmaceutical composition for preventing or treating Behçet's disease, the pharmaceutical composition comprising a peptide containing the amino acid sequence represented by SEQ ID NO: 1, a peptide containing the amino acid sequence represented by SEQ ID NO: 2, a peptide containing the amino acids represented by SEQ ID NO: 3, a peptide containing the amino acids represented by SEQ ID NO: 4, a peptide containing the amino acids represented by SEQ ID NO: 5, or a mixture thereof as an active ingredient.

[0011] Exemplary embodiments of the present disclosure provide a pharmaceutical composition for preventing or treating rheumatoid arthritis, the pharmaceutical composition comprising a peptide containing the amino acid sequence represented by SEQ ID NO: 1, a peptide containing the amino acid sequence represented by SEQ ID NO: 2, a peptide containing the amino acid represented by SEQ ID NO: 3, a peptide containing the amino acid represented by SEQ ID NO: 4, a peptide containing the amino acid represented by SEQ ID NO: 5, or a mixture thereof as an active ingredient.

[0012] Beneficial effects

[0013] According to exemplary embodiments of the present disclosure, peptides of SEQ ID NOs: 1 to 5, or mixtures thereof, were administered intraperitoneally or applied as a topical skin preparation to a mouse model of Behçet's disease or rheumatoid arthritis induced by an autoimmune response. The results showed that skin ulcers and inflammation were alleviated in the mouse model of Behçet's disease, and a therapeutic effect on arthritis was observed in an animal model of rheumatoid arthritis. Therefore, compositions containing peptides of SEQ ID NOs: 1 to 5, or mixtures thereof, as active ingredients can be provided as therapeutic agents for Behçet's disease or rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of identifying the effects of each peptide or peptide mixture on skin ulcers and skin inflammation after administration of each peptide or peptide mixture once a day for 10 consecutive days in the form of a topical skin preparation (SKIN) or once a day for 10 consecutive days by intraperitoneal administration (IP) to a mouse animal model with Behçet's disease are shown, wherein (A) shows the observation results 5 days and 10 days after the initial administration of the mixture (P1234) of P1, P2, P3 and P4, (B) shows the observation results 2 weeks and 4 weeks after the administration of the mixture (P124), and (C) shows the observation results 5 days and 10 days after the administration of P5.

[0015] Figure 2 Shown are the results of FACS analysis identifying the frequency of CD4+IL-17+ cells after treatment of cells isolated from the spleen of a mouse animal model with Behcet's disease with a peptide mixture (P124).

[0016] Figure 3 Shown are the results of real-time PCR analysis identifying IL-17 and RORγT mRNA levels after treatment of cells isolated from the spleen of a mouse animal model with Behçet's disease with a peptide mixture (P124).

[0017] Figure 4 The results of examining changes in arthritis severity in a mouse model with collagen-induced arthritis are shown, specifically, the results of examining changes in arthritis severity for 10 weeks after 10-day treatment with PBS control, MTX (methotrexate, 1 μg / mouse, which is an arthritis therapeutic agent), a control peptide, and a peptide mixture (P124, 3 μg / mouse) in the form of an external skin preparation.

[0018] Figure 5The results of examining changes in arthritis severity after treating a mouse model with collagen-induced arthritis with a peptide mixture in the form of a topical skin preparation are shown. Specifically, the results of examining changes 1 week and 5 weeks after continuous treatment with the peptide (P124, 3 μg / mouse) once a day for 10 days in the form of a topical skin preparation are shown.

[0019] Figure 6 The results of identifying the frequencies of CD4+CD25+ cells, CD4+CD25+FoxP3+ cells and IL-17+ cells in a mouse model of collagen-induced arthritis after treatment with a control peptide (3 μg / mouse), MTX (1 μg / mouse) and a peptide mixture (P124, 3 μg / mouse), respectively, are shown.

[0020] Figure 7 Shown are the results of real-time PCR analysis identifying IL-17 mRNA levels in lymph node cells and bone marrow of a mouse model with arthritis treated with a control peptide, MTX (1 μg / mouse), and a peptide mixture (P124, 3 μg / mouse).

[0021] Figure 8 The results of real-time PCR analysis for identifying RORγT and IL-17 mRNA levels in each experimental group divided into a group consisting of splenocytes isolated from normal mice and treated with anti-CD3 antibodies and anti-CD28 antibodies (CD), a CD group treated with cytokines IL-23, IL-6, and TGFb that stimulate Th17 cell expansion (CD-Th), and a CD-Th group treated with a peptide mixture (P124 0.9 μg, P124 0.1 μg, and P124 0.01 μg).

[0022] Figure 9 Shown are the results of examining the in vivo retention times of the peptides.

