Compositions comprising peptides or peptide mixtures as active ingredients and their medical uses

Abstract

The present invention relates to a composition for preventing or treating inflammatory diseases induced by an autoimmune response, the composition comprising a peptide or peptide mixture as an active ingredient. More specifically, a composition comprising peptides P1 to P5 or a mixture thereof as an active ingredient can be provided as a therapeutic agent for inflammatory diseases induced by an autoimmune response. This is because, as a result of intraperitoneal injection or external application of peptides P1 to P5 or a mixture thereof to mouse animal models of psoriasis and inflammatory bowel disease (inflammatory diseases induced by an autoimmune response), it was confirmed that skin ulcers and skin inflammation in the mouse model of psoriasis and inflammatory bowel disease were alleviated, and inflammatory symptoms in the model of bowel inflammation were alleviated.

Description

Technical Field

[0001] The present disclosure relates to a composition comprising a peptide or a mixture thereof as an active ingredient for preventing or treating a disease selected from the group consisting of psoriasis, inflammatory bowel disease, Crohn's disease, allergic asthma, atopic dermatitis and systemic inflammatory response syndrome induced by an autoimmune response. 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] Inflammatory response is a recovery mechanism that attempts to regenerate the damaged area caused by chemical substances, bacterial infection and physical action (which causes organic changes in organisms or tissues). When harmful stimulation, infection or external injury trigger inflammation, local release of vasoactive substances (such as inflammatory components) triggers inflammation. This type of continuous inflammation may promote mucosal damage, thereby developing into inflammatory diseases (such as rheumatoid arthritis, arteriosclerosis, gastritis and asthma) due to redness, fever, swelling, pain and dysfunction.

[0004] A variety of mediators are involved in inflammatory reactions, including cytokines, prostaglandin E2 (PGE2), and free radicals. In particular, nuclear factor-κB, a transcription factor involved in inflammatory reactions, is activated in macrophages by stimuli such as cytokines, tumor necrosis factor-α (TNF-α), and lipopolysaccharide (LPS). As a result, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are expressed, and the resulting excess nitric oxide (NO) and prostaglandin E2 trigger inflammation.

[0005] Psoriasis is a non-contagious, chronic inflammatory skin disease characterized by excessive proliferation of keratinocytes and an increase in scaly plaques. While the etiology remains unclear, it is believed to be caused by immune abnormalities resulting from chronic activation of inflammatory cell infiltration in the skin and dysregulation of epidermal keratinocytes. Research suggests that psoriasis is an autoimmune disease in which T lymphocytes are involved, and some reports suggest that the primary cause of psoriasis is excessive activation of T lymphocytes.

[0006] Inflammatory bowel disease is a disease of unknown cause that causes chronic inflammation in the intestines and can be divided into ulcerative colitis and Crohn's disease. Both ulcerative colitis and Crohn's disease are chronic, intractable diseases in which symptoms temporarily improve but recur. Although the cause or pathophysiology of inflammatory bowel disease is still unclear, it is estimated that a combination of genetic factors, environmental factors (such as intestinal bacteria or food), and immune factors may be involved in the development mechanism. Although the incidence of ulcerative colitis and Crohn's disease is rapidly increasing, due to the unknown cause, a radical treatment method has not yet been established. Therefore, drugs that delay and alleviate the progression of symptoms are being used instead of radical treatment methods. Summary of the Invention

[0007] Technical problem to be solved by the invention

[0008] The object of the present disclosure is to provide a pharmaceutical composition for preventing or treating a disease selected from the group consisting of psoriasis, inflammatory bowel disease, Crohn's disease, allergic asthma, atopic dermatitis and systemic inflammatory response syndrome induced by an autoimmune response by providing a composition comprising a peptide or a mixture thereof as an active ingredient.

[0009] Technical means to solve technical problems

[0010] Exemplary embodiments of the present disclosure provide a pharmaceutical composition for preventing or treating a disease selected from the group consisting of psoriasis, inflammatory bowel disease, Crohn's disease, allergic asthma, atopic dermatitis, and systemic inflammatory response syndrome, the pharmaceutical composition comprising a peptide having an amino acid sequence represented by SEQ ID NO: 1, a peptide having an amino acid sequence represented by SEQ ID NO: 2, a peptide having amino acids represented by SEQ ID NO: 3, a peptide having amino acids represented by SEQ ID NO: 4, a peptide having amino acids represented by SEQ ID NO: 5, or a mixture thereof as an active ingredient.

[0011] Beneficial effects

[0012] According to exemplary embodiments of the present disclosure, peptides P1 to P5 or a mixture thereof were administered intraperitoneally or applied as a topical skin preparation to mouse animal models of psoriasis and inflammatory bowel disease (inflammatory diseases induced by autoimmune responses). The results showed that skin ulcers and skin inflammation were alleviated in the psoriasis mouse model, and inflammatory symptoms were reduced in the enteritis model. Therefore, a composition comprising peptides P1 to P5 or a mixture thereof as an active ingredient can be provided as a therapeutic agent for diseases induced by autoimmune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1Shown are the results of FACS analysis identifying the frequencies of CD83+ cells and CD40+ cells, which are markers of dendritic cell activation, in peripheral blood cells after administration of a peptide mixture to a mouse animal model with psoriasis.

