Composition for preventing or treating fungal infections comprising wkymvm peptide or analog or derivative thereof

Abstract

The present invention relates to: an antifungal composition and a pharmaceutical composition for preventing or treating fungal infections, comprising a WKYMVm peptide or an analog or derivative thereof; and a pharmaceutical composition for combination therapy for preventing or treating fungal infections, the pharmaceutical composition comprising a WKYMVm peptide or an analog or derivative thereof and an immune enhancer. The compositions induce an increase in immune cells such as neutrophils and B cells, reduce inflammation by a decrease in pro-inflammatory cytokines and an increase in anti-inflammatory cytokines, and significantly increase fungal death, thus having the effect of inhibiting lung damage or kidney damage caused by fungal infections, and therefore can be effectively used as an antifungal agent or a therapeutic agent for fungal infections.

Description

Composition for preventing or treating fungal infections comprising WKYMVM peptide, analogue or derivative thereof

[0001] The present invention relates to an antifungal composition comprising a WKYMVm peptide, an analogue or derivative thereof, a pharmaceutical composition for preventing or treating fungal infections, and a pharmaceutical composition for combined administration for preventing or treating fungal infections comprising a WKYMVm peptide, an analogue or derivative thereof, and an immunostimulant.

[0002]

[0003] Patients with compromised immune systems due to cancer, organ transplants, and bone marrow transplants are at risk for life-threatening opportunistic infections. In particular, the frequency of fungal infections has steadily increased over the past two decades. Fungal infections are known to cause sepsis, meningitis, pneumonia, invasive candidiasis, and acute nephritis in major organs such as the brain, lungs, and kidneys. Invasive fungal infections can cause serious organ damage and death, with mortality rates reaching 20-30% and 50% in solid organ transplant recipients and hematopoietic stem cell transplant recipients, respectively. The global incidence of fungal infections is also steadily increasing, with cryptococcal meningitis causing approximately 950,000 new cases and 620,000 deaths annually, and Pneumocystis pneumonia causing approximately 400,000 new cases and 150,000 deaths annually.

[0004] Current antifungal treatments include allylamines, azoles, and polyenes, which inhibit the ergosterol synthesis pathway, a class of drugs that selectively exist in the fungal cell membrane. Echinocandins, which inhibit the synthesis of beta-glucan, a major component of the fungal cell wall, are also available. However, existing treatments have problematic side effects, including renal toxicity and interactions with medications for underlying conditions. Furthermore, the misuse of existing antifungal agents is leading to the spread of antifungal-resistant fungi, posing a serious problem. Therefore, the development of novel targeted treatments to control antifungal-resistant fungal infections is urgently needed.

[0005] Accordingly, the inventors of the present invention completed the present invention by confirming that the WKYMVm peptide, which is a formyl peptide receptor (FPR) agonist that can regulate the activity of FPR, a cell membrane receptor that plays a key role in innate immunity, has antifungal efficacy and is effective in treating antifungal drug-resistant infections.

[0006]

[0007] The purpose of the present invention is to provide an antifungal composition comprising a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), or an analogue or derivative thereof, as an active ingredient.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating fungal infection, which comprises as an active ingredient a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), or an analogue or derivative thereof.

[0009] Another object of the present invention is to provide a pharmaceutical composition for combination administration for the prevention or treatment of fungal infections, comprising a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), an analogue or derivative thereof; and an immunostimulant as active ingredients.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating neutropenia, which comprises as an active ingredient a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), or an analogue or derivative thereof.

[0011]

[0012] To achieve the above purpose, the present invention provides an antifungal composition comprising, as an active ingredient, a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), or an analogue or derivative thereof.

[0013] In addition, the present invention provides a pharmaceutical composition for preventing or treating fungal infection, which comprises as an active ingredient a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), or an analogue or derivative thereof.