[0023] Figure 10 It is the result of identifying the biological stability of the peptide. DETAILED DESCRIPTION

[0024] Hereinafter, exemplary embodiments of the present disclosure will be described in more detail.

[0025] Exemplary embodiments of the present disclosure can provide a pharmaceutical composition for preventing or treating Behçet's disease, comprising a peptide containing the amino acid sequence represented by SEQ ID NO: 1, a peptide containing the amino acid sequence represented by SEQ ID NO: 2, a peptide containing the amino acid represented by SEQ ID NO: 3, a peptide containing the amino acid represented by SEQ ID NO: 4, and a peptide containing the amino acid represented by SEQ ID NO: 5, or a mixture thereof as an active ingredient.

[0026] The pharmaceutical composition may be in a dosage form selected from the group consisting of oral preparations, external preparations, suppositories and injections, but is not limited thereto.

[0027] In addition, exemplary embodiments of the present disclosure can provide a pharmaceutical composition for preventing or treating rheumatoid arthritis, which comprises a peptide containing the amino acid sequence represented by SEQ ID NO: 1, a peptide containing the amino acid sequence represented by SEQ ID NO: 2, a peptide containing the amino acid represented by SEQ ID NO: 3, a peptide containing the amino acid represented by SEQ ID NO: 4, and a peptide containing the amino acid represented by SEQ ID NO: 5, or a mixture thereof as an active ingredient.

[0028] The pharmaceutical composition may be in a dosage form selected from the group consisting of oral preparations, external preparations, suppositories and injections, but is not limited thereto.

[0029] In an exemplary embodiment of the present disclosure, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of the following suitable additives commonly used in the preparation of pharmaceutical compositions: carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, glidants, Flavoring agents, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders and lubricants.

[0030] Specifically, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate / ester, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum powder, magnesium stearate and mineral oil can be used as carrier, excipient and diluent.Solid preparation for oral administration can comprise tablet, pill, powder, granule and capsule, wherein can prepare this type of solid preparation by mixing at least one excipient (for example starch, calcium carbonate, sucrose or lactose and gelatin) in compositions.In addition, except simple excipient, lubricant (for example magnesium stearate and talcum powder) can also be used. Liquid dosage forms for oral administration may include suspensions, solutions, emulsions and syrups, and in addition to commonly used simple diluents (such as water and liquid paraffin), may also contain a variety of excipients (such as wetting agents), sweeteners, flavorings and preservatives. Dosage forms for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable esters (such as ethyl oleate) can be used as non-aqueous solvents and suspensions. Witepsol, polyethylene glycol, vegetable oils (such as olive oil) and injectable esters (such as ethyl oleate) can be used. Tween 61, cocoa butter, laurel butter and glycerin gelatin are used as the base for suppositories.

[0031] According to exemplary embodiments of the present disclosure, the pharmaceutical composition can be administered to a subject in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes.

[0032] In exemplary embodiments of the present disclosure, the "subject" may be a mammal including a human, but is not limited to these examples.

[0033] Example

[0034] Hereinafter, embodiments will be described in detail to help understand the present disclosure. However, the following embodiments are only used to illustrate the content of the present disclosure, and the scope of the present disclosure is not limited to the following embodiments. The embodiments of the present disclosure are provided to more fully explain the present disclosure to those skilled in the art.

[0035] <Experimental Example>

[0036] The following experimental examples are intended to provide experimental examples that are commonly applicable to various exemplary embodiments according to the exemplary embodiments of the present disclosure.

[0037] 1. Experimental Animals

[0038] As previously reported (S. Sohn et al., Eur J Dermatol, 8(1): 21-23, 1998), HSV type 1 (1×10 6 Male ICR mice (4-5 weeks old) were infected with 20 μL of 5% cytochrome P60 (C16-9) (Figure 5—figure supplement 1). Ten days after the initial viral inoculation, mice were reinoculated with the virus and monitored for 32 weeks. Animals were handled according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Ajou University School of Medicine. The study protocol was approved by the IACUC (approval number: AMC-2016-0010).

[0039] 2. Mouse Model of Behçet's Disease (BD)

[0040] Fifteen percent of HSV-infected mice exhibited symptoms similar to BD. Symptoms included skin ulcers, skin pustules, genital ulcers, erythema, oral ulcers, ear inflammation, arthritis, red eyes (left and right), decreased visual acuity (left and right), weight loss, and facial swelling. Oral ulcers, genital ulcers, skin ulcers, and eye symptoms were categorized as major symptoms, while the other symptoms were considered mild.