[0014] Figure 2 Shown are the results of FACS analysis identifying the frequency of RORγt-expressing cells in peripheral blood cells after administration of a peptide mixture to a mouse animal model with psoriasis.

[0015] Figure 3 Shown are the results of FACS analysis identifying the frequency of regulatory T cells in peripheral blood cells after administration of a peptide mixture to a mouse animal model with psoriasis.

[0016] Figure 4 Shown are the results of FACS analysis identifying the frequency of Ly6G-expressing neutrophils in abdominal macrophages after administration of a peptide mixture to a mouse animal model with psoriasis.

[0017] Figure 5 Shown are the results of FACS analysis identifying the frequency of CD8+NK1.1+ cells in lymphocytes isolated from lymph nodes after administration of a peptide mixture to a mouse animal model with psoriasis.

[0018] Figure 6 Shown are the results of examining inflammatory symptoms after administering a control, a control cream, and peptide P124 as an external skin preparation to the skin of a mouse animal model having psoriasis for 5 days.

[0019] Figure 7 Shown are the results of observing changes in spleen size after oral administration of a peptide mixture to a mouse animal model with dextran sodium sulfate (DSS)-induced intestinal inflammation.

[0020] Figure 8 Shown are the results of FACS analysis identifying the frequencies of CD83+ cells and CD40+ cells, which are dendritic cell activation markers, in peripheral blood cells, and the frequencies of CD83+ cells and CD86+ cells in intraperitoneal macrophages after oral administration of a peptide mixture to a mouse animal model with intestinal inflammation.

[0021] Figure 9 The results of FACS analysis are shown, which identify the frequency of CD11b+ cells, a macrophage marker, in intraperitoneal macrophages, the frequency of NK1.1+ cells, a natural killer cell marker, in spleen cells, the frequency of cells expressing Ly6G+, a neutrophil marker, and the frequency of cells expressing RORγT, a gene involved in Th17+ cell differentiation, after oral administration of a peptide mixture to a mouse animal model with intestinal inflammation.

[0022] Figure 10 Shown are the results of identifying the frequencies of fluorescently labeled cells in intraperitoneal macrophages 48 hours and 72 hours after oral administration of peptide 1, peptide 2, and peptide 4, each conjugated with a different fluorescent substance, to normal mice. DETAILED DESCRIPTION

[0023] Hereinafter, exemplary embodiments of the present disclosure will be described in more detail.

[0024] Exemplary embodiments of the present disclosure may provide a pharmaceutical composition for preventing or treating a disease selected from the group consisting of psoriasis, inflammatory bowel disease, Crohn's disease, allergic asthma, atopic dermatitis, and systemic inflammatory response syndrome, the pharmaceutical composition comprising a peptide having an amino acid sequence represented by SEQ ID NO: 1, a peptide having an amino acid sequence represented by SEQ ID NO: 2, a peptide having amino acids represented by SEQ ID NO: 3, a peptide having amino acids represented by SEQ ID NO: 4, a peptide having amino acids represented by SEQ ID NO: 5, or a mixture thereof as an active ingredient.

[0025] The peptides can prevent or alleviate inflammation induced by autoimmune responses.

[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 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.

[0028] 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.

[0029] 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.

[0030] The preferred dosage of the peptide may vary depending on the condition and weight of the subject, the type and severity of the disease, the form of the drug, and the route and duration of administration, and may be appropriately selected by those skilled in the art. According to exemplary embodiments of the present disclosure, the daily dose may be 0.01 μg / kg to 200 mg / kg, specifically 0.1 mg / kg to 200 mg / kg, and more specifically 0.1 mg / kg to 100 mg / kg, but is not limited thereto. Administration may be performed once a day or several times in divided doses, but the scope of the present disclosure is not thereby limited.

[0031] In exemplary embodiments of the present disclosure, the "subject" may be a mammal including a human, but is not limited to these examples.

[0032] Example

[0033] 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.

[0034] <Example 1> Preparation of peptide

[0035] Efforts to develop low-molecular substances (particularly proteins) derived from snake venom for the treatment of inflammation have led to the discovery of pharmaceutically useful peptides. Peptides having the following sequences of 11 to 23 amino acids have been prepared.

[0036] P1:LICPEKYCNKVHT(SEQ ID NO:1)

[0037] P2: YCNKVHTCRNG (SEQ ID NO: 2)

[0038] P3: PREIVECCSTDKCNH (SEQ ID NO: 3) (SEQ ID NO: 3)

[0039] P4: HTCRNGENICF (SEQ ID NO: 4)

[0040] P5: ENICFKRFYEGNLLGKRYPRGCA (SEQ ID NO: 5)

[0041] <Example 2> Identification of the Effects of Peptides in a Mouse Model with Psoriasis

[0042] Imiquimod (IMQ) at 20 μg / day / mouse was administered as a topical skin preparation to C56BL / 6J mice once daily for 6 days to induce symptoms of psoriasis.