[0014] In addition, the present invention provides a pharmaceutical composition for combination administration for the prevention or treatment of fungal infection, comprising a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), an analogue or derivative thereof; and an immunostimulant as active ingredients.

[0015] In addition, the present invention provides a pharmaceutical composition for preventing or treating neutropenia, which comprises as an active ingredient a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), or an analogue or derivative thereof.

[0016]

[0017] The composition according to the present invention has the effect of inducing an increase in immune cells such as neutrophils and B cells, reducing inflammation by decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines, significantly increasing fungal death, and thereby inhibiting lung damage or kidney damage caused by fungal infection, and thus can be usefully used as an antifungal agent or a treatment for fungal infection, especially as a treatment for fungal infection in patients with serious underlying diseases such as immunosuppression.

[0018]

[0019] Figure 1 shows the results of comparing the survival rate after injecting WKYMVm peptide into an animal model of fungal infection under immunocompromised conditions.

[0020] Figure 2 shows the results of confirming the effect of increasing immune cells after injecting WKYMVm peptide into an animal model of fungal infection under immunocompromised conditions.

[0021] Figure 3 shows the results of confirming the effect of increasing fungal killing capacity (CFU) after injecting WKYMVm peptide into a fungal infection animal model under immunocompromised conditions.

[0022] Figure 4 shows the results of confirming the effect of alleviating lung tissue damage after injecting WKYMVm peptide into an animal model of fungal infection under immunocompromised conditions.

[0023] Figure 5 shows the results of confirming the effect of alleviating kidney tissue damage after injecting WKYMVm peptide into an animal model of fungal infection under immunocompromised conditions.

[0024] Figure 6 shows the results of confirming changes in RBC solubility due to WKYMVm peptide.

[0025] Figure 7 shows the results of confirming the change in HepG2 cell viability due to WKYMVm peptide.

[0026] Figure 8 shows the results confirming the regulation of CYP gene expression by WKYMVm peptide.

[0027] Figure 9 shows the results of confirming the immune enhancement effect by injection of G-CSF (Granulocyte colony-stimulating factor), an immune enhancer, under immunosuppressed conditions.

[0028] Figure 10 shows the results of confirming the immune enhancement effect by injection of GM-CSF (Granulocyte-macrophage colony-stimulating factor), an immune enhancer, under immunosuppressed conditions.

[0029] Figure 11 shows the results of confirming the change in the immune cell profile in the body by injection of WKYMVm peptide and GM-CSF under immunosuppressed conditions.

[0030] Figure 12 shows the results of confirming the neutrophil differentiation stage by injection of WKYMVm peptide, GM-CSF, or GM-CSF / WKYMVm combination in the bone marrow of an animal model of fungal infection under immunocompromised conditions.

[0031] Figure 13 shows the results of confirming the cytokine profile by injection of WKYMVm peptide, GM-CSF, or GM-CSF / WKYMVm combination in the blood of an animal model of fungal infection under immunocompromised conditions.

[0032] Figure 14 shows the results of confirming the cytokine profile by injection of WKYMVm peptide, GM-CSF, or GM-CSF / WKYMVm combination in the kidney of an animal model of fungal infection under immunocompromised conditions.

[0033] Figure 15 shows the results of confirming the fungal killing ability by injection of WKYMVm peptide, GM-CSF, or GM-CSF / WKYMVm combination in an animal model of fungal infection under immunocompromised conditions.

[0034] Figure 16 shows the results of lung damage analysis by injection of WKYMVm peptide, GM-CSF, or GM-CSF / WKYMVm combination in an animal model of fungal infection under immunocompromised conditions.

[0035] Figure 17 shows the results of an analysis of the kidney damage and fungal control effect by injection of WKYMVm peptide, GM-CSF, or GM-CSF / WKYMVm combination in an animal model of fungal infection under immunocompromised conditions.

[0036] Figure 18 shows the results of survival analysis by injection of WKYMVm peptide, GM-CSF, or GM-CSF / WKYMVm combination in an animal model of fungal infection under immunocompromised conditions.