[0041] Mice exhibiting one or more major symptoms and one mild symptom were considered BD mice. Each symptom was scored as 1, and the sum of the scores was used to determine BD severity. Symptoms were considered improved when they disappeared or when lesions decreased in size by 20% or more. The BD disease severity score was calculated based on the BD Activity Index described in the BD Activity Form (www.behcet.ws / pdf / BehcetsDiseaseActivityForm.pdf).

[0042] 3. Flow Cytometry

[0043] Peripheral blood cells from mice were isolated and red blood cells removed. The cells were reacted with fluorescently labeled anti-CCR1 antibodies and analyzed by flow cytometry to measure the frequency of CCR1-expressing cells in each mouse. Cells were isolated from the mouse lymph nodes and reacted with the antibodies in the same manner for analysis.

[0044] <Example 1> Preparation of peptide

[0045] Efforts to develop low-molecular-weight substances (particularly proteins) derived from snake venom for the treatment of Behçet's disease and rheumatoid arthritis have led to the discovery of pharmaceutically useful peptides. Peptides having sequences ranging from 11 to 23 amino acids have been prepared.

[0046] P1:LICPEKYCNKVHT(SEQ ID NO:1)

[0047] P2: YCNKVHTCRNG (SEQ ID NO: 2)

[0048] P3: PREIVECCSTDKCNH (SEQ ID NO: 3) (SEQ ID NO: 3)

[0049] P4: HTCRNGENICF (SEQ ID NO: 4)

[0050] P5: ENICFKRFYEGNLLGKRYPRGCA (SEQ ID NO: 5)

[0051] <Example 2> Identification of the therapeutic effect of peptides on Behcet's disease

[0052] In a previously prepared mouse animal model with Behcet's disease, the P1, P2, P3, P4, P5 peptides or a suitable mixture prepared in Example 1 were mixed with physiological saline for injection and injected into the abdominal cavity of mice with inflammatory diseases, or treated in the form of a topical skin preparation. Figure 1 As shown, improvement of symptoms was visually inspected after 5 and 10 days or at 2 and 4 weeks.

[0053] like Figure 1 As shown, it was found that skin ulcers and skin inflammation were alleviated in the mouse model with Behcet's disease when the P1, P2, P4 and P5 peptides were administered separately. In addition, skin ulcers and skin inflammation were effectively alleviated in the mouse model with Behcet's disease when each peptide mixture was injected intraperitoneally.

[0054] Furthermore, after treating cells isolated from the spleen of mice with Behcet's disease with 1 μg of a mixture of P1, P2, and P4 peptides while culturing them, flow cytometry (FACS) was performed, and the results were as follows: Figure 2 shown.

[0055] like Figure 2 As shown, the frequency of CD4+IL-17+ cells was found to be decreased in the peptide-treated group.

[0056] Furthermore, real-time PCR was performed to compare the mRNA levels of IL-17 and RORγT in splenocytes of mice with Behçet's disease cultured by treating with a mixture of P1, P2, and P4 peptides in the same manner. Figure 3 As shown. Figure 3 As shown, the expressions of IL-17 and RORγT were found to be decreased in the peptide-treated group.

[0057] <Example 3> Identification of the therapeutic effect of peptides on rheumatoid arthritis

[0058] To prepare a mouse model with rheumatoid arthritis, a group of 8-week-old male ICR normal mice (average weight: 20 g) were subcutaneously injected into the tail (first immunization) with 50 μg of a mixture obtained by mixing type II collagen with equal amounts of complete Freund's adjuvant (CFA) and paraffin oil; two weeks later, the same substance was subcutaneously injected into the tail in the same manner to prepare an animal model with rheumatoid arthritis.

[0059] In the same manner as in Example 2, the mixture of P1, P2 and P4 peptides prepared in Example 1 (P124) was administered to the skin of mice as an external skin preparation. In addition, as controls, PBS and MTX were administered in the same manner. Figure 4 shown.

[0060] like Figure 4 As shown in Figure 2, the severity score of the group administered with the peptide mixture of the present disclosure was reduced compared to the PBS control group, confirming that the group administered with the peptide mixture showed an excellent effect. In addition, the group administered with the arthritis therapeutic agent MTX also showed a reduction in the severity score of arthritis, but it was found that this effect decreased after 7 weeks.

[0061] From the above results, it was determined that the peptide mixture (P124) of the present disclosure exhibited superior effects in reducing severity compared to MTX.

[0062] In addition, the peptide mixture (P124) was used as an external skin preparation once a day for 10 consecutive days, and the therapeutic effect on arthritis was examined after 5 weeks. The results were as follows Figure 5 shown.

[0063] like Figure 5 As shown, it was found that the group treated with the peptide mixture of the present disclosure (P124) had an excellent arthritis therapeutic effect.