[0043] A peptide mixture (P124) obtained by mixing 1 μg each of the peptides P1, P2, and P4 isolated in Example 1 with physiological saline for injection and a control cream were administered as a skin external preparation daily for 5 days to examine the characteristics of immune cells.

[0044] The results of peripheral blood cell analysis by flow cytometry (FACS) after peptide administration are as follows: Figure 1 As shown in Figure 2, the frequencies of CD40 positive (+) cells and CD83+ cells, which are activation markers of dendritic cells, were found to be decreased. Figure 2 As shown, the frequency of cells expressing RORγt was detected to be reduced. RORγt is a marker of Th17 cells, and an increase in Th17 cells is associated with exacerbated psoriasis.

[0045] In addition, if Figure 3As shown, the frequency of regulatory T cells having the function of suppressing inflammatory response was found to increase in the peptide-administered group.

[0046] The results of flow cytometry (FACS) analysis of intraperitoneal macrophages after peptide administration are shown in Figure 2. Figure 4 As shown, the frequency of neutrophils expressing Ly6G was found to be reduced. It is known that excessive expression of neutrophils causes excessive inflammatory response due to excessive secretion of cytokines.

[0047] The results of FACS analysis of lymphocytes isolated from lymph nodes after peptide administration are as follows: Figure 5 As shown, the frequency of CD8+NK1.1+ cells was found to be reduced. CD8+NK1.1+ cells secrete IFNγ and participate in the innate immune response to control microbial pathogens. However, their overexpression is known to be associated with the induction of inflammation.

[0048] However, the results of comparing the changes in symptoms after daily administration of the control, control cream, and peptide mixture (P124) as topical skin preparations to a mouse animal model induced with psoriasis symptoms for 5 days were as follows: Figure 6 As shown, inflammatory symptoms appeared on the skin in the control and control cream groups after 6 days, but it was found that almost no psoriasis symptoms remained in the peptide-treated group.

[0049] <Example 3> Identification of the Effects of Peptides in Animal Models with Inflammatory Bowel Disease

[0050] A peptide mixture (P124) obtained by mixing 1 μg each of the peptides P1, P2, and P4 isolated in Example 1 with physiological saline for injection was orally administered once a day for 4 consecutive days to a mouse animal model with dextran sulfate sodium (DSS)-induced intestinal inflammation.

[0051] The results of spleen extraction from mice orally administered with the peptide mixture (P124) are shown in Figure 2. Figure 7 As shown, the spleen was found to be reduced in size.

[0052] In addition, the results of observation of immune cells in a mouse model with intestinal inflammation to which the peptide mixture was administered were as follows: Figure 8 As shown, the frequencies of CD40+ dendritic cells and CD86+ dendritic cells decreased in peripheral blood cells, and the frequencies of CD83+ cells and CD86+ cells in intraperitoneal macrophages also decreased due to peptide administration.

[0053] Overexpression of dendritic cell activation markers is known to be associated with long-term chronic inflammation. Based on the above results, it was found that administration of the peptide reduced dendritic cell activation markers, thereby alleviating the symptoms of inflammatory bowel disease through administration of the peptide.

[0054] In addition, if Figure 9 As shown, it was found that after administering the peptide to a mouse model with intestinal inflammation, the frequency of CD11b+ cells, a macrophage marker, decreased in peritoneal macrophages, and a decrease in the frequency of CD11b+ cells was also observed in splenocytes. In splenocytes, the frequency of NK1.1+ cells, a natural killer cell marker, increased, while the frequency of cells expressing Ly6G+, a neutrophil marker, decreased. The frequency of cells expressing RORγT, a gene involved in Th17+ cell differentiation, also decreased.

[0055] Based on the above results, it was found that the peptides of the present disclosure can alleviate the symptoms of colitis by reducing the frequency of inflammation-inducing cells.

[0056] Furthermore, peptide 1, peptide 2, and peptide 4 were each conjugated with a different fluorescent substance and then orally administered to normal mice, and the frequency of fluorescently labeled cells in intraperitoneal macrophages was measured 48 hours and 72 hours later.

[0057] The results are as follows Figure 10 As shown, the P2 and P4 peptides were maintained for up to 48 hours, and P1 was maintained for up to 72 hours.

[0058] Based on the above results, it was found that the orally administered peptide reaches the abdominal cavity to participate in the immune response, thereby inducing relief of colitis symptoms.

[0059] 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 inflammatory diseases <130> ADP-2020-0490 <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 treating psoriasis or inflammatory bowel disease, comprising a peptide mixture as an active ingredient, wherein the peptide mixture consists 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.

2. The pharmaceutical composition according to claim 1, wherein The peptide alleviates inflammation induced by the autoimmune response.

3. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition is in a dosage form selected from the group consisting of oral preparations, external preparations, suppositories and injections.

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