[0037]

[0038] Hereinafter, the present invention will be described in detail.

[0039] The terms used in this invention have been selected from widely used, common terms, taking into account the functionality of the invention. However, these terms may vary depending on the intentions of those skilled in the art or the emergence of new technologies. Furthermore, in certain cases, terms may be arbitrarily selected, and in such cases, their meanings will be described in detail in the description of the relevant embodiments. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their meanings and the overall content of the invention.

[0040] When the present invention is said to “include” a certain component or a certain step, this does not mean that other components or other steps are excluded, but rather that other components or other steps may be further included, unless specifically stated otherwise.

[0041]

[0042] The present invention provides an antifungal composition comprising, as an active ingredient, a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), or an analogue or derivative thereof.

[0043] In one embodiment of the present invention, the peptide, analogue or derivative thereof may induce or promote the activation of a formyl peptide receptor (FPR). That is, the peptide, analogue or derivative thereof is an FPR agonist or agonist. In addition, the formyl peptide receptor may be formyl peptide receptor 1 (FPR 1) or formyl peptide receptor 2 (FPR 2).

[0044] In one embodiment of the present invention, the composition may have antifungal activity against strains selected from the group consisting of strains of the genus Candida spp., genus Cryptococcus spp., genus Trichosporon spp., and genus Aspergillus spp.

[0045] The Candida genus strain may be selected from the group consisting of, but is not limited to, Candida glabrata, Candida albicans, Candida tropicalis, Candida auris, Candida parapsilosis, Candida krusei, Candida guilliermondii, and Candida lusitaniae. In addition, the Cryptococcus genus strain may be, but is not limited to, Cryptococcus neoformans. Additionally, the strain of the genus Trichosporon may be selected from the group consisting of, but is not limited to, Trichosporon asahii, Trichosporon inkin, Trichosporon cutaneum, Trichosporon mucoides, Trichosporon coremiiforme, Trichosporonfaecale, Trichosporonasteroides and Trichosporonovoides. Additionally, the Aspergillus strain may be selected from the group consisting of, but is not limited to, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, and Aspergillus niger.

[0046] Analogs or derivatives of the above peptides may have sequences that differ from the natural amino acid sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues, and may include amino acid side chains or alpha-backbones substituted with one or more other functional groups. Examples of side chain or backbone modified peptide analogs include, but are not limited to, hydroxyproline, in which the pyrrolidine ring is substituted with a hydroxy group, or N-methyl glycine "peptoids." Types of peptide analogs are well known in the art.

[0047] Amino acid substitutions in peptides that do not alter the overall activity of the molecule are also well known in the art (H.Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common substitutions are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In some cases, modifications such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation may also occur.

[0048] In one embodiment of the present invention, the composition may have antifungal activity against antifungal drug-resistant fungi. The above antifungal agents include ciclopirox, ciclopirox olamine, rilopirox, clotrimazole, econazole, isoconazole, ketoconazole, miconazole, tioconazole, bifonazole, fenticonazole, oxiconazole, butoconazole, sertaconazole, sulconazole, luriconazole, omoconazole, nystatin, natamycin, amphotericin, naftafine, terbinafine, butenafine, amorolfine, albaconazole, efinaconazole, fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole, abafungin, caspofungin, anidulafungin, micafungin, benzoic acid, haloprozin, undecylenic acid, griseofulvin, These include, but are not limited to, tolnaftate or flucytosine.

[0049] In one embodiment of the present invention, the composition may induce fungal death through an increase in immune cells, and the immune cells may be selected from the group consisting of neutrophils, monocytes, dendritic cells, T cells, and B cells, and preferably neutrophils and B cells, but are not limited thereto.

[0050] In addition, the present invention provides a pharmaceutical composition for preventing or treating fungal infection, which comprises a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m) as an active ingredient.