[0064] After isolating cells from a mouse model with arthritis treated with the peptide mixture of the present invention (P124), flow cytometry (FACS) was performed, and the results were as follows: Figure 6 and as shown in Table 1 below.

[0065] like Figure 6 As shown in Table 1 , the frequencies of CD4+CD25+ cells and CD4+CD25+FoxP3+ cells were found to be increased, and the frequency of IL-17+ cells was found to be decreased in the peptide mixture (P124)-treated group, compared with the control group or the methotrexate (MTX)-treated group.

[0066] Table 1

[0067]

[0068] In addition, real-time PCR was performed on the lymph nodes and bone marrow of mice with arthritis treated with the peptide mixture of the present disclosure (P124) to examine the IL-17 levels. Figure 7 shown.

[0069] like Figure 7 As shown, it was found that the expression level of IL-17 was decreased in the lymph node cells of the peptide mixture (P124)-treated group compared with the control group.

[0070] At the same time, the cells were divided into a group (CD) of spleen cells isolated from normal mice and treated with anti-CD3 and anti-CD28 antibodies; a group (CD-Th) to which cytokines IL-23, IL-6, and TGFb, which stimulate the expansion of Th17 cells, were added together with anti-CD3 and anti-CD28 antibodies; and a group treated with a mixture of P1, P2, and P4 peptides (0.9 μg, 0.1 μg, and 0.01 μg) on ​​the basis of CD-Th, and then cultured. Thereafter, real-time PCR was performed to examine RORgt and IL-17 mRNA levels, and the results were as follows: Figure 8 shown.

[0071] like Figure 8As shown, the expressions of RORγT and IL-17 were found to be decreased in the peptide-treated group.

[0072] <Example 4> Identification of peptide stability

[0073] After intraperitoneal injection of the fluorescently labeled peptide P4 into mice, intraperitoneal macrophages were isolated and the frequency of the fluorescently labeled macrophages was analyzed by FACS to measure the retention time of the peptide in the peritoneal cavity (e.g., Figure 9 shown).

[0074] like Figure 9 As shown, the frequency of cells containing the peptide was found to be highest at 24 hours and was maintained until 72 hours.

[0075] In addition, the P1, P2, and P4 peptides were each labeled with a different fluorescent marker, and the three types were mixed and administered to normal mice. After 48 hours, intraperitoneal macrophages were isolated and analyzed by FACS. The results were as follows: Figure 10 shown.

[0076] like Figure 10 As shown, all three types of peptides were found to remain in the peritoneal cavity after 48 hours.

[0077] From the above results, it was found that the P1, P2, and P4 peptides were stably maintained in vivo.

[0078] Although the specific parts of the present disclosure have been described in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred exemplary embodiments, and the scope of the present disclosure is not limited thereto. Therefore, the essential scope of the present disclosure will be defined by the appended claims and their equivalents. <110> Asia University Industry-Academic Cooperation Group <120> Pharmaceutical composition comprising snake venom peptide or mixture thereof for preventing or treating Behcet's disease and rheumatoid arthritis <130> ADP‑2020‑0489 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 13 <212> PRT <213> unknown <220> <223> Three-finger toxin <400> 1 Leu Ile Cys Pro Glu Lys Tyr Cys Asn Lys Val His Thr 1 5 10 <210> 2 <211> 11 <212> PRT <213> unknown <220> <223> Three-finger toxin <400> 2 Tyr Cys Asn Lys Val His Thr Cys Arg Asn Gly 1 5 10 <210> 3 <211> 15 <212> PRT <213> unknown <220> <223> Three-finger toxin <400> 3 Pro Arg Glu Ile Val Glu Cys Cys Ser Thr Asp Lys Cys Asn His 1 5 10 15 <210> 4 <211> 11 <212> PRT <213> unknown <220> <223> Three-finger toxin <400> 4 His Thr Cys Arg Asn Gly Glu Asn Ile Cys Phe 1 5 10 <210> 5 <211> twenty three <212> PRT <213> unknown <220> <223> Three-finger toxin <400> 5 Glu Asn Ile Cys Phe Lys Arg Phe Tyr Glu Gly Asn Leu Leu Gly Lys 1 5 10 15 Arg Tyr Pro Arg Gly Cys Ala 20

Claims

1. A pharmaceutical composition for preventing or treating Behçet's disease, comprising as an active ingredient: a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1; a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2; a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4; a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5; or a mixture of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and a peptide consisting of the amino acid sequence represented by SEQ ID NO:

4. in, The pharmaceutical composition is an intraperitoneal injection or an external skin preparation.

2. A pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising a mixture of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 as active ingredients, in, The pharmaceutical composition is a skin preparation for external use.

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