[0051] In one embodiment of the present invention, the fungal infection may be selected from the group consisting of candidiasis, cryptococcosis, aspergillosis, mucormycosis, cryptococcal meningitis, Pneumocystis pneumonia, fungal sepsis, and fungal acute nephritis. In particular, in the present invention, the fungal killing ability of the composition of the present invention was confirmed through an animal model infected with a Candida genus strain under immunocompromised conditions, and it was confirmed that the composition of the present invention has an effect of inhibiting, alleviating, or treating lung and kidney damage caused by fungal infection, thereby proving that it can be used for the prevention or treatment of fungal infections.

[0052] In addition, the present invention provides a pharmaceutical composition for combination administration for the prevention or treatment of fungal infection, comprising a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m); and an immunostimulant as active ingredients.

[0053] In one embodiment of the present invention, the immune enhancer may be selected from the group consisting of G-CSF (Granulocyte colony-stimulating factor) and GM-CSF (Granulocyte-macrophage colony-stimulating factor), but is not limited thereto.

[0054] In particular, in the present invention, it was confirmed that the survival rate of an animal model infected with a Candida genus strain was completely restored through a synergistic effect of the WKYMVm peptide and an immune enhancer, thereby confirming that the present invention can be used for the prevention or treatment of fungal infections through combined administration.

[0055] In one embodiment of the present invention, the WKYMVm peptide and the immunostimulant may be administered simultaneously or sequentially.

[0056] In addition, the present invention provides a pharmaceutical composition for preventing or treating neutropenia, which comprises a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m) as an active ingredient.

[0057] In one embodiment of the present invention, the composition may increase the number of neutrophils decreased due to a fungal infection or drug treatment.

[0058] The pharmaceutical composition of the present invention may additionally include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means non-toxic to cells or humans exposed to the composition. Any carrier known in the art, such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, etc., may be used without limitation.

[0059] In addition, the pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. Furthermore, it can be used in the form of external preparations for skin in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or gels. Carriers, excipients, and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, the composition is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0060] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the copper sap extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0061] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" as used herein refers to introducing a given substance into a subject through an appropriate method, and the composition may be administered via any common route as long as it can reach the target tissue. Examples of such routes include, but are not limited to, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0062] The term "subject" above refers to all animals, including rats, mice, and livestock, including humans. Preferably, it may be a mammal, including humans.

[0063] The above term, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be easily determined by those skilled in the art based on factors including the patient's sex, age, weight, health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and excretion rate, duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field. Administration may be administered once a day at the above recommended dosage or may be administered in several divided doses.

[0064]

[0065] In addition, the present invention provides a method for preventing or treating a fungal infection, comprising administering to a subject a therapeutically effective amount of a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), an analogue or derivative thereof.

[0066] In addition, the present invention provides a method for preventing or treating a fungal infection, comprising administering to a subject a therapeutically effective amount of a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), an analogue or derivative thereof; and a therapeutically effective amount of an immunostimulant.

[0067] In addition, the present invention provides a method for preventing or treating neutropenia, comprising administering to a subject a therapeutically effective amount of a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), an analogue or derivative thereof.

[0068] The above therapeutically effective amount is preferably applied differently depending on various factors including the type and degree of the response to be achieved, the specific composition including whether other agents are used in some cases, the age, body weight, general health, sex and diet of the subject, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field. Therefore, the effective amount of the composition suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters.

[0069] The above object is applicable to any mammal, which includes not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs and cats.

[0070]

[0071] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0072]

[0073] Example 1. Establishment of an animal model of fungal infection under immunocompromised conditions.

[0074] In the present invention, an immunocompromised and fungal infection animal model was established to simulate the environment in which antifungal drug-resistant fungal infections pose a serious threat to patient health. Briefly, mice were injected with the anticancer drug cyclophosphamide (CY) to establish an immunocompromised animal model. This animal model was then infected with the antifungal drug-resistant fungus Candida glabrata to establish an immunocompromised fungal infection animal model.

[0075]

[0076] Example 2. Analysis of therapeutic efficacy by WKYMVm peptide

[0077] 2.1. Analysis of the efficacy of increasing survival rate

[0078] An experiment was conducted to analyze the survival rate of mice for 14 days after injecting WKYMVm peptide, a formyl peptide receptor (FPR) agonist, at different doses (1 mg / kg, 2 mg / kg, or 4 mg / kg) into an animal model of fungal infection under the established immunosuppressed conditions. As a result, it was confirmed that the survival rate increased in a dose-dependent manner in the group injected with WKYMVm peptide compared to the control group (Vehicle) (Fig. 1).

[0079]

[0080] 2.2. Analysis of immune cell increase efficacy

[0081] Next, the WKYMVm peptide, a formyl peptide receptor (FPR) agonist, was injected at 4 mg / kg into the established fungal infection animal model under immunocompromised conditions, and the immune cell profile of neutrophils, B220 B cells, and precursor cells (CMP, Common myeloid progenitor; and GMP, Granulocyte / monocyte progenitor) was analyzed using a flow cytometer (Cytek™ Aurora). As a result, it was confirmed that in the group treated with WKYMVm peptide, compared to the control group (Vehicle), the precursor cells GMP and CMP+GMP increased, and neutrophils and B cells significantly increased in the bone marrow (BM) and blood, which are immune-active sites (Fig. 2). This confirmed that the WKYMVm peptide induces an increase in immune cells in the fungal infection animal model under immunocompromised conditions.

[0082]

[0083] 2.3. Fungal killing activity analysis

[0084] Next, an experiment was conducted to analyze the fungal killing capacity (CFU) in the peritoneal cavity, kidney, and blood after injecting 4 mg / kg of WKYMVm peptide, a formyl peptide receptor (FPR) agonist, into an animal model of fungal infection under the established immunocompromised conditions. As a result, it was confirmed that the group injected with WKYMVm peptide showed a significant decrease in fungi in the kidney, peritoneal fluid (PF), and blood compared to the control group (Vehicle) (Fig. 3). This confirmed that WKYMVm peptide induces an increase in fungal killing capacity.

[0085]

[0086] 2.4. Analysis of efficacy in alleviating lung and kidney tissue damage

[0087] Next, after injecting 4 mg / kg of WKYMVm peptide, a formyl peptide receptor (FPR) agonist, into the established fungal infection animal model under immunocompromised conditions, an experiment was performed to check for damage to lung and kidney tissues using H&E staining or PAS staining. As a result, it was confirmed that lung tissue damage was alleviated in the group injected with WKYMVm peptide compared to the control group (Vehicle) (Fig. 4). In addition, it was confirmed that kidney tissue damage was also alleviated in the group injected with WKYMVm peptide (Fig. 5). Thus, it was confirmed that WKYMVm peptide has an effect in alleviating lung damage or kidney damage caused by fungal infection.

[0088]

[0089] 2.5. Results

[0090] From the above results, it was confirmed that the WKYMVm peptide of the present invention has the effect of inducing an increase in immune cells, inducing fungal death, and thereby alleviating lung damage or kidney damage caused by fungal infection, thereby increasing the survival rate of an animal model, thereby confirming that it can be used as an antifungal agent or a treatment for fungal infection.

[0091]

[0092] Example 3. Preliminary toxicity test

[0093] To determine whether the WKYMVm peptide, an FPR agonist, exhibits cytotoxicity, the WKYMVm peptide was treated at various concentrations, and the hemolysis of mouse red blood cells and the viability of human hepatocyte HepG2 cells were determined using the Hemolysis Assay and the CCK-8 Viability Assay, respectively. As a result, it was confirmed that there was no change in hemolysis or cell viability at all concentrations of the WKYMVm peptide (Fig. 7). This confirms that the WKYMVm peptide of the present invention is not cytotoxic.

[0094] In addition, in order to confirm whether the WKYMVm peptide, which is an FPR agonist, affects the induction of CYP (Cytochrome) genes, which are drug-metabolizing enzymes that affect various drug metabolism in human hepatocyte HepG2 cells, an experiment was performed to confirm the expression levels of CYP1A2, CYP2B6, and CYP3A4 genes through Real-Time PCR after treating with WKYMVm peptide at various concentrations. As a result, it was confirmed that the WKYMVm peptide did not change the expression levels of CYP1A2, CYP2B6, and CYP3A4 genes (Fig. 8). This confirmed that the WKYMVm peptide of the present invention does not affect drug metabolism.

[0095]

[0096] Example 4. Efficacy analysis by immune enhancers

[0097] After injecting G-CSF (Granulocyte colony-stimulating factor, 250 μg / kg), an immunostimulatory agent that induces granulocytes, into an immunosuppressed animal model induced by anticancer drug injection, the immune cell profile of neutrophil precursors, neutrophils, and B cells in the body was analyzed using a flow cytometer (Cytek™ Aurora). As a result, in the group injected with the immunosuppressant G-CSF, the neutrophil precursor cells, Metamyelocytes (MM) and Banded Cells (BC), both in the bone marrow (BM), blood, and spleen, increased compared to the immunosuppressed animal model (CY), and the number of neutrophils also increased (Fig. 9). However, there was no significant difference in B cells due to the immunosuppressant G-CSF.

[0098] In addition, after injecting GM-CSF (Granulocyte-macrophage colony-stimulating factor, 500 μg / kg), an immune booster that induces granulocytes, monocytes, and dendritic cells into an immunocompromised animal model induced by injection of an anticancer drug, the in vivo immune cell profile of monocytes, neutrophils, and dendritic cells was analyzed using a flow cytometer (Cytek™ Aurora). As a result, in the group injected with the immune booster GM-CSF, monocytes, neutrophils, and dendritic cells in the bone marrow (BM) were confirmed to have slightly increased compared to the immunocompromised animal model (CY), and neutrophils were confirmed to have increased in the blood (Fig. 10). However, there was no significant difference in monocytes in the blood and spleen due to the immune booster GM-CSF.

[0099] Thus, it was confirmed that the number of immune cells in the body, such as neutrophils, increased through injection of G-CSF or GM-CSF, which are immune boosters, into an immunocompromised animal model.

[0100]

[0101] Example 5. Analysis of the efficacy of combined treatment with WKYMVm peptide and an immune enhancer.

[0102] 5.1. Analysis of immune cell increase efficacy

[0103] An experiment was conducted to determine the effect of co-administration of WKYMVm peptide and an immunostimulant in an animal model of fungal infection under immunosuppressed conditions. The immunostimulant GM-CSF (500 μg / kg) was injected before infection with the antifungal drug-resistant fungus Candida glabrata, and the WKYMVm peptide (4 mg / kg) was injected after infection with Candida glabrata. First, the immune cell profile of monocytes, neutrophils, neutrophil precursors, and B cells was analyzed using a flow cytometer (Cytek™ Aurora).

[0104] As a result, in the group injected with the immune booster GM-CSF, the population of cells that had CD16 / 32 and highly expressed Ly6C, a monocyte marker, and Ly6G, a neutrophil marker, increased, confirming an increase in monocytes and neutrophils (Fig. 11). In addition, in the group injected with the WKYMVm peptide alone, it was confirmed that both monocytes and neutrophils increased (Fig. 11). Additionally, in the group injected with the GM-CSF / WKYMVm combination, it was confirmed that both monocytes and neutrophils increased significantly (Fig. 11).

[0105] In addition, in the group injected with WKYMVm peptide alone, it was confirmed that the neutrophil precursor cells MB (Metamyeloblast) and MM (Metamyelocyte) significantly increased compared to the control group (CY + Vehicle) (Fig. 12). In addition, in the group injected with the immune booster GM-CSF and the group injected with GM-CSF / WKYMVm together, it was confirmed that the MM (Metamyelocyte) and BC (Banded cell) significantly increased compared to the control group (CY + Vehicle) (Fig. 12). Thus, it was confirmed that injection of WKYMVm peptide alone or GM-CSF / WKYMVm together induced an increase in immune cells in an animal model of fungal infection under immunocompromised conditions.

[0106]

[0107] 5.2. Analysis of cytokine reduction efficacy

[0108] After injecting WKYMVm peptide (4 mg / kg), the immunostimulatory agent GM-CSF (500 μg / kg), and a combination of GM-CSF and WKYMVm into an animal model of fungal infection under immunosuppressed conditions, the cytokine profile was analyzed. As a result, it was confirmed that the blood cytokines IL-6, CCL2, and IL-10 were significantly reduced in the group injected with WKYMVm peptide alone compared to the control group (CY+Vehicle) (Fig. 13). Similarly, the groups injected with GM-CSF or a combination of GM-CSF and WKYMVm also showed a significant decrease in IL-6, CCL2, and IL-10 cytokines (Fig. 13).

[0109] In addition, it was confirmed that the pro-inflammatory cytokines IL-6, CCL2, and IL-1β were significantly reduced in the kidney groups injected with WKYMVm peptide alone, GM-CSF alone, or GM-CSF / WKYMVm combination compared to the control group (CY+Vehicle) (Fig. 14). In addition, it was confirmed that the anti-inflammatory cytokines IL-10 and IFN-γ were increased in the groups injected with WKYMVm peptide alone and GM-CSF alone compared to the control group (CY+Vehicle) (Fig. 14).

[0110] Thus, it was confirmed that administration of WKYMVm peptide alone or in combination with WKYMVm peptide and GM-CSF had the effect of reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines in an animal model of fungal infection under immunocompromised conditions.

[0111]

[0112] 5.3. Fungal killing activity analysis

[0113] An experiment was conducted to analyze the fungal killing capacity (CFU) in the blood and kidney after administering WKYMVm peptide (4 mg / kg), the immunostimulatory agent GM-CSF (500 μg / kg), and the combination of WKYMVm peptide and GM-CSF to an animal model of fungal infection under immunocompromised conditions. As a result, it was confirmed that fungi were significantly reduced in the blood and kidney in all groups administered WKYMVm peptide alone, GM-CSF alone, or the combination of WKYMVm peptide and GM-CSF compared to the control group (CY+Vehicle) (Fig. 15). This confirmed that administration of WKYMVm peptide alone or the combination of WKYMVm peptide and GM-CSF significantly increased the fungal killing capacity.

[0114]

[0115] 5.4. Analysis of efficacy in alleviating lung and kidney tissue damage

[0116] After injecting WKYMVm peptide (4 mg / kg), the immunostimulator GM-CSF (500 μg / kg), and a combination of GM-CSF and WKYMVm into an immunocompromised fungal infection animal model, the extent of lung and kidney damage was analyzed using H&E staining. As a result, it was confirmed that Candida glabrata infection induced severe lung and kidney damage in the immunocompromised fungal infection animal model (CY) (Figs. 16 and 17). However, in the group injected with GM-CSF, lung and kidney damage was suppressed, and in particular, in the group injected with WKYMVm peptide or GM-CSF / WKYMVm combination, lung and kidney damage was almost completely suppressed (Figs. 16 and 17).

[0117] In addition, PAS staining was used to analyze Candida glabrata colonies in the kidney. As a result, Candida glabrata colonies were confirmed in large numbers in an animal model of fungal infection (CY) under immunocompromised conditions, whereas Candida glabrata colonies were almost completely eliminated in all groups injected with WKYMVm peptide alone, GM-CSF alone, or a combination of GM-CSF and WKYMVm (Fig. 17). This result is consistent with the measurement value of the fungal killing activity mentioned above.

[0118] Thus, it was confirmed that injection of WKYMVm peptide alone or in combination with GM-CSF / WKYMVm significantly increases fungal killing ability, thereby significantly alleviating or treating fungal-induced lung and kidney damage.

[0119]

[0120] 5.5. Analysis of the efficacy of increasing survival rate

[0121] An experiment was conducted to analyze the survival rate after injecting WKYMVm peptide (4 mg / kg), the immunostimulant GM-CSF (500 μg / kg), and a combination of GM-CSF and WKYMVm into an immunocompromised fungal infection animal model. As a result, it was confirmed that the survival rate was significantly reduced in the immunocompromised fungal infection animal model (CY+Vehicle), whereas the survival rate was significantly increased in the groups injected with WKYMVm peptide alone or GM-CSF alone (Fig. 18). In particular, the group injected with GM-CSF / WKYMVm together showed a survival rate of 100%, confirming that the survival rate was completely restored by the synergistic effect of WKYMVm peptide and GM-CSF (Fig. 18).

[0122]

[0123] 5.6. Results

[0124] From the above results, it was confirmed that the combined administration of WKYMVm peptide and an immune enhancer in the present invention induces an increase in immune cells, reduces inflammation by decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines, significantly increases fungal death, thereby suppressing lung damage or kidney damage caused by fungal infection, and increasing the survival rate of animal models, thereby confirming that it can be used as an antifungal agent or a treatment for fungal infection.

[0125]

[0126] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0127] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. An antifungal composition comprising a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), or an analogue or derivative thereof, as an active ingredient.

2. In paragraph 1, An antifungal composition, wherein the peptide, an analogue or derivative thereof, induces or promotes activation of a formyl peptide receptor (FPR).

3. In paragraph 1, An antifungal composition having antifungal activity against strains selected from the group consisting of strains of the genus Candida spp., genus Cryptococcus spp., genus Trichosporon spp., and genus Aspergillus spp.

4. In paragraph 1, An antifungal composition having antifungal activity against antifungal drug-resistant fungi.

5. In paragraph 1, The above composition is an antifungal composition that induces fungal death through an increase in immune cells or activation of immune cells.

6. In paragraph 1, An antifungal composition, wherein the immune cells are selected from the group consisting of neutrophils, monocytes, dendritic cells, T cells, and B cells.

7. A pharmaceutical composition for preventing or treating fungal infection, comprising as an active ingredient a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), or an analogue or derivative thereof.

8. In paragraph 7, A pharmaceutical composition, wherein the fungal infection is selected from the group consisting of candidiasis, cryptococcosis, aspergillosis, mucormycosis, cryptococcal meningitis, Pneumocystis pneumonia, fungal sepsis, and fungal acute nephritis.

9. A pharmaceutical composition for combination administration for the prevention or treatment of fungal infection, comprising a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), an analogue or derivative thereof; and an immunostimulant as active ingredients.

10. In paragraph 9, A pharmaceutical composition, wherein the above-mentioned immune enhancer is selected from the group consisting of G-CSF (Granulocyte colony-stimulating factor) and GM-CSF (Granulocyte-macrophage colony-stimulating factor).

11. In paragraph 9, A pharmaceutical composition wherein the WKYMVm peptide, an analogue or derivative thereof, and an immunostimulant are administered simultaneously or sequentially.

12. A pharmaceutical composition for preventing or treating neutropenia, comprising as an active ingredient a WKYMVm peptide consisting of an amino acid sequence of Trp(W) - Lys(K) - Tyr(Y) - Met(M) - Val(V) - D-Met(m), or an analogue or derivative thereof.

13. In paragraph 12, A pharmaceutical composition, wherein the composition increases the number of neutrophils decreased due to a fungal infection or drug treatment.

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